TNFR2-conjugated polypeptide and method for its use

Abstract

The present invention relates to an engineered modified stephin A polypeptide variant that binds to TNFR2, a polynucleotide encoding the engineered modified TNFR2-binding stephin A polypeptide variant, cells expressing the polypeptide variant, a pharmaceutical formulation of the polypeptide variant, and therapeutic applications of the polypeptide variant for various human diseases, including inflammatory diseases, autoimmune diseases, and cancer.

Description

[Technical Field]

[0001] Controlling cell-mediated and humoral immune responses is a crucial part of a healthy immune system. Abnormal regulation of immune responses, particularly those involving T cells and B cells, is associated with a variety of human diseases, and increased inappropriate immune responses to various autoantigens or external antigens play a causal role in the pathology of other immune disorders, including autoimmune diseases, asthma, allergic reactions, GvHD, graft rejection, and cancer.

[0002] These diseases are mediated by T and B lymphocytes that react to autoantigens, allergens, or harmless derived antigens such as grafts. Regulatory T cells (Tregs) suppress the activity of immune cells that cross-react with autologous major tissue complex (MHC) proteins and other benign antigens. The best-known Treg populations include CD4+, CD25+, FoxP3+ Tregs and CD17+ Tregs.

[0003] Tumor necrosis factor receptor (TNFR) subtypes 1 and 2 are specifically expressed on the surface of Treg cells. Activation of TNFR1 by soluble TNF-α enhances the caspase signaling chain reaction, inducing Treg cell apoptosis. On the other hand, activation of TNFR2, mainly by membrane-bound TNF-α, induces signaling via the extracellular signal-modulating phosphorylation enzyme (MAPK) signaling pathway. This pathway regulates TRAF2 / 3 and NF-κB-mediated gene transcription, promoting cell proliferation and evasion of cell apoptosis. [Background technology]

[0004] The importance of TNFR2's role in Treg proliferation and function has been demonstrated in various studies. TNFR2-deficient mice show reduced thymic and peripheral Treg counts (2013_Chen X, et al. PMID:23277487), indicating that TNFR2- / -Tregs cannot regulate inflammatory responses in vivo (2008_Van Mierlo GJ, et al. PMID:18292492). In humans, Treg cells have been shown to express higher levels of TNFR2 than T effector cells (2013_Okubo Y, et al. PMID:24193319 & 2002_Annunziato F, et al. PMID:12163566), and TNFR2+Treg cells have been reported to most strongly suppress the proliferation and cytokine production of co-cultured T-responder cells (2010_2013_Chen X, et al. PMID:20127680). Furthermore, TNFR2 has been reported to play an important role in carcinogenesis and tumor growth by mediating the TNF response in immunosuppressive cells, thereby allowing immune evasion and tumor development (Sheng Y., et al. doi:10.3389 / fimmu.2018.01170).

[0005] Furthermore, in autoimmune diseases affecting the central nervous system (CNS), particularly multiple sclerosis (MS), pathogenic lymphocytes are induced in the periphery, penetrate the CNS, and induce local inflammation and demyelination. Demyelination refers to damage to the protective layer (myelin sheath) covering the nerve fibers of the brain. Strategies to prevent or reverse demyelination are being studied as treatments for multiple sclerosis. Interestingly, several studies have reported that TNFR2 is involved in neuroprotection. For example, in a demyelinating mouse model induced by cuprizone, TNFR2 plays an important role in the regeneration of oligodendrocytes, which are cells primarily responsible for maintaining and generating the myelin sheath covering axons, while TNFR1-mediated TNF signaling has been reported to promote neuronal demyelination (2001_HA Arnett et al. PMID:11600888).

[0006] TNFR2, which is involved in cell survival or growth, is an attractive target for treating diseases such as autoimmune diseases, GvHD, graft rejection, allergic reactions, asthma, and cancer.

[0007] AFFIMER® is a small, highly stable protein molecule developed by Avacta Life Sciences Limited, engineered from the endogenous protein, stephin A protein. AFFIMER contains two short peptide sequences with arbitrary sequences and an N-terminal sequence, and can bind to target substances with high affinity and specificity in a manner similar to monoclonal antibodies. AFFIMER exhibits significantly improved binding affinity and specificity compared to free peptide libraries, and is extremely small and highly stable compared to antibodies, making it a highly promising next-generation alternative drug platform (US Patents 9,447,170, 885,3131, etc.). [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention relates to a polypeptide that is engineered based on the natural stephin A protein and can specifically bind to TNFR2, thereby providing an AFFIMER polypeptide that can bind to TNFR2 with excellent affinity and specificity. [Means for solving the problem]

[0009] In one view, the present invention is 1x10 -6 We provide an engineered polypeptide that specifically binds to TNFR2 in Kd M, and the polypeptide is a variant of the stephin A protein.

[0010] In other words, the present invention provides a fusion protein comprising a dimer, trimer, or tetramer of an engineered polypeptide, which may optionally contain one or more linkers.

[0011] In another aspect, the present invention provides a fusion protein in which one or more of the engineered polypeptides are linked to a therapeutic or diagnostic moiety.

[0012] In another aspect, the present invention provides a polynucleotide or a set of polynucleotides encoding an engineered polypeptide or a fusion protein.

[0013] In one aspect, the present invention provides a delivery vehicle comprising the polynucleotide.

[0014] In another aspect, the present invention provides a plasmid or a minicircle comprising the polynucleotide.

[0015] In another aspect, the present invention provides an mRNA comprising an open reading frame encoding an engineered polypeptide.

[0016] In one aspect, the present invention provides a lipid nanoparticle, optionally a cationic lipid nanoparticle, comprising the mRNA.

[0017] In another aspect, the present invention provides a pharmaceutical composition comprising an engineered polypeptide, a fusion protein, or a delivery vehicle, optionally comprising a pharmaceutically acceptable excipient.

[0018] In one aspect, the present invention provides a conjugate in which an engineered polypeptide or a fusion protein is linked to a pharmacologically active moiety.

[0019] In another aspect, the present invention provides a method of administering the pharmaceutical composition to a subject.

[0020] In one aspect, the present invention provides an engineered polypeptide, a fusion protein, or a delivery vehicle for use as a medicament.

[0021] In another respect, the present invention provides an engineered polypeptide that competes with an engineered polypeptide in binding to TNFR2.

[0022] In another respect, the present invention provides an engineered polypeptide that binds to the same TNFR2 epitope. [Brief explanation of the drawing]

[0023] [Figure 1] This outlines the selection strategy for the human TNFR2 selection campaign. Both libraries were screened through solution and passive selection processes. The hTNFR2 concentration was decreased with each round, and the number of washing steps between rounds 1 and 2 was increased. In passive selection, Avacta's exclusive Type 3 Affimer phage library (US Pat. No. 9.932.575) was used, and deselection and competition methods were employed simultaneously. In the deselection method, the Fc concentration in round n+1 was the hTNFR2 concentration in round n. In the competition method, the Fc concentration was set to a molar ratio 10 times that of the hTNFR2 concentration specified in that round. In the case of passive selection using Avacta's exclusive Type 1 Affimer phage library (US Pat. No. 8,841,491), only a deselection method was performed, and a round 4 was added because the initial screening in round 3 showed high diversity and hit rate. In the case of solution selection, both libraries were subjected to competitive methods to reduce the enrichment of Fc-conjugated phage-displayed Affimer polypeptides. Streptavidin- and neutraavidin-coated beads were used alternately in each round to reduce the enrichment of streptavidin- and / or neutraavidin-conjugated phage-displayed peptides. [Figure 2] This shows the AFFIMER polypeptide that binds to Expi293FTM cells that overexpress human TNFR2. [Figure 3] This shows titration test data indicating the inhibition rate (%) and IC50 at 3 μM for the best inhibitor. [Figure 4] This shows the results of two repeated SEAP trials, ranking 20 clones. [Figure 5] This shows the cross-reactivity of the hTNFR2AFFIMER clone and Cynomolgus TNFR2 repeated three times. [Modes for carrying out the invention]

[0024] AFFIMER Stefin polypeptides are a subgroup belonging to the cystatin family of proteins, and include proteins containing numerous cystatin-like sequences. The cystatin-based Stefin subgroup consists of relatively small single-domain proteins comprising approximately 100 amino acids. These proteins do not undergo known post-translational modifications and lack disulfide bonds, and are presumed to be capable of identical folding in diverse extracellular and intracellular environments. Stefin A itself is a single-domain protein consisting of 98 amino acids and exists as a monomer. The structure of Stefin A has already been elucidated, and Stefin A can be rationally modified into an AFFIMER polypeptide. Since the only known biological activity of cystatin is the inhibition of cathepsin activity, it is possible to thoroughly evaluate the presence or absence of residual biological activity in genetically modified proteins.

[0025] "AFFIMER polypeptide" (hereinafter also referred to as "AFFIMER protein") refers to a small, highly stable protein that is a variant of the stephin polypeptide. AFFIMER proteins contain two peptide loops and an N-terminal sequence, all of which can be randomized to bind to target proteins with high affinity and specificity. This is similar to the approach of monoclonal antibodies. Stabilization of the two peptides by the stephin A protein scaffold restricts the structural sequences the peptides can take, resulting in improved binding affinity and specificity compared to free peptide libraries. Such engineered, modified non-antibody-binding proteins are designed to mimic the molecular recognition properties of monoclonal antibodies for a variety of applications. Mutations can also be made to other parts of the stephin A polypeptide sequence, which can improve the properties of affinity reagents, such as stability and tolerance over a wide temperature and pH range.

[0026] In some embodiments, the AFFIMER polypeptide may include a sequence having substantial identity with a stephin A-derived sequence, such as a sequence having substantial identity with human stephin A. Those skilled in the art will understand that modifications to the scaffold sequence can be made from this specification without departing the scope of this disclosure. In particular, the AFFIMER polypeptide may include an amino acid sequence having at least 25%, 35%, 45%, 55%, or 60% or more identity with the sequence corresponding to human stephin A, and may have, for example, 70% or more, 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, or 95% or more identity. In such cases, the sequence mutation does not adversely affect the scaffold's ability to bind to its intended target (e.g., TNFR2), nor does the biological function of wild-type stephin A removed by the mutation described herein be restored or newly generated.

[0027] An "AFFIMER agent" refers to a protein containing the AFFIMER polypeptide sequence and other modifications (e.g., conjugation, post-translational modification, etc.) that has therapeutic activity for administration to an individual.

[0028] An "AFFIMER-linked conjugate" refers to an AFFIMER formulation containing an AFFIMER polypeptide sequence to which at least one moiety is linked via chemical conjugation other than the formation of a continuous peptide bond via the C-terminus or N-terminus of the AFFIMER polypeptide portion. An AFFIMER-linked conjugate can also be an "AFFIMER-drug conjugate," which refers to an AFFIMER formulation to which at least one pharmacologically active moiety is linked. An AFFIMER-linked conjugate can also be an "AFFIMER-tag conjugate," which refers to an AFFIMER formulation to which at least one detectable group (e.g., a detectable label) is linked.

[0029] An "encoded AFFIMER polynucleotide" refers to a nucleic acid construct that, when introduced into cells and expressed, generates the intended AFFIMER formulation.

[0030] TNFR2 Tumor necrosis factor receptor 2 ("TNFR2"), along with tumor necrosis factor receptor 1 (TNFR1), is a type 1 membrane-bound receptor that binds to tumor necrosis factor α (TNFα). TNFR2 is also known as p75, TNF Receptor Superfamily Member 1B, or CD120b, and is encoded in humans by the TNFRSF1B gene. Human TNFR2 is expressed as a protein with a length of 461 amino acids and a molecular weight of 48 kDa (UniProt P20333). In mice, the TNFR2 protein has a length of 474 amino acids and a molecular weight of 50 kDa.

[0031] A "TNFR2 AFFIMER agent" refers to an AFFIMER agent containing an AFFIMER polypeptide that binds to at least one TNFR2 molecule. In particular, it binds to human TNFR2 and selectively to macaque (cynomolgus) TNFR2, with a dissociation constant (Kd) of 10. ―6 This refers to cases where M is less than or equal to M. In some embodiments, the TNFR2 AFFIMER formulation has a Kd of 1 × 10 ―7 M or less, 1×10 ―8 M or less, 1×10 ―9 M or less or 1 × 10 ―10 It binds to TNFR2 at a Kd value of 1 × 10⁻¹⁶ or less. It should be understood that in this specification, the terms “TNFR2 AFFIMER polypeptide,” “TNFR2 AFFIMER protein,” and “engineered modified TNFR2-binding steroidin A polypeptide variant” are used interchangeably. Therefore, “TNFR2 AFFIMER polypeptide” means a Kd of 1 × 10⁻¹⁶ ―6 This refers to an engineered modified polypeptide that specifically binds to TNFR2 at M or less, and the engineered modified polypeptide is a variant of the stephin A protein.

[0032] Polypeptides A polypeptide (including peptides and proteins) refers to a polymer of amino acids of any length.

[0033] The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also includes naturally or artificially modified amino acid polymers, including, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other operations such as conjugation with labeling components. Furthermore, the term includes, for example, polypeptides containing one or more amino acid analogs (e.g., non-natural amino acids) and a variety of other modifications known in the art.

[0034] Amino acids (also referred to herein as amino acid residues) are involved in one or more peptide bonds in a polypeptide. Generally, the abbreviations for amino acids used herein are based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). For example, Met, Ile, Leu, Ala, and Gly represent the "residues" of methionine, isoleucine, leucine, alanine, and glycine, respectively. A "residue" refers to a radical obtained by removing the OH portion of the carboxyl group (-COOH) and the H portion of the α-amino group (-NH2) from the α-amino acid in question. An "amino acid side chain" refers to the amino acid portion excluding the -CH(NH2)COOH portion, as defined in KDKopplePeptides and Amino Acids, WABenjamin Inc., New York and Amsterdam, 1966, pp. 2 and 33.

[0035] In many cases, the amino acids described herein may be naturally occurring amino acids in proteins, or products of anabolic or catabolic reactions of amino acids containing amino and carboxyl groups. Particularly suitable amino acid side chains include those selected from the following amino acid side chains: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, tryptophan, and peptidylglycan, amino acids and amino acid analogs identified as components of bacterial cell walls.

[0036] Amino acid residues with "basic sidechains" include arginine (Arg), lysine (Lys), and histidine (His). Amino acid residues with "acidic sidechains" include glutamic acid (Glu) and aspartic acid (Asp). Amino acid residues with "neutral polar sidechains" include serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), cysteine ​​(Cys), and tyrosine (Tyr). Amino acid residues with "neutral non-polar sidechains" include glycine (Gly), alanine (Ala), valine (Val), isoleucine (Ile), leucine (Leu), methionine (Met), proline (Pro), tryptophan (Trp), and phenylalanine (Phe). Amino acid residues with "non-polar aliphatic sidechains" include glycine (Gly), alanine (Ala), valine (Val), isoleucine (Ile), and leucine (Leu). Amino acid residues with "hydrophobic sidechains" include alanine (Ala), valine (Val), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp). Amino acid residues with "small hydrophobic sidechains" include alanine (Ala) and valine (Val). Amino acid residues with "aromatic sidechains" include tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe).

[0037] The specific amino acid residues described herein include not only natural amino acids but also their analogs, derivatives, and congeners. For example, when the StefinA protein variant of the present invention is produced by chemical synthesis, it may include, but is not limited to, amino acid analogs such as cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxyphenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring amino acid metabolites or precursors having side chains suitable for this specification are recognized by those skilled in the art and are included within the scope of this disclosure.

[0038] Furthermore, where the structure of an amino acid allows for stereoisomerization, the (D) and (L) stereoisomers of such amino acids are also included herein. In this specification, the configuration of amino acids and amino acid residues is indicated by the appropriate symbol (D), (L), or (DL), and where coordination is not explicitly stated, the amino acid or residue may be (D), (L), or (DL) coordinated. It is noteworthy that some structures of the compounds herein contain asymmetric carbon atoms. Therefore, isomers arising from such asymmetry are also included in the scope of this disclosure. Such isomers can be obtained in substantially pure form by classical separation techniques and stereochemically controlled synthesis. Unless otherwise specified in this application, the names of specific amino acids are interpreted to include all (D) and (L) stereoisomers.

[0039] In the amino acid or nucleic acid sequences described herein, “identical” or “% identity” means that two or more sequences are identical or match to a certain percentage when aligned for comparison and maximum match. For maximum match, conserved amino acid substitutions may not be treated as part of sequence identity. Percent identity can be measured by sequence comparison software or algorithms or by visual examination. A variety of algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and their variants. In some embodiments, two nucleic acids or polypeptides according to this disclosure are substantially identical, meaning that when aligned for comparison and maximum match, they have at least 70%, 75%, 80%, 85%, 90%, or in some embodiments, at least 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue percentage identity when measured through a sequence comparison algorithm or visual examination. In some embodiments, % identity exists in amino acid sequence regions corresponding to lengths of at least about 10 residues, about 20 residues, about 40-60 residues, about 60-80 residues, or any integer length in between. In other embodiments, % identity may exist in regions longer than 60-80 residues, for example, at least about 80-100 residues, and in some embodiments, it may be substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or antibody. In some embodiments, % identity exists in nucleotide sequence regions corresponding to lengths of at least about 10 bases, about 20 bases, about 40-60 bases, about 60-80 bases, or any integer length in between. In other embodiments, % identity may exist in regions longer than 60-80 bases, for example, at least about 80-1000 bases, and in some embodiments, it may be substantially identical over the full length of the sequences being compared, such as the nucleotide sequence coding a protein of interest.

[0040] When calculating % identity, residues defined as 'Xaa' or 'X' in the reference sequence herein are included in the % identity calculation; that is, all amino acids at the corresponding positions in the comparison sequence are considered to be identical to those in the reference sequence.

[0041] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are defined in the art and are well known. These groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Generally, conservative substitutions in the sequences of polypeptides, soluble proteins, and / or antibodies as described herein do not cause loss of binding to target binding sites of polypeptides, soluble proteins, or antibodies, including amino acid sequences. Methods for confirming non-loss of binding amino acid conservative substitutions are well known in the art.

[0042] An "isolated" protein, polypeptide, antibody, polynucleotide, vector, cell, or composition is a protein, polypeptide, antibody, polynucleotide, vector, cell, or composition that is not found in nature. An isolated protein, polypeptide, antibody, polynucleotide, vector, cell, or composition includes those that have been purified to the extent that they are no longer found in nature. For example, an isolated protein, polypeptide, antibody, polynucleotide, vector, cell, or composition is substantially pure. A substance is considered substantially pure if it is at least 50% pure (e.g., free from contaminants), 60% pure, 70% pure, 80% pure, 90% pure, 95% pure, 98% pure, or 99% pure.

[0043] A fusion polypeptide (e.g., a fusion protein) refers to a hybrid polypeptide expressed by a nucleic acid molecule containing at least two open reading frames (e.g., two separate molecules, e.g., two separate genes).

[0044] A linker (also called a linker region) may be inserted between a primary polypeptide (e.g., a copy of the TNFR2 AFFIMER polypeptide) and a secondary polypeptide (e.g., another AFFIMER polypeptide, an Fc domain, a ligand-binding domain, etc.). In some embodiments, the linker may be a peptide linker. It is desirable that the linker does not adversely affect polypeptide expression, secretion, or biological activity. In some embodiments, the linker is non-antigenic and does not induce an immune response.

[0045] A transmembrane domain (also called a TM domain) is a sequence added to an AFFIMER polypeptide. For example, it can be added by adding a polynucleotide sequence encoding the TM domain to the polynucleotide sequence encoding the AFFIMER polypeptide. This allows the AFFIMER polypeptide to be retained on the surface of the cell expressing it. A variety of TM domains are available and can be easily used with AFFIMER polypeptides.

[0046] An "AFFIMER-antibody fusion" refers to a fusion protein containing an AFFIMER polypeptide portion and an antibody variable region. An "AFFIMER-antibody fusion" may contain a full-length antibody, for example, when at least one AFFIMER polypeptide sequence is attached to the C-terminus or N-terminus of at least one VH and / or VL chain of the assembled antibody chain. That is, it may be a fusion protein in which at least one chain of the assembled antibody is fused with an AFFIMER polypeptide. An "AFFIMER-antibody fusion" may also contain at least one AFFIMER polypeptide sequence as part of the fusion protein, along with the antigen-binding site or variable region of the antibody section.

[0047] An antibody, as an immunoglobulin molecule, means a molecule that recognizes a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof and specifically binds to it through at least one antigen-binding site. The antigen-binding site is usually located within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” includes complete (poly)clonal antibodies, complete monoclonal antibodies, antibody sections (Fab, Fab', F(ab')2, Fv sections), single-chain Fv(scFv) antibodies formatted to include an Fc or other FcγRIII binding domain, polyspecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antibody antigen-binding site (formatted to include an Fc or other FcγRIII binding domain), antibody mimetic molecules, and all other modified immunoglobulin molecules containing an antigen-binding site as long as the antibody exhibits the desired biological activity.

[0048] The antibody may be one of the five major immunoglobulin systems: IgA, IgD, IgE, IgG, IgM, or their isotypes (e.g., IgG2, IgG3, IgG4, IgA1, IgA2). This is based on the identity of heavy chain constant domains called alpha, delta, epsilon, gamma, and mu. In some embodiments, Fc may be in a null-binding form that does not bind to FcγR2b. In some embodiments, the antibody may not be IgG1Fc.

[0049] The variable region of an antibody may be the variable region of the antibody light chain or the variable region of the antibody heavy chain, and may exist individually or in combination. Generally, the variable regions of the heavy and light chains contain four framework regions (FRs) and three complementary determination regions (CDRs, also called hypervariable regions). Each intrachain CDR is maintained in close proximity by the framework region and, together with the CDRs of other chains, contributes to the formation of the antibody's antigen-binding site. There are at least two methods for determining CDRs: (1) an approach based on interspecific sequence variability (Kabat et al., 1991, Sequences of Proteins of Immunological Interset, 5th edition, National Institutes of Health, Bethesda Md.) and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al Lazikani et al., 1997, J.Mol.Biol., 273:927-948). A combination of these two approaches is also used in the industry to determine CDRs.

[0050] A humanized antibody refers to a non-human (e.g., mouse) antibody, specific immunoglobulin chain, chimeric immunoglobulin, or section containing minimal non-human sequences. Generally, a humanized antibody is a human immunoglobulin in which CDR residues are replaced with CDR residues from a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and / or binding ability. In some cases, residues in the Fv framework region of the human immunoglobulin may be replaced with corresponding residues from the non-human antibody. Humanized antibodies can be further modified to purify and optimize the antibody's specificity, affinity, and / or binding ability by substituting additional residues within the Fv framework region and / or the substituted non-human residues. A humanized antibody may include a variable domain containing all or substantially all of the CDRs corresponding to the non-human immunoglobulin, and the framework region may be all or substantially all of the human immunoglobulin sequence. In some embodiments, the variable domain includes the framework region of the human immunoglobulin sequence. In some embodiments, the variable domain includes the framework region of the human immunoglobulin consensus sequence. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region or domain (Fc), which is generally that of human immunoglobulin. Humanized antibodies are usually distinguished from chimeric antibodies.

[0051] An epitope (also called an antigenic determinant) is a portion of an antigen that can be recognized and specifically bound by a specific antibody, a specific AFFIMER polypeptide, or another specific binding domain. When the antigen is a polypeptide, the epitope may be formed from a continuous sequence of amino acids, or it may be formed from discontinuous amino acids arranged in close proximity by the tertiary structure folding of the protein. Epitopes formed from continuous amino acids (also called linear epitopes) are conserved during protein denaturation, while epitopes formed by tertiary structure folding (also called structural epitopes) are generally lost during protein denaturation. Epitopes generally contain at least three, and more commonly at least five, six, seven, or eight to ten, amino acids in a unique three-dimensional sequence.

[0052] The terms "specifically binds to" or "specific for" refer to a measurable and reproducible interaction between an AFFIMER polypeptide, antibody, or other binding partner and a target (e.g., TNFR2) that can determine the presence or absence of the target within a heterogeneous molecular population, including biological molecules. For example, an AFFIMER polypeptide that specifically binds to TNFR2 is an AFFIMER polypeptide that binds to TNFR2 with higher affinity, affinity (if in a multi-form), easier binding, and / or longer duration than it would to other targets.

[0053] The terms "conjugate," "conjugation," and their grammatical variations mean that two or more compounds are joined or linked together by any bonding or linking method known in the art, thereby forming another compound. This may also refer to a compound produced by the bonding or linking of two or more compounds. For example, a TNFR2 AFFIMER polypeptide directly or indirectly linked to at least one chemical group or polypeptide is one exemplary conjugate. Such conjugates include fusion proteins, those produced by chemical conjugation, and those produced by other means.

[0054] Polynucleotides Polynucleotides (also referred to herein as nucleic acids or nucleic acid molecules) may include DNA, RNA (e.g., messenger RNA (mRNA)), or combinations of DNA and RNA as polymers of nucleotides of any length. Nucleotides may also be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, and may be any substrates that are included in the polymer by DNA or RNA polymerases.

[0055] A polynucleotide encoding a polypeptide refers to the sequence or order of deoxyribonucleotides arranged along a deoxyribonucleic acid chain. Such a sequence of deoxyribonucleotides determines the order of amino acids arranged along a polypeptide (e.g., protein) chain. Therefore, a nucleic acid sequence encodes an amino acid sequence.

[0056] When used in relation to a nucleotide sequence, "sequence" may include DNA and / or RNA (e.g., messenger RNA) and may be single-stranded or double-stranded.

[0057] Nucleic acid sequences can be modified (e.g., mutated) from naturally occurring nucleic acid sequences, for example.

[0058] Nucleic acid sequences may have any length, but for example, they may have 21,000,000 or more nucleotides (or any integer length greater than or equal to that). For example, they may have a nucleotide length of about 100, about 10,000, or about 200 to about 500.

[0059] Transfection refers to the process of introducing exogenous nucleic acids into eukaryotic cells. Transfection can be achieved by a variety of methods known to the industry, such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybren-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, plasmide fusion, retroviral infection, and biolytic techniques.

[0060] A vector is a structure that can transmit and normally express one or more genes or sequences of interest within a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, coarsmids, phage vectors, DNA or RNA expression vectors associated with cationic condensants, and DNA or RNA expression vectors encapsulated in liposomes. In some embodiments, a vector may be an isolated nucleic acid that can be used to deliver a composition into the cell. A wide variety of vectors are known in the art, such as linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, a vector may be an autonomously replicating plasmid or virus. Furthermore, the term should be interpreted to include cases where non-plasmids and non-viral compounds (e.g., polylysine compounds, liposomes, etc.) facilitate intracellular delivery of nucleic acids. Examples of unrestricted viral vectors include adenovirus vectors, adeno-associated virus vectors, and retroviral vectors.

[0061] An expression vector is a vector containing recombinant polynucleotides in which an expression regulatory sequence and the nucleotide sequence to be expressed are functionally linked. An expression vector contains sufficient cis-acting elements necessary for expression, with other expression elements potentially provided by host cells or in vitro expression systems. Examples of expression vectors include coarsmids, plasmids (e.g., naked or liposome-containing), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, adeno-associated viruses).

[0062] "Operafully linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, if a promoter influences the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are contiguous and can link two protein coding regions within the same read frame.

[0063] A promoter is a DNA sequence recognized by the cell's transcriptional synthesis mechanism and is necessary for the transcription of a specific polynucleotide sequence or a sequence that has been introduced into it.

[0064] Inducible expression refers to expression under specific conditions, including, for example, activation (or deactivation) of an intracellular signaling pathway or contact of a cell containing an expression vector with a small molecule that regulates the expression, causing the inducible promoter to be sensitive to the concentration of the small molecule and regulate the expression (or degree of expression) of the linked gene. This is in contrast to constitutive expression, which is expressed under physiological conditions without being restricted by specific conditions.

[0065] Electroporation refers to the use of membrane electrical pulses to induce tiny passages (pores) in biological membranes, through which biomolecules such as plasmids and other oligonucleotides can move from one side of the cell membrane to the other.

[0066] Treatments "Treatment" means (1) therapeutic measures to treat, delay, alleviate symptoms of, and / or suppress the progression of a diagnosed pathological condition or disorder, and (2) preventive measures to prevent or delay the onset of the target pathological condition or disorder. Therefore, those who require treatment include those who already have the disorder, those who are susceptible to the disorder, and those who need to be prevented from developing the disorder.

[0067] "Subject," "individual," and "patient" are used interchangeably herein to refer to all animals, including but not limited to humans, non-human primates, dogs, cats, and rodents (e.g., mammals).

[0068] A "sustained response" refers to a sustained effect on reducing inflammation after treatment is discontinued. In some embodiments, the duration of the sustained response may be the same as the duration of treatment, or at least 1.5, 2.0, 2.5, or 3.0 times the duration of treatment.

[0069] A "complete response (CR)" means the disappearance of all target lesions. A "partial response (PR)" means that the sum of the longest diameters (SLD) of the target lesions decreases by at least 30% relative to the baseline SLD. A "stable disease (SD)" means that, after the start of treatment, the SLD, relative to the smallest SLD, does not shrink enough to be classified as a PR, nor does it increase enough to be classified as a PD.

[0070] "Progression-free survival (PFS)" refers to the period during and after treatment during which the treated disease (e.g., inflammatory or autoimmune disease) does not worsen. PFS may include not only the period during which the patient experiences a complete response (CR) or partial response (PR), but also the period during which the patient experiences stable disease (SD).

[0071] The "Overall response rate (ORR)" is the sum of the complete response rate (CR rate) and the partial response rate (PR rate).

[0072] "Overall survival" refers to the proportion of individuals within a group who are likely to survive beyond a specific period.

[0073] "Agonist" and "Agonistic" refer to formulations that can directly or indirectly robustly induce, activate, promote, increase or enhance the biological activity of a target or target pathway. In this specification, an agonist includes any formulation that partially or completely induces, activates, promotes, increases or enhances the activity of a protein but other target of interest. TNFR2 is a type I membrane-bound receptor that binds to TNFα together with TNFR1.

[0074] "Antagonist" and "antagonistic" refer to formulations that can directly or indirectly partially or completely block, inhibit, reduce or neutralize the biological activity of a target and / or pathway. In this specification, "antagonist" includes any formulation that partially or completely blocks, inhibits, reduces or neutralizes the activity of a protein but other target of interest.

[0075] "Modulation" and "modulate" mean a change or alteration of biological activity. Modulation includes, but is not limited to, stimulating or inhibiting activity. Modulation may include an increase or decrease in activity, a change in binding properties, or a different change in the biological, functional, or immunological properties related to the activity of a protein, pathway, system, or other biological target of interest.

[0076] An immune response includes reactions from both the innate and adaptive immune systems. This includes cell-mediated and / or humoral immune responses, and encompasses not only T cell and B cell responses but also responses from other cells in the immune system, such as spontaneous killer (NK) cells, mononuclear cells, and macrophages.

[0077] "Pharmaceutically acceptable" means a substance that has been approved or may be approved by a federal or state regulatory agency, is listed in the United States Pharmacopeia or a generally accepted pharmacopoeia, and is available for use in animals (including humans).

[0078] "Pharmaceutically acceptable excipient" means an excipient, carrier, or immunosuppressant that can be administered to a subject with at least one of the formulations herein and is non-toxic when administered in a dose sufficient to exert a therapeutic effect without impairing its pharmacological activity. Generally, those skilled in the art and the U.S. FDA consider a pharmaceutically acceptable excipient, carrier, or immunosuppressant to be an inactive component of the formulation.

[0079] An "effective amount" (also referred to herein as a "therapeutically effective amount") means an amount of a formulation, such as a TNFR2 AFFIMER agent, that is effective in treating a disease or disorder in a target (e.g., a mammal). In the case of inflammatory or autoimmune diseases, a therapeutically effective amount of a TNFR2 AFFIMER agent may have a therapeutic effect, for example, by reducing inflammation, alleviating to some extent at least one of the symptoms associated with the inflammatory or autoimmune disease, reducing morbidity and mortality, improving quality of life, or achieving a combination of these.

[0080] Other (Miscellaneous) In the present invention, embodiments described using the term "includes" are understood to also include similar embodiments described as "constituted" and / or "essentially constituted." Furthermore, embodiments described as "essentially constituted" are understood to also include other embodiments described as "constituted."

[0081] The descriptions of values ​​or parameters expressed as "about" in this specification include examples of such values ​​or parameters. For example, "about X" includes a description of X.

[0082] In this specification, "and / or" as in "A and / or B" is used to mean A and B, A or B, or A (alone) and B (alone). Similarly, "A, B and / or C" is used to mean A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A; B; and C.

[0083] In this specification, "at least one" may be used interchangeably with "one or more".

[0084] TNFR2 AFFIMER polypeptide (TFNR2 AFFIMER Polypeptide) AFFIMER polypeptides are scaffolds based on the stephin A protein, and represent sequences derived from the stephin A protein, such as mammalian, more preferably human, stephin A protein.

[0085] In one aspect, the present invention provides an AFFIMER polypeptide that binds to TFNR2 (also known as "TFNR2 AFFIMER polypeptide"), derived from a wild-type StefinA protein having at least one solvent-accessible loop capable of binding to TFNR2, preferably selectively binding, and preferably having a dissociation constant (Kd) of 10 -6 This includes those that are M or less.

[0086] In one embodiment of the present invention, the TFNR2 AFFIMER polypeptide is derived from wild-type human stefin A polypeptide, has a backbone sequence, and one or both of loop 2 [represented by (Xaa)n] and loop 4 [represented by (Xaa)m] are replaced with alternative loop sequences (Xaa)n and (Xaa)m, and has the following general formula (I). [Formula I] FR1-(Xaa)n-FR2-(Xaa)m-FR3 Here, FR1 is a polypeptide sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity with the polypeptide sequence MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TGETYGKLEA VQYKTQVX (SEQ ID NO: 1) or the amino acid sequence of SEQ ID NO: 1, wherein X is any number of independently selected amino acids, more preferably three or fewer independently selected amino acids, or more preferably X may be V, and / or

[0087] The above FR2 is a polypeptide sequence containing an amino acid sequence that has at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) identity with the amino acid sequence represented by GTNYYIKVRA GDNKYMHLKV FKSL (Sequence ID 2) or the amino acid sequence of Sequence ID 2.

[0088] The above FR3 is a polypeptide sequence containing an amino acid sequence that has at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100%) identity with the amino acid sequence represented by EDLVLTGYQV DKNKDDELTG F (Sequence ID 3) or the amino acid sequence of Sequence ID 3, and

[0089] In each case, Xaa is an arbitrary amino acid residue, and n and m are integers between 3 and 20, respectively.

[0090] In one embodiment of the present invention, FR1 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% homology with SEQ ID NO: 1. In one embodiment of the present invention, FR1 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO: 1.

[0091] In one embodiment of the present invention, FR2 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% homology with SEQ ID NO: 2. In one embodiment of the present invention, FR2 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO: 2.

[0092] In one embodiment of the present invention, FR3 may be an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% homology with SEQ ID NO: 3.

[0093] In one embodiment of the present invention, the NFR2 AFFIMER polypeptide may include an amino acid sequence represented by formula II: [Formula II] MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-Xaa2-TNYYIKV RAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa)m-Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4). Here, Xaa is an amino acid residue in each individual case, and n and m are integers between 3 and 20, respectively.

[0094] In some embodiments of the present invention, the TNFR2 AFFIMER polypeptide may include an amino acid sequence having at least 90% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) identity with the following amino acid sequence: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF(Sequence ID 5). Xaa is an amino acid residue in each case, and n and m are integers between 3 and 20, respectively.

[0095] In one embodiment of the present invention, Xaa1 may be Gly, Ala, Val, Arg, Lys, Asp, or Glu, preferably Gly, Ala, Arg, or Lys, more preferably Gly or Arg; Xaa2 may be Gly, Ala, Val, Ser, or Thr, preferably Gly or Ser; and Xaa3 may be Arg, Lys, Asn, Gln, Ser, or Thr, preferably Arg. Xaa4 may be Lys, Asn, or Gln, more preferably Lys or Asn; Xaa5 may be Ala, Val, Val, Ser, or Thr, preferably Gly or Ser; Xaa6 may be Gly, Ala, Val, Asp, or Glu, more preferably Ala, Val, Asp, or Glu, more preferably Ala or Glu; and Xaa7 may be Ala, Val, Ile, Leu, Arg, or Lys, more preferably Ile, Leu, or Arg, more preferably Leu or Arg.

[0096] In one embodiment of the present invention, n may be an integer between 3 and 15, 3 and 12, 3 and 9, 3 and 7, 5 and 7, 5 and 9, 5 and 12, 5 and 15, 7 and 12, or 7 and 9.

[0097] In one embodiment of the present invention, m can be an integer between 3 and 15, 3 and 12, 3 and 9, 3 and 7, 5 and 7, 5 and 9, 5 and 12, 5 and 15, 7 and 12, or 7 and 9.

[0098] In another embodiment of the present invention, the TNFR2 AFFIMER polypeptide may have an amino acid sequence represented by formula (IIb). MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV(Sequence code 484)Xaa2-(Xaa)n-Xaa3-TNYYIKVRAGDNKYMHLKVF(Sequence code 485)-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF(Sequence code 486) Here, Xaa is any number of amino acid residues in each case, and more appropriately, three or fewer (preferably one or two) independently selected amino acids. n and m are each independent integers between 3 and 20.

[0099] In one embodiment of the present invention, the TNFR2 AFFIMER polypeptide comprises an amino acid sequence having at least 90% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) identity with the following amino acid sequence: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV(Sequence ID 484)Xaa2-(Xaa)n-Xaa3-TNYYIKVRAGDNKYMHLKVF(Sequence ID 485)-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF(Sequence ID 486), Alternatively, MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD(sequence code 487)-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL(sequence code 488)-(Xaa)m-EDLVLTGYQVDKNKDDELTGF(sequence code 489), Xaa is an arbitrary number of amino acid residues in each case, more preferably three or fewer (more preferably one or two) independently selected amino acids. n and m are each independent integers between 3 and 20.

[0100] In one embodiment of the present invention, Xaa1 is Gly, Ala, Val, Arg, Lys, Asp, or Glu, more preferably Gly, Ala, Arg, or Lys, and even more preferably Gly or Arg; Xaa2 is Val, Asp, or 'Leu-Ala'; Xaa3 is Gly, Ala, Val, Ser, or Thr, more preferably Gly or Ser; Xaa4 is Arg, Lys, Asn, Gln, Ser, Thr, more preferably Arg, Lys, Asn, or Gln, and even more preferably Lys or Asn; Xaa5 is Gly, Al a, Val, Ser or Thr, more preferably Gly or Ser, Xaa6 is Ala, Val, Ile, Leu, Gly or Pro, more preferably Ile, Leu or Pro and even more preferably Leu or Pro, Xaa7 is Gly, Ala, Val, Asp or Glu, more preferably Ala, Val, Asp or Glu and even more preferably Ala or Glu, and Xaa8 may be Ala, Val, Ile, Leu, Arg or Lys, more preferably Ile, Leu or Arg and even more preferably Leu or Arg.

[0101] In one embodiment of the present invention, n can be an integer between 3 and 15, 3 and 12, 3 and 9, 3 and 7, 5 and 7, 5 and 9, 5 and 12, 5 and 15, 7 and 12, or 7 and 9.

[0102] In one embodiment of the present invention, m can be an integer between 3 and 15, 3 and 12, 3 and 9, 3 and 7, 5 and 7, 5 and 9, 5 and 12, 5 and 15, 7 and 12, or 7 and 9.

[0103] In one embodiment of the present invention, Xaa is characterized in that each of the amino acids that can be independently added to a polypeptide by recombinant expression in prokaryotic or eukaryotic cells, preferably one of 20 naturally occurring amino acids.

[0104] In the sequences and formulas of the present invention, (Xaa)n is an amino acid sequence selected from sequence numbers 6 to 102. In one embodiment of the present invention, (Xaa)n may be a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with the amino acid sequence selected from sequence numbers 6 to 102.

[0105] In the sequence and formula of the present invention, (Xaa)m is an amino acid sequence selected from sequence numbers 103 to 199. In one embodiment of the present invention, (Xaa)m may be a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with the amino acid sequence selected from sequence numbers 103 to 199.

[0106] In one embodiment of the present invention, the TNFR2 AFFIMER polypeptide has an amino acid sequence selected from SEQ ID NOs. 200 to 296, wherein the sequence may selectively exclude one or more of the 21 carboxy-terminal residues. In another embodiment of the present invention, the TNFR2 AFFIMER polypeptide has an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% % identity with the sequence selected from SEQ ID NOs. 200 to 296, wherein the sequence may selectively exclude one or more of the 21 carboxy-terminal residues.

[0107] [Table 1] JPEG2026520086000002.jpg195153JPEG2026520086000003.jpg194153JPEG2026520086000004.jpg195153JPEG2026520086000005.jpg194153JPEG2026520086000006.jpg195153JPEG2026520086000007.jp g194153JPEG2026520086000008.jpg194153JPEG2026520086000009.jpg195153JPEG2026520086000010 .jpg193153JPEG2026520086000011.jpg194153JPEG2026520086000012.jpg194153JPEG2026520086000 013.jpg194153JPEG2026520086000014.jpg194153JPEG2026520086000015.jpg195153JPEG202652008 6000016.jpg193153JPEG2026520086000017.jpg194153JPEG2026520086000018.jpg195153JPEG202652 0086000019.jpg194153JPEG2026520086000020.jpg194153JPEG2026520086000021.jpg194153JPEG202 6520086000022.jpg195153JPEG2026520086000023.jpg194153JPEG2026520086000024.jpg145153 (Table 1) The TNFR2 AFFIMER polypeptide sequence' - The last 21 amino acids are sequences added for cloning and analysis. The 3xAla linker, 10-amino acid Myc tag, 2xAla linker, and 6xHis tag are all optional and not required.

[0108] In one embodiment of the present invention, the TNFR2 AFFIMER polypeptide has an amino acid sequence encoded by a nucleic acid that includes a coding sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity with a sequence selected from SEQ ID NOs. 297 to 393, and can selectively exclude nucleotides encoding 21 carboxy-terminal amino acids.

[0109] In one embodiment of the present invention, the TNFR2 AFFIMER polypeptide has an amino acid sequence encoded by a nucleic acid, which includes a coding sequence that is hybridized under strict conditions, complementary to a sequence selected from SEQ ID NOs. 297-393 (selectively excluding nucleotides encoding 21 carboxy-terminal amino acids). Here, strict conditions include, for example, hybridization at 45°C in the presence of 6x sodium chloride / sodium citrate (SSC) followed by washing with 0.2x SSC at 65°C.

[0110] [Table 2] JPEG2026520086000026.jpg198153JPEG2026520086000027.jpg199153JPEG2026520086000028.jpg199153JPEG2026520086000029.jpg199153JPEG2026 520086000030.jpg199153JPEG2026520086000031.jpg199153JPEG20265200 86000032.jpg198153JPEG2026520086000033.jpg198153JPEG2026520086000 034.jpg198153JPEG2026520086000035.jpg198153JPEG2026520086000036.jpg199153JPEG2026520086000037.jpg199153JPEG2026520086000038.jpg1 99153JPEG2026520086000039.jpg199153JPEG2026520086000040.jpg19915 3JPEG2026520086000041.jpg199153JPEG2026520086000042.jpg138153 (Table 2) The nucleic acid sequence of the present invention, with 63 nucleic acids at the 5'-terminus encoding 21 amino acids at the C'-terminus, is a sequence added for cloning and analysis: the 3xAla linker, 10-amino acid Myc tag, 2xAla linker, and 6xHis tag are all selective and not required.

[0111] Furthermore, minor modifications may extend beyond the loop 2 and loop 4 insertions described above and may include small deletions or additions to the Stephin A or Stephin A-derived sequences disclosed herein, for example, the addition or deletion of up to 10 amino acids to Stephin A or Stephin A-derived AFFIMER polypeptides.

[0112] In one embodiment of the present invention, the AFFIMER formulation has a dissociation constant (Kd) of about 1 μM or less, about 00 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less, as a TNFR2 AFFIMER formulation comprising an AFFIMER polypeptide moiety that binds to human TNFR2 in the monomeric state.

[0113] In one embodiment of the present invention, the AFFIMER formulation, as a TNFR2 AFFIMER formulation comprising an AFFIMER polypeptide moiety that binds to human TNFR2 in the monomeric state, for example BIACORE TM has a dissociation rate constant (Koff) of about 10-3 s-1 or less (e.g., in units of 1 / second), about 10-4 s-1 or less, or moreover about 10-5 s-1 or less, measured by analysis.

[0114] In one embodiment of the present invention, the AFFIMER formulation, as a TNFR2 AFFIMER formulation comprising an AFFIMER polypeptide moiety that binds to human TNFR2 in the monomeric state, for example BIACORE TM has an association rate constant (Kon) of at least about 103 M-1 s-1 or more, about 104 M-1 s-1 or more, about 105 M-1 s-1 or more, or even at least about 106 M-1 s-1 or more, measured by analysis.

[0115] In one embodiment of the present invention, the AFFIMER formulation is a TNFR2 AFFIMER formulation comprising an AFFIMER polypeptide moiety that binds to human TNFR2 with an IC50 of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less in a competitive binding assay with human TNFR2 in the monomeric state.

[0116] In one embodiment of the present invention, the AFFIMER formulation has a melting temperature (Tm, e.g., the temperature at which folded and unfolded states exist in equal proportions) of 65°C or higher, preferably at least 70°C, 75°C, 80°C, or even 85°C or higher. The melting temperature is a particularly useful indicator of protein stability. The relative ratio of folded to unfolded protein can be determined by a number of techniques known to those skilled in the art, such as differential scanning calorimetry, UV difference spectroscopy, fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) (Pace et al. (1997) "Measuring the conformational stability of a protein", Protein structure: A Practical approach 2: 299-321).

[0117] Fusion protein - general In one embodiment of the present invention, the AFFIMER polypeptide may include additions, substitutions, and / or deletions of peptides containing one or more amino acids for purposes such as regulating the biological activity of the AFFIMER polypeptide or adding additional biological activity. For example, additions, substitutions, and / or deletions may regulate at least one property or activity of the modified AFFIMER polypeptide. For example, such additions, substitutions, and / or deletions may regulate the affinity (e.g., binding to and inhibition of TNFR2), circulating half-life, therapeutic half-life, stability, protease cleavage, dose, release, or bioavailability of the modified AFFIMER polypeptide, or reduce deamidation, improve shelf-life, or alter the route of administration. Similarly, the AFFIMER polypeptide may include protease cleavage sequences, reactive groups, antibody-binding domains (e.g., not limited to FLAG or poly-His) or other affinity-based sequences (e.g., not limited to FLAG, poly-His, GST, etc.) that improve detection, purification, or other properties, and may also include binding molecules not limited to biotin.

[0118] In some cases, such additional sequences are added to one and / or the other end of an AFFIMER polypeptide in the form of a fusion protein. Thus, in certain aspects of the present invention, an AFFIMER formulation is a fusion protein comprising at least one AFFIMER polypeptide sequence and at least one heterologous polypeptide sequence (hereinafter referred to as a “fusion domain”). The fusion domain may be selected to confer desired properties such as secretion from cells or maintenance on the cell surface (e.g., in the case of an encoded AFFIMER polynucleotide), action on substrates or other recognition sequences for post-translational deformation, multi-body formation through protein-protein interactions, alteration of serum half-life (generally prolongation), tissue localization or tissue exclusion, and other alterations of ADME properties.

[0119] For example, some fusion domains are particularly useful for the isolation and / or purification of fusion proteins, such as in affinity chromatography. Well-known examples of fusion domains that facilitate expression or purification include, but are not limited to, polyhistidine tags, Strep II tags, streptavidin-binding peptide (SBP) tags, calmodulin-binding peptide (CBP) tags, S-tags, HA tags, c-Myc tags, thioredoxin, protein A, and protein G affinity tags.

[0120] For AFFIMER to be secreted, the protein may contain a signal sequence that directs it to translocate to the lumen of the endoplasmic reticulum and ultimately be secreted (or retained on the cell surface, if it has a transmembrane domain or cell surface retention signal). The signal sequence (also called a signal peptide or leader sequence) may be located at the N-terminus of the nascent polypeptide. The signal sequence targets the nascent polypeptide to the endoplasmic reticulum, and the protein is classified to a destination, such as the internal space of an organelle, the internal membrane, the extracellular membrane, or the extracellular space through secretion. Generally, the signal sequence may be cleaved after being transported to the endoplasmic reticulum, and may be characterized by cleavage at residues within the signal sequence. The signal sequence (also called a signal peptide or leader sequence) may be located at the N-terminus of the nascent peptide. The signal sequence targets the polypeptide to the endoplasmic reticulum, and functions to classify the protein to a destination, such as the internal space of an organelle, the internal membrane, the extracellular membrane, or the extracellular space through secretion. Generally, the signal sequence can be cleaved by signal peptidases after being transported to the endoplasmic reticulum, and the cleavage of the signal sequence generally occurs at specific sites in the amino acid sequence, varying depending on the amino acid residue within the signal sequence.

[0121] In one embodiment of the present invention, the signal sequence may have an amino acid length of about 5 to 40 (for example, an amino acid length of about 5 to 7, about 7 to 10, about 10 to 15, about 15 to 20, about 20 to 25, about 25 to 30, about 30 to 35, or about 35 to 40).

[0122] In one embodiment of the present invention, the signal sequence may be, for example, a native signal peptide derived from humans. In another embodiment of the present invention, the signal sequence may be a non-native signal peptide. For example, in one embodiment of the present invention, the non-native signal peptide may be, for example, a signal peptide derived from a corresponding natively secreted human protein and may include one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten or more) substitutions, insertions, and / or deletions.

[0123] In one embodiment of the present invention, the signal sequence may be, but is not limited to, an immunoglobulin (e.g., IgG heavy chain or IgG-copper light chain), a cytokine (e.g., IL-2 or CD33), a plasma albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, a signal sequence derived from luciferase, trypsinogen, chymotrypsinogen or other secreted proteins, or other signal peptides. Exemplary signal sequences include, but are not limited to, the following:

[0124] [Table 3] (Table 3) Signal peptide sequence

[0125] Many natural linkers have an α-helical structure. The α-helical structure is rigid and stable due to hydrogen bonds within the segments and a tightly packed backbone. Therefore, stiff α-helical linkers can be used as rigid spacers between protein domains (George et al. (2002) "An analysis of protein domain linkers: their classification and role in protein folding" Protein Eng. 15(11):871-9). In general, rigid linkers adopt an α-helical structure or exhibit a relatively rigid structure containing a large number of Pro residues. Rigid linkers can separate functional fusion domains more efficiently than flexible linkers. The length of the linker can be easily optimized by adjusting the number of repeats to achieve the optimal distance between domains. Consequently, rigid linkers can be used when spatial separation of domains is important for maintaining the stability or bioactivity of the fusion protein. From this perspective, the α-helix-forming linker having the sequence (EAAAK)n (SEQ ID NO: 421) is commonly used in the construction of many fusion proteins, and other examples of rigid linkers include, but are not limited to, the Pro-rich sequence (XP)n, where X is any amino acid, preferably Ala, Lys, or Glu.

[0126] For illustrative purposes, representative linkers include GRA, poly(Gly), poly(Ala), and the linkers shown in Table 4 below, but are not limited to these.

[0127] [Table 4] (Table 4) Linker array

[0128] AFFIMER polypeptides may also further contain transmembrane domains (TM domains), for example in the form of fusion proteins. Such TM domains function to ensure that the AFFIMER lipeptide is retained on the surface of the cell expressing it. Such TM domains are known and include, but are not limited to, CD3, CD8, CD28, and PDGFR TMDs.

[0129] Furthermore, the AFFIMER polypeptide sequence or adjacent polypeptide portion provided in part of the fusion protein may be modified to include one or more sequences that serve as sites for enzymatic post-translational modification. These include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, and glycolipid-linkage modification.

[0130] Multiple specific fusion proteins In one embodiment of the present invention, the AFFIMER formulation may be, for example, a multispecific polypeptide comprising a first TNFR2 AFFIMER polypeptide and at least one additional binding domain. The additional binding domain may be a polypeptide sequence selected from the group consisting of, for example, a second AFFIMER polypeptide (e.g., which may be the same as or different from the first AFFIMER polypeptide), an antibody or this antigen-binding polypeptide, a ligand-binding site of a receptor (e.g., a receptor trap polypeptide), a receptor-binding ligand (e.g., a cytokine, a growth factor or analog thereof), an engineered modified T-cell receptor, and an enzyme or a catalytic fragment thereof.

[0131] In one embodiment of the present invention, the AFFIMER formulation may include at least one additional TNFR2-targeting AFFIMER polypeptide sequence. The additional TNFR2 AFFIMER polypeptide may be identical to or different from (or a mixture thereof) the first TNFR2 AFFIMER polypeptide in order to generate a multi-specific AFFIMER fusion protein. The AFFIMER formulation may bind to the same site, to overlapping sites, to two sites (biparatopic), or to two or more sites (multiparatopic).

[0132] In one embodiment of the present invention, the AFFIMER formulation may include at least one antigen-binding site derived from an antibody. The resulting AFFIMER formulation may be a single chain (e.g., scFv) containing all of the TNFR2 AFFIMER polypeptide and antigen-binding sites, or it may be a multimeric protein complex, such as an antibody assembled from the heavy and / or light chains of the antibody.

[0133] In one embodiment of the present invention, if the AFFIMER preparation contains a full-length immunoglobulin, the fusion protein may retain the Fc function of the immunoglobulin's Fc region. For example, the AFFIMER preparation may bind to the Fc receptor on an Fc receptor-positive cell through its Fc region. In one embodiment of the present invention, the AFFIMER preparation may bind to the Fc receptor and activate an Fc receptor-positive cell, thereby initiating or increasing the expression of cytokines and / or co-stimulating antigens. Cytokines and / or co-stimulating antigens may transmit secondary activation signals or higher signals necessary for T cell activation.

[0134] In one embodiment of the present invention, the fusion protein may possess antibody-dependent cell-mediated cytotoxicity (ADCC) activity by binding its Fc region to other cells expressing Fc receptors present on the surface of effector cells of the immune system, such as immune cells, hepatocytes, and endothelial cells. This is a cell-mediated immune defense mechanism in which effector cells of the immune system induce ADCC-mediated cell death (e.g., tumor cell death) by actively lysing target cells to which membrane surface antigens have been bound by antibodies. In one embodiment of the present invention, the ADCC function of the fusion protein has been demonstrated.

[0135] As mentioned above, the Fc region of the AFFIMER formulation may have a beneficial effect on maintaining the serum level of the fusion protein, independently of Fc-mediated cytotoxicity, which may contribute to improved stability and persistence in the body. For example, when the Fc portion binds to Fc receptors on endothelial cells and phagocytic cells, the AFFIMER formulation is internalized, recycled, and returned to the bloodstream, thereby improving its half-life in the body.

[0136] Examples of targets for additional AFFIMER polypeptides include, but are not limited to, other immune checkpoint proteins, immune co-stimulatory receptors (especially if the additional AFFIMER can act as an agonist on such co-stimulatory receptors), receptors, cytokines, growth factors, and tumor-associated antigens. In one embodiment of the present invention, the immunoglobulin-derived region may be a monoclonal antibody against at least one autoimmune target (e.g., TNFR2 or IL6-R). In one embodiment of the present invention, the TNFR2AFFIMER polypeptide may be part of an AFFIMER formulation comprising one or more binding domains that bind to a protein whose expression is upregulated in an autoimmune disease (e.g., TNFR2 or IL6-R).

[0137] For a detailed description of the various TNFR2 AFFIMER formulation formats included herein, please refer to William BA et al. J. Clin. Med. 2019;8(8):1261.

[0138] In one embodiment of the present invention, the multispecific TNFR2 AFFIMER formulation may further comprise a half-life extension moisture as described herein. For example, the TNFR2 AFFIMER formulation may comprise at least one TNFR2 AFFIMER polypeptide linked via a peptide linker to a binding domain (e.g., CD3ε chain or CD16) specific to the binding domain of at least one immune cell (e.g., T cell and / or NK cell), and further linked to a half-life extension moisture, such as a fragment crystallizable (Fc) domain (e.g., FcyR null-binding Fc), human serum albumin (HSA), or HSA AFFIMER polypeptide. In one embodiment of the present invention, the half-life extension moisture may be a fragment crystallizable (Fc) domain. In one embodiment of the present invention, the half-life extension moisture may be human serum albumin (HSA). In one embodiment of the present invention, the half-life extension moisture may be an HSA AFFIMER polypeptide.

[0139] Engineering of PK and ADME properties In one embodiment of the present invention, the AFFIMER formulation may not exhibit a desirable half-life and / or pharmacokinetic profile (PK profile) for a route of administration such as parenteral therapeutic administration. In the present invention, the term "half-life" means the time required for the active ingredient, such as the AFFIMER formulation or fusion protein of the present invention, to lose half of its pharmacological or physiological activity or concentration. Biological half-life can be affected by the removal, excretion, degradation (e.g., enzymatic degradation) of the substance or its absorption and concentration in a particular organ or tissue of the body. In one embodiment of the present invention, biological half-life can be assessed by determining the time required for the plasma concentration of the substance to reach half of its steady-state level (plasma half-life). To address these shortcomings, a common method used to extend the half-life in other protein therapeutics is to integrate a half-life extension moiety as part of the AFFIMER formulation.

[0140] As used herein, the term “half-life extending moiety” means a pharmaceutically acceptable moiety, domain, or molecule covalently bonded (chemically joined or fused) to the AFFIMER polypeptide of the present invention to form an AFFIMER formulation. The moiety, domain, or molecule may be joined or fused directly or via a linker through selectively unnaturally encoded amino acids. The half-life extending moiety extends the half-life by preventing or mitigating deformation from degradation by endogenous proteolytic enzymes or other degradation of activity, and may further improve or modify pharmacokinetic or biophysical properties such as increased absorption rate, reduced toxicity, improved solubility, reduced protein aggregation, increased biological activity and / or target selectivity of the modified AFFIMER polypeptide, improved manufacturability, and reduced immunogenicity of the modified AFFIMER polypeptide, compared to a comparer, such as the unjointed form of the unmodified AFFIMER polypeptide.

[0141] In one embodiment of the present invention, the "half-life extension moisture" is not limited to proteins or peptides, but may include non-proteinaceous half-life extension moisture. Examples include, but are not limited to, water-soluble polymers such as polyethylene glycol (PEG) or discrete PEG, HES (hydroxyethyl starch), lipids, branched or unbranched acyl groups, branched or unbranched C8-C30 acyl groups, branched or unbranched alkyl groups, and unstructured polypeptides such as serum albumin, transferrin, adnectin (e.g., albumin-binding adnectin or pharmacokinetically extended adnectin (PKE adnectin)), Fc domains, XTEN and PAS polypeptides (e.g., structurally disordered polypeptide sequences containing amino acids Pro, Ala and / or Ser), and fragments thereof.

[0142] In one embodiment of the present invention, the half-life-extending moisture can be characterized by extending the half-life of the AFFIMER formulation obtained as a result of circulation in mammalian serum, compared to a protein that is not bound to such moisture (e.g., a single AFFIMER polypeptide).

[0143] For example, the half-life may be extended by approximately 1.2 times, 1.5 times, 2.0 times, 3.0 times, 4.0 times, 5.0 times, or 6.0 times or more, but is not limited thereto. In one embodiment of the present invention, the half-life may be extended by 6 hours or more, 12 hours or more, 24 hours or more, 72 hours or more, 96 hours or more, or 1 week or more after administration, but is not limited thereto.

[0144] Further examples include, but are not limited to, the following, as half-life-extending molecules that may be included in the AFFIMER formulation of the present invention.

[0145] - Genetic fusion of a pharmacologically effective AFFIMER sequence with a protein or protein domain that naturally has a long half-life (e.g., Fc domain fusion, transferrin (Tf) fusion, or albumin fusion). Fusion technology with the aforementioned protein-based half-life extension molecules is well known in this field: for example, Beck et al. (2011) “Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies.” MAbs. 3:1-2; Czajkowsky et al. (2012) “Fc-fusion proteins: new developments and future perspectives.” EMBO Mol Med. 4:1015-28; Huang et al. (2009) “Receptor-Fc fusion therapeutics, traps, and Mimetibody technology” Curr Opin Biotechnol. 2009; 20:692-9; Keefe et al. (2013) “Transferrin fusion protein therapies: acetylcholine receptor-transferrin fusion protein as a model.” In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; p. 345-56; Weimer et al. (2013) “Recombinant albumin fusion proteins. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. p. 297-323; Walker et al.(2013) “Albumin-binding fusion proteins in the development of novel long-acting therapeutics. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: applications and challenges. Hoboken: Wiley; 2013. p. 325-43.

[0146] - Genetic fusion of a pharmacologically effective AFFIMER sequence with an inert polypeptide (e.g., XTEN (also known as recombinant PEG or rPEG), HAP (homoamino acid polymer, HAPylation), proline-alanine-serine polymer (PAS; PASylation), or elastin-like peptide (ELP; ELPylation)).The aforementioned fusion technology with inactive polypeptides is well known in this field: for example, Schellenberger et al. (2009) “A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner.” Nat Biotechnol. 2009; 27:1186-90; Schlapschy et al. “Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life.” Protein Eng Des Sel. 2007; 20:273-84; Schlapschy (2013) “PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins.” Protein Eng Des Sel. 26:489-501. Floss et al. (2012) “Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application.” Trends Biotechnol. 28:37-45. Floss et al. “ELP-fusion technology for biopharmaceuticals. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: application and challenges. Hoboken: Wiley; 2013. p. 372-98.

[0147] - Increase in hydrodynamic radius by chemical conjugation of pharmacologically active peptides or proteins for repeating chemical moieties such as PEG (PEGylation) or hyaluronic acid. Techniques for increasing hydrodynamic radius through chemical conjugation are well-known in this field: for example, Caliceti et al. (2003) “Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates” Adv Drug Delivery Rev. 55:1261-77; Jevsevar et al. (2010) PEGylation of therapeutic proteins. Biotechnol J 5:113-28; Kontermann (2009) “Strategies to extend plasma half-lives of recombinant antibodies” BioDrugs. 23:93-109; Kang et al. (2009) “Emerging PEGylated drugs” Expert Opin Emerg Drugs. 14:363-80; and Mero et al. (2013) “Conjugation of hyaluronan to proteins” Carb Polymers. 92:2163-70.

[0148] -Polysialylation significantly increases or selectively increases the negative charge of the fusion of pharmacologically active peptides or proteins, (b) fusion of negatively charged, highly sialylated peptides (e.g., carboxy-terminal peptide [CTP; of chorionic gonadotropin (CG) b-chain], a well-known method for extending the half-life of native proteins such as the human CG b-subunit). The technique of extending half-life through polysialylation is well known in this field: for example, Gregoriadis et al. (2005) “Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids” Int J Pharm. 2005; 300:125-30; Duijkers et al. “Single dose pharmacokinetics and effects on follicular growth and serum hormones of a long-acting recombinant FSH preparation (FSHCTP) in healthy pituitary-suppressed females” (2002) Hum Reprod. 17:1987-93; and Fares et al. “Design of a longacting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit” (1992) Proc Natl Acad Sci USA. 89:4304-8. 35; and Fares “Half-life extension through O-glycosylation.

[0149] - Non-covalent bonding via peptide or protein-binding domains to proteins that generally have long half-lives, such as HSA, IgG, transferrin, or fibronectin. Such methods are well known in this field: for example, Andersen et al. (2011) “Extending half-life by indirect targeting of the neonatal Fc receptor (FcRn) using a minimal albumin binding domain” J Biol Chem. 286:5234-41; O'Connor-Semmes et al. (2014) “GSK2374697, a novel albumin-binding domain antibody (albudAb), extends systemic exposure of extendin-4: first study in humans-PK / PD and safety” Clin Pharmacol Ther. 2014; 96:704-12; Sockolosky et al. (2014) “Fusion of a short peptide that binds immunoglobulin G to a recombinant protein substantially increases its plasma half-life in mice” PLoS One. 2014; 9:e102566.

[0150] Conventional genetic fusion to long-lived serum proteins offers an alternative method for half-life extension, distinct from chemical conjugation to PEG or lipids. Antibody Fc domains and human serum albumin have been used as traditional fusion domains for half-life extension. Fc fusion involves the fusion of a peptide, protein, or receptor exodomain to the Fc portion of an antibody. Fc-albumin fusion proteins not only increase the size of peptide drugs and extend their half-life, but also utilize the natural biological recycling mechanism of FcRn (neonatal Fc receptor). pH-dependent binding of these proteins to FcRn prevents the degradation of the fusion protein in endosomes. Fusion proteins with such proteins fused can have a half-life of approximately 3 to 16 days, which is significantly longer than the half-life of typical PEGylated or lipid-conjugated peptides. Antibody Fc domain fusion can improve the solubility and stability of peptide or protein drugs. An example of Fc domain fusion is dulaglutide, a GLP receptor agonist. Human serum albumin is the same protein used by lipid acylated peptides, and is yet another fusion domain used for half-life extension. An example based on the HSA binding platform is albiglutide, a GLP-1 receptor agonist. The main difference between the Fc domain and albumin is that Fc is generally used in a dimerized form, while HSA is a monomeric structure, and the fusion protein can be used as a dimer or monomer depending on the choice of fusion partner. The dimeric nature of AFFIMER-Fc fusion may be more effective when the AFFIMER target is located sufficiently close or is itself a dimer, but this may or may not be desirable depending on the target.

[0151] Fc fusion In one embodiment of the present invention, the AFFIMER polypeptide may be part of a fusion protein of an immunoglobulin Fc domain ("Fc domain") or a fragment or variant thereof (e.g., a functional Fc domain). In one embodiment of the present invention, the Fc domain may be a null-binding Fc domain. In this context, an Fc fusion ("Fc-fusion"), for example, a TNFR2 AFFIMER formulation produced as an AFFIMER-Fc fusion protein, is a polypeptide in which at least one TNFR2 AFFIMER sequence is covalently bonded (directly or indirectly) to the Fc domain of an immunoglobulin via a peptide backbone. The Fc fusion may, for example, include a TNFR2 AFFIMER sequence in the same polypeptide as the Fc domain of an antibody (which promotes effector function and pharmacokinetics). The immunoglobulin Fc domain may also be indirectly linked to at least one TNFR2AFFIMER polypeptide. A variety of known linkers can be selectively used to link Fc to a polypeptide containing a TNFR2 AFFIMER sequence to generate an Fc fusion. In one embodiment of the present invention, the Fc fusion may be dimerized to form an Fc fusion homodimer, or different Fc domains may be used to form an Fc fusion heterodimer.

[0152] In one embodiment of the present invention, the Fc-fusion isodimer may include a dimer of a TNFR2 AFFIMER formulation comprising a TNFR2 AFFIMER polypeptide, an Fc domain, and a TNFR2 AFFIMER polypeptide.

[0153] There are several reasons for selecting the Fc region of a human antibody to generate TNFR2 AFFIMER formulations as TNFR2 AFFIMER fusion proteins. The primary rationale is to form a protein that is large and stable enough to exhibit a pharmacokinetic profile similar to that of the antibody, and to utilize the properties conferred by the Fc region. These properties include the salvage neonatal FcRn receptor pathway, which includes FcRn-mediated recycling. This pathway allows the fusion protein to be recirculated to the cell surface after endocytosis, avoiding lysosomal degradation and consequently released back into the bloodstream, contributing to an extended serum half-life. Furthermore, another obvious advantage is that the Fc domain binds to Protein A, simplifying the downstream process in the production of AFFIMER formulations and enabling the acquisition of high-purity AFFIMER formulations.

[0154] Generally, the Fc domain may contain the constant region of an antibody excluding the first constant region immunoglobulin domain. Therefore, the Fc domain can include the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and flexible hinges located at the N-terminus of these domains. In the case of IgA and IgM, the Fc domain can include the J chain. In the case of IgG, the Fc domain can include the immunoglobulin domains Cγ2, Cγ3, and the hinge between Cγ1 and Cγ2. While the boundaries of the Fc domain can be diverse, the human IgG heavy chain Fc region is generally defined to include the carboxyl-terminal residues C226 or P230, where the amino acid numbers follow the EU index presented by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). While the boundary of the Fc domain can be diverse, the Fc region of human IgG heavy chains is generally defined as containing the carboxyl terminus from residue C226 or P230, where the amino acid numbers follow the EU index presented by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). Fc refers to the region described above separately, or to this region in the context of antibodies, this fragment, or fusion proteins. Polymorphisms reported at various Fc positions are also considered to be included in the Fc domain category as used herein.

[0155] In one embodiment of the present invention, the term "functional Fc region" as used herein means an Fc domain or fragment thereof that can bind to an FcRn. The functional Fc region may be characterized by binding to an FcRn but not having an effector function. The ability of an Fc region or fragment thereof to bind to an FcRn can be determined by standard binding tests known in the art. Here, "effector function" may include, for example, C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptors). These effector functions can be evaluated using a variety of validation methods known in the art.

[0156] In one embodiment of the present invention, the Fc domain is derived from the IgG1 subclass, but other subclasses (e.g., IgG2, IgG3, or IgG4) may also be used. Examples of usable IgG1 immunoglobulin Fc domains are as follows: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 442).

[0157] In one embodiment of the present invention, the Fc region included in the fusion protein may include a hinge region. For example, the hinge region may include core hinge residues located at sequence positions 1-16 of the human IgG1 Fc domain (e.g., DKTHTCPPCPAPELLG (SEQ ID NO: 536)). In one embodiment of the present invention, the fusion protein may partially form a multimeric structure (e.g., a dimer) by cysteine ​​residues at positions 6 and 9 within the hinge region of the human IgG1 immunoglobulin Fc domain sequence. In one embodiment of the present invention, the hinge region may further include residues derived from CH1 and CH2 regions located on the sides of the core hinge sequence of the IgG immunoglobulin Fc domain sequence. In one embodiment of the present invention, the hinge sequence may include or consist of GSTHTCPPCPAPELLG (SEQ ID NO: 443) or EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 444).

[0158] In one embodiment of the present invention, the hinge region may preferably include one or more substitutions that confer pharmacokinetic, biophysical, and / or biological properties. For example, the hinge region may include or consist of the following sequences: EPKSCDKTHTCPPCPAPELLGGPS (Sequence ID 445), EPKSSDKTHTCPPCPAPELLGGPS(SEQ ID NO: 446), EPKSSDKTHTCPPCPAPELLGGSS (Sequence ID 447), EPKSSGSTHTCPPCPAPELLGGSS (Sequence ID 448), DKTHTCPPCPAPELLGGPS (SEQ ID NO: 449), and DKTHTCPPCPAPELLGGSS(SEQ ID NO: 450).

[0159] In one embodiment of the present invention, the 18th residue P of the human IgG1 immunoglobulin Fc domain sequence may be replaced with S to remove the Fc effector function, and examples of such substitutions are as follows: EPKSSDKTHTCPPCPAPELLGGSS (sequence number 451), EPKSSGSTHTCPPCPAPELLGGSS (sequence number 452), and DKTHTCPPCPAPELLGGSS (sequence number 453).

[0160] In one embodiment of the present invention, the DKs at positions 1-2 of the human IgG1 immunoglobulin Fc domain may be replaced with GSs to remove a potential clip site. An example of such a substitution is shown in the following sequence: EPKSSGSTHTCPPCPAPELLGGSS (Sequence ID 448). In one embodiment of the present invention, the 103rd residue C in the heavy chain constant region of human IgG (e.g., CH1-CH3) may be substituted with S to prevent the formation of an improper cysteine ​​bond in the absence of the light chain, and an example of such substitution is shown in the following sequence: EPKSSDKTHTCPPCPAPELLGGPS(SEQ ID NO: 446), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 451), and EPKSSGSTHTCPPCPAPELLGGSS (Sequence ID 452).

[0161] In one embodiment of the present invention, the Fc domain may be a mammalian-derived Fc domain, preferably a human Fc domain. In one embodiment of the present invention, the Fc domain may be an Fc domain derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment of the present invention, the Fc domain may have about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with a natural Fc domain and / or the Fc region of the parent polypeptide. In one embodiment of the present invention, the Fc domain may have 90% or more sequence identity with a natural Fc domain and / or the Fc domain of the parent polypeptide.

[0162] In one embodiment of the present invention, the Fc domain may include an amino acid sequence selected from SEQ ID NOs. 454 to 467, or the Fc domains of SEQ ID NOs. 454 to 467 described in the examples. In one embodiment of the present invention, the lysine at the C-terminus of the Fc domain should be interpreted as a selective component of the fusion protein containing the Fc domain. In one embodiment of the present invention, the Fc domain may include an amino acid sequence selected from SEQ ID NOs. 454 to 467. In one embodiment of the present invention, the Fc domain may include an amino acid sequence selected from SEQ ID NOs. 454 to 467 in which the lysine present at its C-terminus is omitted.

[0163] [Table 5] JPEG2026520086000046.jpg199153JPEG2026520086000047.jpg94153

[0164] In the present invention, the terms "antibody-dependent cell-mediated toxicity" or "ADCC" refer to a form of cytotoxicity in which cytotoxic effector cells exhibit cytotoxicity and kill cells by specifically binding to antigen-possessed targets via secreted immunoglobulins bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages).

[0165] In one embodiment of the present invention, the AFFIMER formulation may include an Fc domain that does not have ADCC and / or complement activation, or effector function, or has reduced function. For example, the Fc domain may be characterized by including a naturally deactivated constant region derived from an IgG2 or IgG4 isotype, or a mutated IgG1 constant region. Suitable examples of modifications are described in EP0307434, etc. For example, the Fc domain may include, but is not limited to, substitutions of alanine residues at positions 235 and 237 (EU index numbers).

[0166] In one embodiment of the present invention, the AFFIMER formulation may include an Fc domain that retains some or all of the function of Fc. For example, if the fusion protein includes an Fc domain of human IgG1 or IgG3, it may exhibit ADCC and / or CDC activity. The level of effector function of the Fc domain of the present invention can be regulated by known methods. For example, it may include mutations in the CH2 domain, specifically one or more mutations selected from the 239, 332, and 330 positions of CH2 in IgG1. For example, the mutations may be one or more selected from S239D, I332E, and A330L. In one embodiment of the present invention, the glycosylation profile of the Fc domain may be modified so that fucosylation of the Fc region is reduced.

[0167] Albumin Fusion In one embodiment of the present invention, the AFFIMER formulation may include at least one AFFIMER sequence in addition to an albumin sequence or a fragment thereof. In other embodiments of the present invention, the AFFIMER formulation may be conjugated to an albumin sequence or albumin fragment via chemical bonding, as well as being integrated into a polypeptide sequence containing an AFFIMER polypeptide. In one embodiment of the present invention, the albumin, albumin variant, or albumin fragment may be human serum albumin (HSA), a variant thereof, or a fragment thereof. In one embodiment of the present invention, the albumin serum protein other than HSA may be characterized by being derived from, for example, cynomolgus monkeys, cattle, dogs, rabbits, and mice, with BSA being the most structurally similar to HSA (Kosa et al., (2007) J Pharm Sci. 96(11):3117-24). In one embodiment of the present invention, the albumin may be, but is not limited to, cynomolgus monkey serum albumin or a non-human serum albumin such as bovine serum albumin. Mature HSA, a 585-amino acid polypeptide (approximately 67 kDa) with a serum half-life of about 20 days, is primarily responsible for maintaining colloidal osmotic pressure, blood pH, and the transport and distribution of numerous endogenous and exogenous ligands. Serum albumin protein is composed of three structurally homologous domains (domains I, II, and III), mostly exhibiting an α-helical configuration, and is highly stabilized by 17 disulfide bonds.

[0168] In one embodiment of the present invention, the AFFIMER formulation may be characterized by comprising at least one AFFIMER polypeptide and mature human serum albumin (e.g., SEQ ID NO: 468) or a variant or fragment thereof. In one embodiment of the present invention, the albumin of the fusion protein may be characterized by maintaining a desired level of PK and / or biodistribution properties.

[0169] In one embodiment of the present invention, the albumin sequence can be separated from the AFFIMER polypeptide sequence or other flanking sequences via a linker sequence.

[0170] Unless otherwise specified, "albumin" or "mature albumin" in this invention refers to human serum albumin (HSA). However, full-length HSA is known to contain a signal peptide (MKWVTFISLLFLFSSAYS (SEQ ID NO: 483)) composed of 18 amino acids and a prodomain sequence (RGVFRR (SEQ ID NO: 469)) composed of the following 6 amino acids. The above total of 24 amino acids may be referred to as the pre-prodomain. In one embodiment of the present invention, the AFFIMER-HSA fusion protein may be characterized by being expressed and secreted by the HSA pre-prodomain. In one embodiment of the present invention, the fusion protein containing HSA may be characterized by containing the above signal sequence and being expressed and secreted by it.

[0171] In other embodiments of the present invention, instead of being provided as part of a fusion protein with an AFFIMER polypeptide, the serum albumin polypeptide may be covalently bonded to the AFFIMER-containing polypeptide via a linkage other than a backbone amide bond. For example, this may be done through crosslinking by chemical conjugation between the amino acid side chains of the albumin polypeptide and the AFFIMER-containing polypeptide, respectively.

[0172] In one embodiment of the present invention, a chemical modification applicable to producing an AFFIMER formulation to increase the protein half-life is lipidization, which involves covalently bonding fatty acids to the peptide side chain. This was developed as a method for extending the half-life of insulin and shares the same half-life extension mechanism as PEGylation. Specifically, it increases the hydrodynamic radius to reduce renal filtration. However, the lipid moisture itself is relatively small, and its effect is mediated indirectly through non-covalent bonding of the lipid moisture to circulating albumin. One consequence of lipidization is reduced water solubility of the peptide, but this can be modulated by engineering the linker between the peptide and the fatty acid. For example, glutamic acid or mini-PEG can be used in the linker. Modifications using linker engineering and lipid moisture can affect autoaggregation, delay biodistribution independently of albumin, and increase the half-life (Jonassen et al. (2012) Pharm Res. 29(8):2104-14).

[0173] In one embodiment of the present invention, the albumin-binding domain may be albumin-binding adnectins (see WO2011140086 "Serum Albumin Binding Molecules", WO2015143199 "Serum albumin-binding Fibronectin Type III Domains" and WO2017053617 "Fast-off rate serum albumin binding fibronectin type iii domains"), albumin-binding domain 3 (ABD3) of Protein G of Streptococcus G148, an albumin-binding domain antibody (e.g., GSK2374697, AlbudAb), or an albumin-binding nanobody (e.g., ATN103, Ozolralizumab).

[0174] AFFIMERXT In one embodiment of the present invention, molecules that bind to serum proteins such as HSA may include HSA AFFIMER polypeptides. Examples of such HSA AFFIMER polypeptides are described in WO2022 / 023540. The HSA AFFIMER polypeptides provided herein may, in one embodiment of the present invention, bind to other molecules to extend the half-life of those molecules (e.g., therapeutic polypeptides). These HSA AFFIMER polypeptides have been demonstrated in in vivo pharmacokinetic (PK) studies to extend the serum half-life of other AFFIMER polypeptide therapeutics bound within a single genetic fusion, for example, that can be produced in E. coli. AFFIMERXT TM Polypeptides can also be used to extend the half-life of other peptides or protein therapeutic agents, such as the TNFR2 AFFIMER of the present invention.

[0175] In one embodiment of the present invention, the HSA AFFIMER polypeptide extends the in vivo serum half-life of the TNFR2 AFFIMER polypeptide. For example, the HSA AFFIMER polypeptide can extend the half-life of the TNFR2 AFFIMER polypeptide by at least two times compared to the half-life when it is not bound to the TNFR2 AFFIMER polypeptide. In one embodiment of the present invention, the HSA AFFIMER polypeptide can extend the half-life of the TNFR2 AFFIMER polypeptide by at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least twenty times, or at least thirty times. In one embodiment of the present invention, the HSA AFFIMER polypeptide can extend the half-life of the TNFR2 AFFIMER polypeptide by approximately two to five times, two to ten times, three to five times, three to ten times, four to ten times, or five to ten times compared to the half-life when it is not bound to the TNFR2 AFFIMER polypeptide. In one embodiment of the present invention, the HSA AFFIMER polypeptide can extend the half-life of the TNFR2 AFFIMER polypeptide to at least 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or, for example, up to 1 week, after in vivo administration, compared to the half-life of the TNFR2 AFFIMER polypeptide when it is not bound to the HSA AFFIMER polypeptide.

[0176] The HSA AFFIMER polypeptide comprises an AFFIMER polypeptide derived from wild-type stephin A protein having an amino acid sequence that allows at least one solvent-accessible loop to selectively bind to HSA, and in some embodiments, the dissociation constant (Kd) is 10 ―6 It may be less than M.

[0177] In one embodiment of the present invention, the HSA AFFIMER polypeptide is derived from a wild-type human stepin A protein having a backbone sequence, and one or both of loop 2 [represented by (Xaa)n] and loop 4 [represented by (Xaa)m] can be replaced with alternative loop sequences (Xaa)n and (Xaa)m to have the following formula (I). FR1-(Xaa)n-FR2-(Xaa)m-FR3 (I)

[0178] Here, FR1 is an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity with the amino acid sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 470),

[0179] The aforementioned FR2 is an amino acid sequence represented by GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2), or an amino acid sequence having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100%) identity with the amino acid sequence of SEQ ID NO: 2.

[0180] The FR3 is an amino acid sequence represented by EDLVLTGYQV DKNKDDELTG F (Sequence ID 3), or an amino acid sequence having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100%) identity with the amino acid sequence of Sequence ID 3, and

[0181] In each case, Xaa is an arbitrary amino acid residue individually, and n and m are integers from 3 to 20, independently of each other. Additional variations of this structure are described in WO2022 / 023540.

[0182] In all embodiments, each amino acid in (Xaa)n may be the same or selected from any amino acids, and this also applies to (Xaa)m.

[0183] In one embodiment of the present invention, the TNFR2 AFFIMER polypeptide may include an HSA AFFIMER polypeptide containing an amino acid sequence selected from SEQ ID NOs. 471-477 (Table 6). In one embodiment of the present invention, the TNFR2 AFFIMER polypeptide has an extended serum half-life and may contain an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to the amino acid sequence selected from SEQ ID NOs. 471-477 (Table 6). HSA AFFIMER polypeptides for use in the present invention are described in WO2022 / 023540.

[0184] [Table 6] (Table 6) Examples of AFFIMER polypeptide sequences that specifically bind to HSA

[0185] Conjugates The AFFIMER formulation of the present invention may also include at least one functional moiety intended to confer detectability or additional pharmacological activity to the AFFIMER formulation. A functional moiety for detection means that the AFFIMER formulation can be used to detect binding to cells or tissues in vivo. A functional moiety having pharmacological activity means a formulation designed to be delivered to tissues expressing TNFR2, which is the target of the AFFIMER formulation, as in the case of the TNFR2 AFFIMER formulation of the present invention, thereby potentially inducing pharmacological results in the targeted tissue or cells.

[0186] The present invention provides AFFIMER formulations containing conjugates of substances having diverse functional groups, substituents, or moieties. The above functional materials include labels, dyes, immunoadhesion molecules, radionuclides, cytotoxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, proteins and polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelators, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, saccharides, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biomaterials, nanoparticles, and spin labels. Label, fluorophore, metal-containing moiety, radioactive moiety, novel functional group, group that interacts covalently or non-covalently with other molecules, photocaged moiety, actinic radiation-excitable moiety, photoisomerizable moiety, biotin, biotin derivative, biotin analogue, heavy atom-containing moiety, chemically cleavable group(group), photocleavable group, elovgated side chain, carbon-linked sugar, redox-active agent, aminothioacid, toxic moiety, isotope-labeled moiety, biophysical probe, phosphorescent group, chemiluminescent group, electron-dense group, magnetic group, intercalating group, chromophore, energy transfer agent, biologically active agent, detectable label, small molecule, quantum dot This may include, but is not limited to, dots, nanotransmitters, radionucleotides, radiotransmitters, neutron-capture agents, and other preferred compounds or substances.

[0187] Labels and Detectable Moieties If the moisture is a detectable label, it may be a fluorescent label, a radioactive label, an enzyme label, or any other label known to those skilled in the art. In one embodiment of the present invention, the functional moisture may be a detectable label that can be included as part of a conjugate to form a specific AFFIMER formulation suitable for medical imaging. Here, “medical imaging” means all techniques for visualizing internal parts of the human or animal body for diagnostic, research, or therapeutic purposes. For example, the AFFIMER formulation may be detected (and quantified) by radioscintigraphy, magnetic resonance imaging (MRI), computed tomography (CT scan), nuclear imaging, positron emission tomography (PET) contrast agents containing metal, fluorescence imaging (e.g., fluorescence imaging including near-infrared fluorescence (NIRF) imaging), bioluminescence imaging, or a combination thereof. The functional moisture of the present invention may selectively be a contrast agent for X-ray imaging. Formulations useful for improving such techniques are substances that enable visualization of specific sites, organs, or diseased areas within the body, or that improve the quality of the generated image, thereby facilitating or improving the interpretation of the image. Such formulations are referred to herein as contrast agents, which facilitate the distinction of different regions in an image by increasing the "contrast" between different parts of the image. Accordingly, the term "contrast agents" includes not only formulations that improve the quality of images that can be generated in the absence of such formulations (e.g., in the case of MRI), but also formulations that are essential for image generation (e.g., in the case of nuclear imaging).

[0188] In one embodiment of the present invention, the detectable label may include a chelate moiety for chelating a metal, and may be a chelator for, for example, a radiometal or a paramagnetic ion.

[0189] In one embodiment of the present invention, the detectable label may be a chelator for radionuclides useful in radiotherapy or imaging procedures. Useful radionuclides in the present invention include gamma (γ) emitters, positron emitters, Auger electron emitters, X-ray emitters, and fluorescence emitters, with beta (β)- or α (α)- emitters being used for therapeutic purposes.

[0190] Examples of radionuclides useful for treating toxins in radiation therapy include, but are not limited to, the following: 43K, 47Sc, 51Cr, 57Co, 58Co, 59Fe, 64Cu, 67Ga, 67Cu, 68Ga, 71Ge, 75Br, 76Br, 77Br, 77As, 81Rb, 90Y, 97Ru, 99mTc, 100Pd, 101Rh, 103Pb, 105Rh, 109Pd, 111Ag, 111In, 113In, 119Sb, 121Sn, 123I, 125I, 127Cs. 128Ba, 129Cs, 131I, 131Cs, 143Pr, 153Sm, 161Tb, 166Ho, 169Eu, 177Lu, 186Re, 188Re, 189Re, 191Os, 193Pt, 194Ir, 197Hg, 199Au, 203Pb, 211At, 212Pb, 212Bi, and 213Bi. The conditions under which a metal and a chelator form a coordinate bond are described, for example, in U.S. Patents 4,831,175, 4,454,106, and 4,472,509, by Gansow, among others. Examples of chelators, mentioned simply for illustrative purposes, include 1,4,7,-triazacyclononane-N,N′,N′′-triacetic acid (NOTA), 1,4,7,10-tetraazacyalododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA), and 1,4,8,11-tetraazacyalotetradecane-N,N′,N′′,N′′′-tetraacetic acid (TETA).

[0191] In one embodiment of the present invention, other detectable isotopes that can be directly introduced into amino acid residues of AFFIMER polypeptides without the need for a chelator include: 3 H, 14 C, 32 P, 35 S and 36 Cl may be included.

[0192] In one embodiment of the present invention, paramagnetic ions useful in the diagnostic procedure may also be administered. Examples of paramagnetic ions may include chromium(III), manganese(II), iron(III), cobalt(II), nickel(II), copper(II), neodymium(II), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), erbium(III), or combinations of these paramagnetic ions.

[0193] Examples of fluorescent labels may include, but are not limited to, organic dyes (e.g., cyanine, fluorescein, rhodamine, Alexa Fluors, Dylight Fluors, ATTO dyes, BODIPY dyes, etc.), biological fluorophores (e.g., green fluorescent protein (GFP), R-phycorerythrin, etc.), and quantum dots.

[0194] Examples of non-limiting fluorescent compounds that can be used in this invention include Cy5, Cy5.5 (also known as Cy5++), Cy2, Fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7, Fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++), Texas Red, LightCycler-Red 640, LightCycler Red 705, tetramethylrhodamine (TMR), rhodamine, rhodamine derivative (ROX), hexachlorofluorescein (HEX), and rhodamine 6G (rhodamine This may include 6G, R6G), rhodamine derivative JA133, Alexa fluorescent dyes (e.g., Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, Alexa Fluor 647), 4′,6-diamidino-2-phenylindole (DAPI), propidium indole, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, lissamine, and fluorescent transition metal complexes such as europium.Fluorescent compounds that can be used in the invention may also include fluorescent proteins, such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and its derivatives (BFP, EBFP, EBFP2, Azurite, Mkalamal), cyan fluorescent protein and its derivatives (CFP, ECFP, Cerulean, CyPet), and yellow fluorescent protein and its derivatives (YFP, Citrine, Venus, YPet). WO2008142571, WO2009056282, WO9922026.

[0195] Examples of enzyme-labeled enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and β-galactosidase.

[0196] Another well-known label is biotin. Biotin-labeled proteins generally consist of a biotinyl group, a spacer arm, and a reactive group responsible for binding to the target site of the protein. Biotin can be useful for binding proteins labeled with other molecules, including the avidin molecule.

[0197] AFFIMER polypeptide-drug conjugate In one embodiment of the present invention, the AFFIMER formulation may contain at least one therapeutic agent to form, for example, an AFFIMER polypeptide-drug conjugate. As used herein, the term "therapeutic agent" means a substance that can be used to treat, alleviate, manage or prevent a disease in humans or other animals. Such therapeutic agents include, but are not limited to, substances listed in the United States Pharmacopeia, the Homeopathic Pharmacopeia of the United States, the National Formulary, or their supplements, and include small molecules, nucleotides, oligopeptides, polypeptides, and the like.Examples of therapeutic agents that can bind to polypeptides containing AFFIMER include cytotoxic agents, anti-metabolites, alkylating agents, antibiotics, growth factors, cytokines, anti-angiogenic agents, anti-mitotic agents, toxins, and apoptotic agents. Examples include DNA alkylating agents, topoisomerase inhibitors, microtubule inhibitors (e.g., DM1, DM4, MMAF, MMAE), endoplasmic reticulum stress inducing agents, plantinum compounds, anti-metabolites, and vinca alkaloids. This includes, but is not limited to, alkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, radiosensitizers, and chemotherapeutic combination therapies.

[0198] To produce the conjugate of the present invention, any known method in the art for conjugating antibodies and other proteins may be used, including, for example, the methods described by Hunter et al. (1962, Nature 144:945), David et al. (1974, Biochemistry 13:1014), Pain et al. (1981, J.Immunol. Methods 40:219), and Nygren (1982, Histochem. and Cytochem. 30:407). Methods for conjugating peptides, polypeptides, organic and inorganic molecules to antibodies and other proteins are known and common techniques and can be readily applied to produce variants of the AFFIMER formulation of the present invention.

[0199] In one embodiment of the present invention, if the conjugated moisture is a peptide or polypeptide, the moisture may be chemically crosslinked to the polypeptide containing the AFFIMER, or may be included as part of a fusion protein with the polypeptide containing the AFFIMER. An example is the diphtheria toxin-AFFIMER fusion protein. In the case of non-peptide entities, the addition to the polypeptide containing the AFFIMER may generally occur through chemical conjugation, and may include chemical conjugation, for example, through the active group of an amino acid side chain, a C-terminal carboxyl group, or an N-terminal amino group. In one embodiment of the present invention, whether a fusion protein or a chemically crosslinked moisture, the conjugated moisture may include at least one site that is sensitive to environmental conditions (e.g., pH), thereby allowing the conjugated moisture to be released from the polypeptide containing the AFFIMER. For example, this may occur in diseased tissue (or, if the conjugated moisture has a function of protecting healthy tissue, the tissue that needs to be protected).

[0200] Spacers In one embodiment of the present invention, the AFFIMER polypeptide-drug conjugate may include a spacer or binding (L1) between a half-life extension moiety that can be cleaved by an enzyme (e.g., an enzyme present in the inflammatory microenvironment) and a substrate recognition sequence (SRS).

[0201] The spacer described above can be any molecule, and may include, for example, one or more nucleotides, amino acids, or chemical action groups. In one embodiment of the present invention, the spacer may be a peptide linker (e.g., two or more amino acids). It is desirable that the spacer does not adversely affect polypeptide expression, secretion, or biological activity. In one embodiment of the present invention, the spacer must not be antigenic and must not induce an immune response. The immune response may include responses from the innate immune system and / or adaptive immune system. Thus, the immune response may be a cell-mediated immune response and / or a humoral immune response. The immune response may be, for example, a T cell response, a B cell response, a spontaneous killing cell (NK cell) response, a mononuclear cell response, and / or a macrophageous cell response. Other cell responses may also be included in the present invention. In one embodiment of the present invention, the linker may be non-protein-coding.

[0202] In one embodiment of the present invention, L1 may be a hydrocarbon (linear or cyclic) such as 6-maleimidocaproyl, maleimidopropanoyl, and maleimidomethyl cyclohexane-1-carboxylate, or L1 may be N-Succinimidyl 4-(2-pyridylthio)pentanoate, N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, or N-Succinimidyl(4-iodo-acetyl)aminobenzoate.

[0203] In one embodiment of the present invention, L1 may be a polyether, for example, poly(ethylene glycol) or other hydrophilic linker. For example, if the CBM contains a thiol group (e.g., a cysteine ​​residue), L1 may be poly(ethylene glycol) bonded to the thiol group via a maleimide moiety.

[0204] Examples of non-limiting linkers that may be used within the scope of the present invention are described in International Publication WO2019 / 236567, published on 12 December 2019, which is included herein by reference.

[0205] Encoded AFFIMER polynucleotide for in vivo delivery Another approach of the present invention involves allowing the human body itself to produce therapeutic polypeptides for the delivery of therapeutic AFFIMER formulations, such as TNFR2 AFFIMER formulations. Various clinical studies have demonstrated the usefulness of in vivo gene transfer using various different transfer systems. In vivo gene transfer aims to administer encoded AFFIMER polynucleotides to the patient, rather than the AFFIMER formulation itself. Through this, the patient's body can produce the therapeutic AFFIMER formulation of interest over a long period and secrete it systemically or locally depending on the site of production. Genetically encoded AFFIMER polynucleotides offer a more efficient alternative in terms of effort and cost compared to producing, purifying, and administering polypeptide versions of AFFIMER formulations in the conventional manner. Numerous antibody expression platforms pursued in vivo can be applied to the delivery of encoded AFFIMER polynucleotides, including viral vectors, naked DNA, and RNA.

[0206] The success of gene therapy has been primarily driven by improvements in nonviral and viral gene transfer vectors. A variety of physical and chemical nonviral methods have been used to transfer DNA and mRNA to mammalian cells, a considerable number of which have been developed into clinical-stage technologies for ex vivo and in vivo gene therapy and can be readily applied to the TNFR2 AFFIMER polynucleotide transfer encoded herein.

[0207] An effective approach to the transmission of encoded AFFIMER genes is to ensure that expression is achieved at an appropriate level for the specific application. The promoter, as a major cis-acting element in vector genome design, can determine not only the overall intensity of expression but also cell specificity. Similarly, polyadenylation of the transcribed and encoded AFFIMER transcript can be important for nuclear export, translation, and mRNA stability. Therefore, in one embodiment of the present invention, the encoded AFFIMER polynucleotide may include not only the promoter and / or polyadenylation signaling sequence, but also other regulatory elements known to the art, such as enhancers, intron sequences, and post-transcriptional regulatory agents.

[0208] Viral vectors Exemplary viral gene therapy systems readily applicable to the present invention include plasmids, adenoviruses, adeno-associated viruses (AAVs), retroviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, poxviruses, reoviruses, measles viruses, and Semliki Forest viruses. Preferably, non-cytopathic eukaryotic virus-based viral vectors are used, in which non-essential genes can be replaced by nucleic acid constructs containing nucleic acid sequences encoding epitopes and target sequences of interest.

[0209] More specifically, the encoded AFFIMER polynucleotide can be transmitted into the body using double-stranded DNA viruses such as adenoviruses and adeno-associated viruses (AAVs), which have already been approved for use in human gene therapy. The TNFR2 AFFIMER polypeptide can be encoded and transmitted into the body using retroviral vectors such as gamma retroviruses or lentiviruses. In one embodiment of the present invention, the viral vector can be pseudotyped into one of the following: amphotropic, polytropic, xenotropic, 10A1, GALV, VSV-G, baboon endogenous virus, RD114, rhabdovirus, alphavirus, measles virus, or influenza virus coat.

[0210] Non-viral vectors Exemplary nucleotides or polynucleotides that may be used in the encoded TNFR2 AFFIMER formulation of the present invention include, but are not limited to, ribonucleotides (RNA), deoxyribonucleotides (DNA), treosnucleotides (TNA), glycol nucleotides (GNA), peptide nucleotides (PNA), locked nucleotides (including LNA, LNA with a β-D-ribo structure, α-LNA with an α-L-ribo structure (α-LNA, an isomer of LNA), 2'-amino-LNA functionalized with a 2'-amino group, and 2'-amino-α-LNA functionalized with a 2'-amino group), ethylene nucleotides (ENA), cyclohexenyl nucleosides (CeNA), or hybrids and combinations thereof. Accordingly, the encoded TNFR2 AFFIMER polynucleotides may be transmitted via plasmid DNA, minicircle DNA, mRNA, RNA replicons, etc. The means of transduction may include electroporation-based transduction, lipofection and the use of other transduction reagents, naked DNA transduction, gold particle transduction and other means known in the art.

[0211] Expression Methods and Systems for Protein Production The TNFR2 AFFIMER formulation proteins described herein may be produced by any suitable method known in the art. Such methods range from direct protein synthesis to constructing a DNA sequence encoding a polypeptide sequence and expressing it in a suitable host. Furthermore, in the case of recombinant AFFIMER formulation proteins that involve additional modifications such as chemical modification or conjugation, the recombinant AFFIMER formulation proteins may be further manipulated chemically or enzymatically after isolation from host cells or chemical synthesis. The AFFIMER polypeptide may be secreted or conjugated to the membrane of the expressing cell by anchoring or tethering, for example, through the inclusion of a transmembrane domain.

[0212] The present invention comprises a recombination method and nucleic acid for recombinantly expressing the protein of a recombinant AFFIMER formulation, the method comprising: (i) introducing a polynucleotide encoding the amino acid sequence of the AFFIMER formulation into a host cell (e.g., the polynucleotide is present in a vector and / or operably ligated to a promoter); (ii) culturing the host cell (e.g., a eukaryotic or prokaryotic host cell) under suitable conditions for the expression of the polynucleotide; and (iii) selectively separating the AFFIMER formulation from the host cell and / or the culture medium in which the host cell was cultured. See, for example, WO04 / 041862, WO2006 / 122786, WO2008 / 020079, WO2008 / 142164 or WO2009 / 068627.

[0213] In one embodiment of the present invention, the DNA sequence encoding the protein of the recombinant AFFIMER formulation of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. The oligonucleotide may be designed based on the amino acid sequence of the desired polypeptide, and may be designed by selecting codons preferred in the host cell from which the recombinant polypeptide of interest is produced. Standard methods may be applied when synthesizing the polynucleotide sequence encoding the isolated polypeptide of interest. For example, it may be used to construct a back-translated gene from which the entire amino acid sequence has been back-translated. Alternatively, a DNA oligomer containing the nucleotide sequence encoding a specific isolated polypeptide may be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide may be synthesized and then ligated together. Individual oligonucleotides generally contain a 5′ or 3′ overhang for complementary assembly.

[0214] Once the nucleotide sequence encoding the protein of a recombinant AFFIMER preparation is secured, a vector for producing the protein of the recombinant AFFIMER preparation can be fabricated using recombinant DNA technology known to the art. An expression vector containing the coding sequence of the recombinant AFFIMER preparation and appropriate transcriptional and translational control signals can be constructed using methods well known to experts. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. For example, one can refer to the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al. des., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY.

[0215] An expression vector containing the nucleotide sequence encoding the recombinant AFFIMER formulation protein can be transmitted to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transformed cell can be cultured by conventional techniques to produce the recombinant AFFIMER formulation protein of the present invention. In certain embodiments, the expression of the recombinant AFFIMER formulation protein can be regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0216] The expression vectors described above may include a replication origin, which can be selected based on the type of host cell used for expression. For example, the replication origin of plasmid pBR322 (Product No. 303-3s, New England, Beverly, Mass.) is useful for many Gram-negative bacteria, while diverse replication origins from SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or papillomaviruses (e.g., HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, components of the replication origin are not required in mammalian expression vectors (for example, the SV40 replication origin is commonly used because it contains an early promoter).

[0217] The vector described above may contain at least one selectable marker gene, which may be a genetic element encoding a protein necessary for the survival and growth of host cells cultured in the selective medium. Typical selectable marker genes encode either (a) proteins that confer resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin in prokaryotic host cells), (b) proteins that compensate for cellular auxotrophic deficiencies, or (c) proteins that supply essential nutrients not available in the complex medium. Preferred selectable markers include the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. The neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells. Other selectable genes can be used for amplification of the expressed gene. Amplification is the process by which a gene is repeatedly arranged within the chromosomes of successive generations of recombinant cells when there is a high demand for the production of growth-critical proteins. Examples of selection markers used in mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. Mammalian cell transformants are exposed to selection pressure that is uniquely adapted to ensure their survival by markers present in the vector. Selection pressure is applied by continuously changing the concentration of the selector in the culture medium while culturing the transformed cells, resulting in the amplification of all DNA encoding the selection gene and the recombinant AFFIMER protein. As a result, a larger amount of recombinant AFFIMER protein is synthesized from the amplified DNA.

[0218] The vector may also contain at least one ribosome binding site, which can be transcribed into mRNA containing the coding sequence encoding the recombinant AFFIMER protein. For example, such a site may be characterized by a Shine-Dalgarno sequence (prokaryotic) or a Kozak sequence (eukaryotic). These elements are generally located between the 5′ end of the coding sequence of the expressed polypeptide and the 3′ end of the promoter. Shine-Dalgarno sequences can be diverse but generally consist of numerous polypurines containing high concentrations of adenine (A) and guanine (G). Numerous Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using the methods described above and used in prokaryotic vectors.

[0219] The expression vectors described above generally contain an operable-linked promoter that is recognized by a host organism and operably linked to the nucleic acid molecule encoding the recombinant AFFIMER preparation protein. Depending on the host cell used for expression and the desired yield, native or heterologous promoters may be used. Examples of promoters used in prokaryotic hosts include the beta-lactamase and lactose promoter system, the alkaline phosphatase, the tryptophan (trp) promoter system, and hybrid promoters such as the TAC promoter. Other known bacterial promoters are also suitable, and their sequences have already been published and can be linked to the desired nucleic acid sequence using a linker or adapter while providing restriction enzyme cleavage sites.

[0220] Promoter used in yeast hosts is also well known in the industry. Yeast enhancers are advantageous when used in conjunction with yeast promoters. Promoter suitable for use in mammalian host cells is also well known, and examples include promoters derived from the genomes of polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, and hepatitis B virus. Most preferably, there is a promoter derived from Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat-shock promoters and actin promoters.

[0221] Additional promoters that may be used to express the selective binding agent of the present invention include, but are not limited to, the following:

[0222] SV40 early promoter region (Bernoist and Chambon, Nature, 290:304-310, 1981), CMV promoter, promoter included in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980), Cell 22:787-97), herpes thymidine kinase promoter (Wagner et al. (1981), Proc.Natl.Acad.Sco.USA78:1444-5), regulatory sequences of the metallothionine gene (Brinster et al., Nature, 296;39-42, 1982), prokaryotic expression vector. The beta-lactamase promoter (Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. USA, 75; 3727-3731, 1978), or the tac promoter (DeBoer, et al. (1983), Proc. Natl. Acad. Sci. USA, 80:21-5). Additionally, it has been used in transgenic animals exhibiting tissue specificity in animal transcriptional control regions of interest: the elastase I gene control region, which shows activity in pancreatic acinar cells (Seift et al. (1984), Cell 38:639-46; Ornitz et al. (1986), Cold Spring Harbor Symp. Quant. Biol.).50:399-409; MacDonald (1987), Hepatology 7:425-515); insulin gene control region showing activity in pancreatic beta cells (Hanahan (1985), Nature 315:115-22); immunoglobulin gene control region showing activity in lymphoid cells (Grosschedl et al. (1984), Cell 38;647-58; Adames et al. (1985), Nature 318;533-8; Alexander et al. (1987), Mol.Cell.Biol.7:1436-44); mouse mammary tumor virus regulatory region showing activity in testicular, mammary, lymphocyte and mast cells (Leder et al. Albumin gene control region active in the liver (Pinkert et al. (1987), Genes and Devel. 1:268-76), alpha-fetoprotein gene control region active in the liver (Krymlauf et al. (1985), Mol.Cell.Biol.5:1639-48; Hammer et al. (1987), Science, 235:53-8), alpha 1-antitrypsin gene control region active in the liver (Kelsey et al. (1987), Genes and Devel. 1:161-71), beta-globin gene control region active in myeloid cells (Mogram et al. (1987) al., Nature, 315,338-340, 1985;Kollias et al.(1986), Cell 46:89-94), the myelin basic protein gene control region that is active in oligodendrocytes of the brain (Readhead et al. (1987), Cell, 48:703-12), the myosin light chain-2 gene control region that is active in skeletal muscle (Sani (1985), Nature, 314:283-6), and the gonadotropin-releasing hormone gene control region that is active in the hypothalamus (Mason et al. (1986), Science 234:1372-8).

[0223] Enhancer sequences may be inserted into the above vectors to increase transcription in eukaryotic host cells. Several enhancer sequences derived from mammalian genes are known (e.g., globin, elastase, albumin, α-fetal protein, and insulin). However, virus-derived enhancers are generally used. Exemplary enhancer elements include the SV40 enhancer, the CMV (cytomegalovirus) early promoter enhancer, the polyoma enhancer, and the adenovirus enhancer, which are used for eukaryotic promoter activation.

[0224] The enhancers described above can be incorporated into the vector at the 5′ or 3′ position of the polypeptide coding region, but are generally positioned at the 5′ position of the promoter.

[0225] Vectors for expressing nucleotides include those suitable for bacterial, insect, and mammalian host cells. Such vectors include pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, San Diego, Calif.), pBSII (Stratagene Company, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Niotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BlueBacII; Invitrogen), pDSR-alpha (PCT Publication No. W090 / 14363), and pFastBacDual (Gibco / BRL, Grand Island, NY).

[0226] Additional usable vectors may include cosmids, plasmids, or modified viruses, but the vector system must be compatible with the selected host cells. Such vectors include, for example, Bluecript plasmid derivatives (high-capy-number ColE1-based phagemids, Stratagene Cloning Systems Inc., La Jolla, Calif.) and PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPO). TM Recombinant molecules include, but are not limited to, TA CloningKit, PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, Calif., and mammalian, yeast, or viral vectors (e.g., baculovirus expression system pBacPAK plasmid derivatives, Clontech, Palo Alto, Calif.). Recombinant molecules can be introduced into host cells through transformation, transfection, infection, electroporation, or other known techniques.

[0227] Eukaryotic and prokaryotic host cells (including mammalian cells for the expression of the recombinant AFFIMER formulations disclosed herein) include many immortalized cell lines well known in the art and available from the ATCC (American Type Culture Collection). These include CHO (Chinese hamster ovary) cells, NSO (NSO mouse myeloma) cells, SP2 (Sp2 / 0 mouse myeloma) cells, HeLa (Henrietta Lacks cervical carcinoma) cells, BHK (Baby hamster kidney) cells, COS (CV-1 in Origin with SV40 genes) cells, human hepatocellular carcinoma (Hep G2) cells, A549 cells, 3T3 cells, HEK-293 (Human embryonic kidney 293) cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Specific cell lines can be selected by identifying cell lines with high expression levels. Other usable cell lines include insect cell lines (e.g., Sf9 cells), amphibian cells, bacterial cells, plant cells, and fungal cells.Fluids are yeast and are filamentous fungus) The following species include Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, and Pichia membraneefaciens、Minuta pichia(Ogataea minuta、Pichia lindneri)、Pichia opuntiae、Pichia thermotolerans、Pichia salictaria、Pichia guercuum、Pichia pijperi、Pichia stiptis、Pichia methanolica、Pichia sp.、Saccharomyces cerevisiae、Saccharomyces sp.、Hansenula polymorpha、Kluyveromyces sp.、Kluyveromyces lactis、Candida albicans、Aspergillus nidulans、Aspergillus niger、Aspergillus oryzae、Trichoderma reesei、Chrysosporium lucknowense、Fusarium sp.、Fusarium gramineum、Fusarium venenatum、Physcomitrella patens and Neurospora crassa.Pichia sp.、any Saccharomyces sp.Hansenula polymorpha、any Kluyveromyces sp albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolutica, and Neurospora crassa.

[0228] A variety of host-expression vector systems can be used to express recombinant AFFIMER proteins. Such host-expression systems not only provide a means for producing and subsequently purifying the coding sequences of recombinant AFFIMER proteins, but also provide cells that can express recombinant AFFIMER proteins in situ when transformed or transduced with appropriate nucleotide coding sequences. Examples of such systems include microorganisms transformed with recombinant bacteriophage DNA, plasmid DNA, or coarsemid DNA expression vectors (e.g., Escherichia coli, Bacillus subtilis), yeast transformed with recombinant yeast expression vectors (e.g., Saccharomyces pichia), insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus), plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid), or mammalian cell systems possessing recombinant expression constructs (e.g., COS cells, CHO cells, BHK cells, 293 cells, 293T cells, 3T3 cells, lymphotic cells; Examples include Per.C6 cells (mouse retinal cells; developed by Crucell), etc. (see USPat. No. 5,807,715). The above mammalian cell systems may contain recombinant expression constructs that include, for example, promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0229] In bacterial systems, numerous expression vectors can be advantageously selected depending on the intended use of the recombinant AFFIMER protein being expressed. For example, when producing large quantities of protein for the creation of a pharmaceutical composition of the recombinant AFFIMER protein, a vector that instructs the expression of a high level of fusion protein that is easily purified may be preferred. Such vectors may include, for example, the Escherichia coli (E. coli) expression vector pUR278 (Ruther et al. (1983) "Easy Identification of cDNA Clones" EMBO J.2:1791-1794), in which the coding sequence of the AFFIMER protein is individually ligated into the vector in a frame such as a lac Z coding region to generate the fusion protein. Other examples include, but are not limited to, pIN vectors (Inouye et al. (1985) "Up-Promotor mutations In The Lpp Gene of Escherichia coli," Nucleic acids Res. 13:3101-3110; Van Heeke et al. (1989) "Expression of Human Asparagine Synthetase in Escherichia coli," J. Biol. Chem. 24:5503-5509). pGEX vectors can also be used to express exogenous polypeptides fused with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified by adsorption and binding from lysed cells to glutathione-agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or Factor Xa rotease cleavage site, allowing the cloned target gene product to be isolated from the GST moiety.

[0230] In insect systems, AcNPV (Autographa californica nuclear polyhedrosis virus) is used as a vector for expressing foreign genes. This virus proliferates in Spodoptera frugiperda cells. The protein coding sequence of the AFFIMER formulation can be individually cloned into the non-essential region of the virus (e.g., the polyhedrin gene) and placed under the regulation of the AcNPV promoter (e.g., the polyhedrin promoter).

[0231] Numerous viral-based expression systems can be used in mammalian host cells. When adenovirus is used as an expression vector, the protein-coding sequence of the AFFIMER formulation of interest can be ligated to the adenovirus transcription / translation regulatory complex (e.g., late promoter and tripartite leader sequence). This chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. If inserted into a non-essential region of the viral genome (e.g., E1 or E3 region), a recombinant virus capable of viable host infection and expressing immunoglobulin molecules is generated (see Logan et al. (1984) "Adenovirus Tripartite Leader Sequence Enhances Translation of mRNAs LateAfter Infection," Proc.Natl.Acad.Sci.(USA)81:3655-3659). A specific initiation signal may also be required for efficient translation of the inserted protein-coding sequence of the AFFIMER formulation. Such signals include the ATG initiation codon and adjacent sequences. Furthermore, the entire insert can only be translated if the initiation codon is phase-matched with the reading frame of the desired coding sequence. Such exogenous translation-regulating signals and initiation codons can take diverse forms, either naturally occurring or synthetically. Expression efficiency can be improved by the inclusion of appropriate transcription enhancer elements and transcription terminators (see Bitter et al. (1987) "Expression and Secretion Vectors for Yeast," Methods in Enzymol. 153:516-544).

[0232] Furthermore, host cell lines capable of regulating the expression of inserted sequences or modifying and processing gene products in a desired manner may be selected. Such modifications (e.g., glycosylation) and processing of protein products (e.g., cleavage) may be important for protein function. Different host cells have unique and specific mechanisms for post-translational processing and modification of proteins and gene products. Therefore, appropriate cell lines or host systems may be selected to ensure proper modification and processing of expressed foreign proteins. For this purpose, eukaryotic host cells possessing cellular mechanisms for proper processing of primary transcripts, glycosylation, and phosphorylation of gene products may be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20, and T47D, CRL7030, and Hs578Bst cells.

[0233] For long-term, high-yield recombinant protein production, stable expression is considered. For example, cell lines that stably express the antibody of the present invention can be engineered. Instead of using expression vectors derived from viral replication, host cells can be transformed into DNA controlled by appropriate expression regulators (e.g., promoters, enhancer sequences, transcription terminaters, polyadenylation sites, etc.) and selectable marker genes. After introduction of the foreign DNA, the engineered cells are cultured in enriched media for 1-2 days, and then transferred to selective media. The selectable marker gene in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into the chromosome, proliferate, and form a foci. This foci can then be cloned again and extended into a cell line. Such a method can be advantageously used to engineer cell lines that express the protein of the recombinant AFFIMER formulation of the present invention. The engineered cell lines described above may be particularly useful for screening and evaluating compounds that directly or indirectly interact with the protein of the recombinant AFFIMER formulation.

[0234] In the present invention, a variety of selection systems can be used. For example, the herpes simplex virus thymidine kinase (Wigler et al. (1977) Cell 11:223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al. (1962), Proc. Natl. Acad. Sci. (USA) 48:2026-2034), and adenine phosphoribosyltransferase (Lowy et al. (1980), Cell 22:817-823) genes can be used for tk-, hgprt-, or aprt- cells, respectively, but are not limited thereto. Furthermore, resistance to antimetabolites can be used as a basis for selection for the following genes: dhfr conferring resistance to methotrexate (Wigler et al. (1980), Proc. Natl. Acad. Sci. (USA) 77:3567-3570; O'Hare et al. (1982), Proc. Natl. Acad. Sci. (USA) 78:1527-1531); gpt conferring resistance to mycophenolate (Mulligan et al. (1981), Proc. Natl. Acad. Sci. (USA) 78:2072-2076); neo conferring resistance to aminoglycoside G-418 (Tachibana et al. (1991), Cytotechnology 6(3):219-226; Tolstoshev (1993), Ann. Rev. Pharmacol. Toxicol.32:573-596; Mulligan (1993) Science 260:926-932; and MOrgan et al. (1993) Ann.Rev.biochem.62:191-217).Commonly known methods in the field of recombinant DNA technology that may be used include those described in Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY; Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CURRENT PROTOCOLS IN HUMAN GENETICS, John Wiley & Sons, NY; Colbere-Garapin et al. (1981) J. Mol. Biol. 150:1-14; and hygro (Santerre et al. (1984) Gene 30:147-156) for conferring resistance to hygromycin.

[0235] The expression level of the recombinant AFFIMER formulation protein of the present invention can be increased by vector amplification (see Bebbington and Hentschel, in DNA CLONING, Vol. 3 (Academic Press, New York, 1987)). When the marker of the vector system expressing the recombinant AFFIMER formulation protein is amplified, an increase in the level of inhibitor present in the host cell culture medium can increase the number of copies of the marker gene. Because the amplified region is linked to the nucleic acid sequence encoding the recombinant AFFIMER formulation protein, the production of the recombinant AFFIMER formulation protein can also be increased (Crouse et al. (1983) Mol. Cell. Biol. 3:257-266).

[0236] If the AFFIMER formulation of the present invention is an AFFIMER antibody fusion or other multiprotein complex, host cells can be co-transfected with two expression vectors, for example, a first vector encoding the heavy chain and a second vector encoding the light chain polypeptide. One or both of the two expression vectors may contain the AFFIMER polypeptide coding sequence of the present invention. The two vectors may contain the same selectable marker that enables the identical expression of the heavy chain and light chain polypeptides. Alternatively, a single vector encoding all of the heavy chain and light chain polypeptides may be used. In such situations, the light chain must be positioned before the heavy chain to prevent excessive expression of the toxic free heavy chain (Proudfoot (1986) "Expression and Amplification of Engineered Mouse Dihydrofolate Reductase Minigenes," Nature 322:562-565; Kohler (1980) "Immunoglobulin Chain Loss in Hybridoma Lines," Proc. Natl. Acad. Sci. (USA) 77:2197-2199). The coding sequences for the heavy and light chains may include cDNA or genomic DNA.

[0237] Generally, glycoproteins produced in specific cell lines or transgenic animals may have glycosylation patterns characteristic of those cell lines or transgenic animals. Therefore, the specific glycosylation pattern of the protein in recombinant AFFIMER formulations may vary depending on the specific cell line or transgenic animal used to produce the protein. In some embodiments of the AFFIMER / antibody fusion of the present invention, a glycosylation pattern containing only unfucosylated N-glycans may be released, as this has been reported to typically exhibit more potent efficacy than fucosylated counterparts in both in vitro and in vivo settings (see, for example, Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003); U.S. Patents 6,946,292 and 7,214,775).

[0238] Furthermore, the expression of AFFIMER formulations from production cell lines can be enhanced using numerous known techniques. For example, glutamine synthetase (GS) gene expression systems are a common approach to improve expression under specific conditions. GS systems are described in whole or in part in connection with European Patent Nos. 0216846, 0256055, 0323997 and European Patent Application No. 89303964.4. Thus, in some embodiments of the present invention, mammalian host cells (e.g., CHO) lack the glutamine synthetase gene and grow in the absence of glutamine in culture medium, but polynucleotides encoding immunoglobulin chains can compensate for the gene deletion in the host cells by containing the glutamine synthetase gene. Not only host cells containing the above-mentioned binder, the nucleic acid or vector encoding it, but also expression methods for producing the above-mentioned binder using such host cells are included in the scope of the present invention.

[0239] Recombinant protein expression in insect cell culture systems (e.g., baculoviruses) provides a powerful method for producing correctly folded and biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art.

[0240] Proteins from recombinant AFFIMER formulations produced by transformed host cells can be purified by any suitable method. Standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and sizing column chromatography), centrifugation, differential lysis, or other standard techniques for protein purification. Affinity tags such as hexahistidine, maltose-binding domains, influenza keratin protein sequences, and glutathione-S-transferase can be attached to the protein and easily purified by passing it through a suitable affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), and X-ray crystallography.

[0241] In one embodiment of the present invention, the protein of the recombinant AFFIMER formulation produced in bacterial culture can be separated by at least one concentration, salting-out, ion-exchange chromatography, or size exclusion (gel filtration) chromatography step, for example, following initial extraction from a cell pellet. High-performance liquid chromatography (HPLC) may be used in the final purification step. Microbial cells used for the expression of the recombinant protein can be disrupted by any suitable method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysants.

[0242] Methods of Use and Pharmaceutical Compositions The AFFIMER formulation of the present invention can be usefully used in a variety of applications, including systemic lupus erythematosus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and ulcerative colitis), graft-versus-host disease (GvHD) (related to stem cell transplantation) (also known as allograft rejection), and transplantation or solid organ transplantation. This includes, but is not limited to, methods for treating transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjögren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, glomerulosclerosis, and / or cancer. The above methods of use may be in vitro, ex vivo, or in vivo.

[0243] Systemic lupus erythematosus Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can cause inflammation (including edema) and pain throughout the body, and is commonly referred to simply as lupus. There are several types of lupus, with systemic lupus erythematosus being the most common. Other types of lupus are as follows: Cutaneous erythematous lupus: This type of lupus affects the skin. "Cutaneous" is a term that refers to the skin. Patients with cutaneous erythematous lupus may exhibit skin-related symptoms, such as increased sensitivity to sunlight and rashes. Hair loss is also a possible symptom associated with this disease. Drug-induced lupus: This type of lupus is caused by certain medications. Patients with drug-induced lupus may exhibit symptoms similar to systemic lupus erythematosus, but it is generally transient. Neonatal lupus: Neonatal lupus is a rare type of lupus that is discovered in infants at birth. Children born with neonatal lupus inherit antibodies from their mothers, meaning the mother either had lupus during pregnancy or may develop the disease in the future. Not all newborns born to mothers with lupus will develop lupus.

[0244] In one embodiment of the present invention, therapies that may be used in combination with the TNFR2 AFFIMER polypeptide include, for example, steroids (including corticosteroids and prednisone), hydroxychloroquine (Plaquenil), azathioprine (Imuran), methotrexate (Rheumatrex), cyclophosphamide (Cytoxan), mycophenolate mofetil (CellCept), belimumab (Benlysta), and / or rituximab (Rituxan).

[0245] Lupus nephritis Lupus nephritis develops as a complication of lupus. It occurs when lupus autoantibodies affect the structure of the kidneys, which filter waste products. This can lead to kidney inflammation, hematuria, proteinuria, hypertension, renal dysfunction, or renal failure. Approximately half of adult patients with systemic lupus develop lupus nephritis. In systemic lupus, immune system proteins damage the kidneys, impairing their ability to filter waste products.

[0246] Rheumatoid arthritis Rheumatoid arthritis is a type of chronic (progressive) arthritis that affects the joints on both sides of the body, such as the hands, wrists, and knees. The short-term goal of rheumatoid arthritis medications is to reduce joint pain and edema and improve joint function. The long-term goal is to slow or prevent disease progression, especially joint damage. Arthritis is a general term that describes inflammation of the joints. Rheumatoid arthritis is a type of chronic (progressive) arthritis (pain- and swelling-inducing) that typically occurs symmetrically in the joints (on both sides of the body, such as the hands, wrists, and knees). This involvement of multiple joints is a distinguishing feature that sets rheumatoid arthritis apart from other types of arthritis. Rheumatoid arthritis affects not only the joints, but in some cases can also affect the skin, eyes, lungs, heart, blood, nerves, or kidneys. Examples of therapies that may be used in combination with the TNFR2 AFFIMER polypeptide of the present invention to treat rheumatoid arthritis include the following: Analgesic and anti-inflammatory drugs: These products include nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen (MOTRIN), naproxen (ALEVE), and similar products. Another type of drug, COX-2 inhibitors, also belongs to this category and alleviates the signs and symptoms of rheumatoid arthritis. Celecoxib (CELEBREX), one type of COX-2 inhibitor, is available in the United States. COX-2 inhibitors are designed to reduce gastrointestinal bleeding side effects.

[0247] Disease-modifying antirheumatic drugs (DMARDs): Unlike other NSAIDs, DMARDs can effectively modify the immune system itself, thereby slowing disease progression. Traditional DMARDs include methotrexate (TREXALL), gold salts, penicillamine (CUPRIMINE), hydroxychloroquine (PLAQUENIL), sulfasalazine (AZULFIDINE), cyclosporine (SANDIMMUNE), cyclophosphamide (CYTOXAN), and leflunomide (ARAVA). Currently, methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine are the most frequently used (cyclosporine, cyclophosphamide, gold salts, and penicillamine are no longer commonly used). Biologics: In addition to these "conventional" DMARDs, new drugs have been approved. Currently, there are seven types of drugs, and depending on the case, each classification contains different types of drugs from one another (some of which have been used since 2000 as anti-TNF agents). Collectively, these DMARDs are known by another name, biologics (or biologics). Compared to conventional DMARDs, these products target the molecules that cause inflammation in rheumatoid arthritis. Inflammatory cells in the joints are involved in the development of rheumatoid arthritis itself. Biologics reduce the inflammatory process that ultimately induces the joint damage seen in rheumatoid arthritis. By attacking cells at a more specific level than inflammation itself, biologics are considered more effective and more specifically targeted. Biologics include etanercept (ENBREL), infliximab (REMICADE), adalimumab (HMMIRA), anakinra (KINARET), abatacept (ORENCIA), rituximab (RITUXAN), certolizumab pegol (CIMZIA), golimumab (SYMPON), tocilizumab (ACTEMRA), and tofacitinib (XELJANJ). Some biologics are used in combination with conventional DMARDs, particularly methotrexate.

[0248] Multiple sclerosis Multiple sclerosis (MS) is an autoimmune disease. In this condition, the immune system mistakenly attacks normal cells. In people with multiple sclerosis, the immune system attacks the myelin cells, which are the protective membranes that cover the nerves in the brain and spinal cord. When myelin is damaged, the nerve signals transmitted from the brain to other parts of the body are blocked. The damage can trigger symptoms that affect the brain, spinal cord, and eyes. There are four types of multiple sclerosis. Clinically isolated syndrome (CIS): At the stage when the first symptoms of MS appear, healthcare providers generally classify it as CIS. Not all cases of CIS progress to multiple sclerosis. Relapsing-remitting MS (RRMS): This is the most frequent form of multiple sclerosis. Patients with RRMS have new or worsening symptoms (flare-ups, also known as relapses or exacerbations). Periods of remission follow (when symptoms stabilize or disappear). Primary progressive MS (PPMS): In patients diagnosed with PPMS, the symptoms progress gradually without relapses or remissions. Secondary progressive MS (SPMS): In many cases, patients initially diagnosed with RRMS eventually progress to SPMS. In secondary progressive multiple sclerosis, nerve damage accumulates continuously and the symptoms gradually worsen. Some relapses or flare-ups (when symptoms worsen) may be experienced, but then remission periods are often no longer seen (when symptoms stabilize or disappear).

[0249] Therapies that can be used in combination with the TNFR2 AFFIMER polypeptide of the present invention include, for example: Disease-modifying therapies (DMT): Many drugs have received FDA approval for long-term MS treatment. Such agents help reduce relapses (also called flare-ups or attacks). They can delay the progression of the disease and prevent the formation of new lesions in the brain and spinal cord. Relapse management pharmacotherapy: In the case of severe attacks, a neurologist may recommend the use of high-dose corticosteroids. These drugs can rapidly reduce inflammation and delay damage to the myelin sheath that covers nerve cells. Physical rehabilitation: Because multiple sclerosis can affect bodily functions, maintaining physical health and muscle strength helps maintain mobility. Mental health consultation: Coping with chronic illness can be emotionally challenging. MS can sometimes affect mood and memory. Working with a neuropsychologist or seeking other emotional support is an essential part of disease management.

[0250] inflammatory bowel disease Inflammatory bowel disease (IBD) is a group of diseases that cause chronic inflammation (pain and edema) in the intestines. Crohn's disease and ulcerative colitis are the major types of IBD. The types are as follows: Crohn's disease causes pain and swelling in the digestive tract. It can affect any part of the digestive tract, from the mouth to the anus. Most frequently, it affects the small and upper parts of the large intestine. Ulcerative colitis is a disease that induces swelling and ulcerative lesions in the large intestine (colon and rectum). Microcolitis induces intestinal inflammation that can only be detected under a microscope. Examples of therapeutic methods that may be used in combination with the TNFR2 AFFIMER polypeptide of the present invention include the following: Aminosalicylic acids (anti-inflammatory agents such as sulfasalazine, mesalamine, or valsalazide) minimize visceral irritation. Antibiotics treat infections and abscesses. Biological agents block immune system signals that induce inflammation. Corticosteroids such as prednisone suppress the immune system and manage redness. Immunomodulators soothe hypersensitive immune systems. Other possible treatments include antidiarrheals; supplements such as nonsteroidal anti-inflammatory drugs (NSAIDs), vitamins, and probiotics may be used.

[0251] Graft-versus-host disease (GvHD) Graft-versus-host disease (GvHD) is a condition that can occur after allogeneic transplantation. In GvHD, donated bone marrow or peripheral hematopoietic stem cells recognize the recipient's body as a foreign object, and the donated cells / bone marrow attack the body. Examples of graft-versus-host disease (GvHD) include acute graft-versus-host disease (aGvHD) and chronic graft-versus-host disease (cGvHD).

[0252] psoriasis Psoriasis is a chronic skin disorder that means a persistent skin condition. People with psoriasis have thick, pink or red skin patches covered with white or silvery scales. These thick, scaly patches are called plaques. Psoriasis generally begins in early adulthood, but it can also begin later in life. In addition to the red, scaly patches, symptoms of psoriasis include itching, cracking, dry skin, a scaly scalp, skin pain, sunken nails, brittle or crumbling nails, and joint pain. Therapeutic methods that may be used in combination with the TNFR2 AFFIMER polypeptide of the invention include, for example: This may include, but is not limited to, steroid creams, moisturizers for dry skin, anthralines (drugs that slow the production of skin cells), medicated lotions, shampoos and bath cleaners for improving scalp psoriasis, vitamin D3 ointments, vitamin A or retinoid creams, phototherapy, PUVA (a treatment combining the drug psoralen with a specific form of ultraviolet light exposure), methotrexate, retinoids, cyclosporine and / or immunotherapy.

[0253] Sjögren's syndrome Sjögren's syndrome is a lifelong autoimmune disorder that reduces the amount of water produced by the sweat glands in the eyes and mouth. It is named after Henrik Sjögren, the Swedish ophthalmologist who first described the disease. Dry mouth and dry eyes are the main symptoms, but most people who experience these problems do not have Sjögren's syndrome. Dry mouth is also called xerostomia. Sjögren's syndrome has two forms: primary Sjögren's syndrome, which occurs spontaneously and not due to other health conditions, and secondary Sjögren's syndrome, which occurs as a result of other autoimmune diseases such as rheumatoid arthritis, lupus, and psoriatic arthritis. Therapies used in combination with the TNFR2 AFFIMER polypeptide of the present invention include, for example: treatment of dry eyes (e.g., artificial tears, prescription eye drops, punctal plugs, surgery, autologous serum eye drops), treatment of dry mouth (e.g., salivary gland agents), and treatment of joint or organ problems (e.g., analgesics, anti-rheumatic agents, immunosuppressants, steroids, antifungal agents, and treatments for dry vaginal conditions).

[0254] Myasthenia gravis Myasthenia gravis (MG) is an autoimmune disease in which the body's immune system mistakenly attacks its own tissues. MG affects signal transmission between nerves and muscles (neuromuscular junction). Patients with myasthenia gravis lose the ability to control their muscles voluntarily. They experience varying degrees of muscle weakness and fatigue. They may also be unable to move the muscles of their eyes, face, neck, and limbs. MG is a neuromuscular disease that lasts a lifetime. Myasthenia gravis affects approximately 20 out of every 100,000 people. Experts estimate that between 36,000 and 60,000 Americans suffer from this neuromuscular disorder. The actual number of people affected may be even higher, as some with milder cases may not even be aware that they have the disease. MG primarily affects women aged 20-40 and men aged 50-80. About one in ten cases of MG occurs in teenagers (adolescent MG). While the disease can affect people of all ages, it is rare in children. Autoimmune myasthenia gravis is the most common form of this neuromuscular disease. Autoimmune myasthenia gravis is as follows: Ocular form: The muscles that move the eyes and eyelids weaken. This can lead to drooping eyelids or difficulty opening the eyes. Some people experience double vision. Weakened vision is often the first sign of MG. Nearly half of people with ocular MG develop the generalized form within two years of the first symptoms. Generalized type: Muscle weakness affects the eyes, face, neck, arms, legs, and other parts of the body. It may become difficult to talk or drink, raise your arms above your head, stand up from a seated position, walk long distances, or climb stairs. Therapies that may be used in combination with the TNFR2 AFFIMER polypeptide of the invention include, for example: drugs, monoclonal antibodies, IV immunoglobulins (IVIG), plasmapheresis, and / or surgery.

[0255] Cancer The binding of TNF and TNFR2 has been reported to promote the activation of tumor cells and tumor-associated cell types, influencing immune responses such as tumor-infiltrating lymphocytes and creating an environment in which tumors can progress. In fact, TNFR2 expression in tumors is associated with disease progression and poor clinical outcomes (Sheng Y, et al.). Therefore, the TNFR2 AFFIMER polypeptide of the present invention can be used in cancer treatment as a monotherapy or in combination with other cancer therapies such as immunotherapy, CAR-T therapy, checkpoint inhibitors, and conventional cancer therapies.

[0256] Pharmaceutical preparations The pharmaceutical formulations and dosage forms of the present invention can be manufactured for the storage or use of genetically modified cells using a pharmaceutically acceptable vehicle (e.g., carrier or excipient). A person ordinarily skilled in the art can generally consider a pharmaceutically acceptable carrier, excipient, and / or stabilizer as the inactive component of the dosage form or pharmaceutical composition.

[0257] In one embodiment of the present invention, the pharmaceutical composition may be stored in a lyophilized form. In one embodiment of the present invention, the dosage form comprising the genetically modified cells of the present invention may be lyophilized.

[0258] Suitable pharmacokinetic vehicles include non-toxic buffers such as phosphates, citrates and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, alkylparabens such as phenol, butyl or benzyl alcohol, methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight polypeptides (e.g., residues of less than approximately 10 amino acids); serum Examples of surfactants include, but are not limited to, proteins such as lubumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and nonionic surfactants such as WEEN or polyethylene glycol (PEG) (Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London).

[0259] The pharmaceutical compositions disclosed herein may be administered in a variety of ways for topical or systemic treatment. Administration may be topical via epidermal or transdermal patches, ointments, lotions, creams, gels, suppositories, sprays, liquids and powders; pulmonary administration via inhalation or infusion of powders or aerosols, including nebulizers, intra-arterial and nasal inhalations; orally; or parenterally, including intravenous, intra-arterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion) or intracranial (e.g., intraspinal cavity or ventricle).

[0260] Also, the AFFIMER formulations described herein can be entrapped in microcapsules. The microcapsules can be produced, for example, by coacervation techniques or interfacial polymerization, such as by hydroxyethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, and can be produced by colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, but are not limited thereto (Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London).

[0261] In one embodiment of the invention, the pharmaceutical composition can be a dosage form comprising an AFFIMER formulation complexed with liposomes. Methods for producing liposomes are well known in the art. For example, some liposomes can be produced by reverse phase evaporation of a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes of a desired diameter can be obtained through a filter of a predetermined pore size.

[0262] In one embodiment of the present invention, a sustained-release dosage form containing the AFFIMER formulation of the present invention can be manufactured. A suitable example of a sustained-release dosage form includes a semipermeable matrix of a solid hydrophobic polymer containing the AFFIMER formulation of the present invention, the matrix of which may be in the form of a molded product (e.g., a film or microcapsule). Examples of sustained-release matrices include, but are not limited to, polyesters, hydrogels such as poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT.™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprorelin acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0263] In one embodiment of the present invention, in addition to administering the AFFIMER formulation of the present invention, the administration of at least one additional therapeutic agent may be further included. Additional treatment may be provided before, concurrently with, and / or after administration of the AFFIMER formulation of the present invention. In one embodiment of the present invention, the pharmaceutical composition may include the AFFIMER formulation of the present invention and the additional therapeutic agent.

[0264] Generally, combination therapy using two or more therapeutic agents is preferable, but not always, to use formulations that act through different mechanisms of action. Combination therapy using formulations with different mechanisms of action can exhibit additive or synergistic effects. Combination therapy can be used at lower doses of each formulation than when used as monotherapy, which can reduce toxicity and side effects and / or increase the therapeutic index of the AFFIMER formulation.

[0265] In one embodiment of the present invention, combination therapy of the AFFIMER formulation of the present invention and at least one additional therapeutic agent may exhibit additive or synergistic effects. In one embodiment of the present invention, combination therapy may also increase the therapeutic index of the additional therapeutic agent and / or the AFFIMER formulation. In one embodiment of the present invention, combination therapy may reduce the toxicity and / or side effects of the additional therapeutic agent and / or the AFFIMER formulation.

[0266] For concomitant administration, single pharmaceutical dosage forms or separate dosage forms may be used for co-administration, and they may be administered sequentially in any order; however, it is generally preferable that all activators be administered within a period during which they can exert their biological activity simultaneously.

[0267] The AFFIMER formulation of the present invention and at least one additional therapeutic agent may be administered in any order or simultaneously. In one embodiment of the present invention, the AFFIMER formulation may be administered to a patient who has previously been treated with a second therapeutic agent. In one embodiment of the present invention, the AFFIMER formulation and the second therapeutic agent may be administered substantially simultaneously or concurrently. For example, a subject may receive the AFFIMER formulation while undergoing a treatment process with a second therapeutic agent (e.g., chemotherapy). In one embodiment of the present invention, the AFFIMER formulation may be administered within one year after treatment with a second therapeutic agent. In one embodiment of the present invention, the AFFIMER formulation may be administered within 10 months, 8 months, 6 months, 4 months, or 2 months after treatment with a second therapeutic agent. In one embodiment of the present invention, the AFFIMER formulation may be administered within 4 weeks, 3 weeks, 2 weeks, or 1 week after treatment with a second therapeutic agent. In one embodiment of the present invention, the AFFIMER formulation may be administered within 5 days, 4 days, 3 days, 2 days, or 1 day after treatment with a second therapeutic agent. Two (or more) agonists or treatments may be administered to the subject within a few hours or minutes (e.g., substantially simultaneously).

[0268] For the treatment of a disease, the appropriate dose of the AFFIMER formulation of the present invention is at the discretion of the treating physician, depending on the type of disease being treated, the severity and course of the disease, the responsiveness to the disease, whether the AFFIMER formulation is administered for therapeutic or preventive purposes, previous treatment options, the patient's clinical history, etc. The AFFIMER formulation may be administered as a single dose or as part of a series of treatments over several days to several months, and may be administered until complete recovery or mitigation of the disease state is achieved. The optimal administration schedule may be calculated through measurements of drug accumulation in the patient's body and may vary depending on the relative efficacy of the individual formulations. The prescribing physician may determine the optimal dose, method of administration, and repetition rate. In one embodiment of the present invention, the above dose may be 0.01 μg to 100 mg, 0.1 μg to 100 mg, 1 μg to 100 mg, 1 mg to 100 mg, 1 mg to 80 mg, 10 mg to 100 mg, 10 mg to 75 mg, or 10 mg to 50 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 0.1 mg to approximately 20 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 0.1 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 0.25 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 0.5 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 1 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 1.5 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 2 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 2.5 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 5 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 7.5 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 10 mg per kg of body weight.In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 12.5 mg per kg of body weight. In one embodiment of the present invention, the dosage of the AFFIMER preparation may be approximately 15 mg per kg of body weight. In one embodiment of the present invention, the above dosage may be administered once or more times a day, once or more times a week, once or more times a month, or once or more times a year. In one embodiment of the present invention, the AFFIMER preparation may be administered once a week, once every two weeks, once every three weeks, or once every four weeks.

[0269] In one embodiment of the present invention, the AFFIMER formulation may be administered in an initial higher “load dose,” followed by at least one lower dose. In one embodiment of the present invention, the frequency of administration may also be modified. In one embodiment of the present invention, the therapy may include administering an additional dose (or “maintenance dose”) once a week, once every two weeks, once every two weeks, or once a month after the initial dose. For example, the therapy may include administering a maintenance dose once a week, for example, half of the initial dose, after the initial load dose has been administered. In one embodiment of the present invention, the therapy may include administering a maintenance dose, for example, half of the initial dose, every other week (every two weeks), after the initial load dose has been administered. In one embodiment of the present invention, the therapy may include administering an initial dose three times over three weeks, for example, the same amount of maintenance dose every other week.

[0270] As is well known in this industry, the administration of any therapeutic agent can cause side effects and / or toxicity. In some cases, these side effects and / or toxicity may be so severe that it becomes impossible to administer a particular formulation in a therapeutically effective dose. In some cases, drug therapy may be discontinued and other formulations may be tried. However, since many formulations within the same therapeutic system often exhibit similar side effects and / or toxicity, patients may have to discontinue treatment or, if possible, tolerate the unpleasant side effects associated with the therapeutic agent.

[0271] In one embodiment of the present invention, the administration schedule may be limited by a specific number of doses or "cycles." In one embodiment of the present invention, the AFFIMER formulation may be administered over 3, 4, 5, 6, 7, 8 or more cycles. For example, this may include administration of the AFFIMER formulation every 2 weeks for 6 cycles, every 3 weeks for 6 cycles, every 2 weeks for 4 cycles, or every 3 weeks for 4 cycles. The administration schedule may be determined by a typical technician in the industry and modified as necessary.

[0272] Accordingly, the present invention provides a method for administering a polypeptide or formulation described herein to a subject, the method comprising using an intermittent dosing strategy for administering at least one formulation (e.g., two or three formulations), thereby reducing side effects and / or toxicity associated with the administration of the AFFIMER formulation. In one embodiment of the present invention, a method for treating an inflammatory or autoimmune disease in a human subject may be administered to the subject in a therapeutically effective dose of the AFFIMER formulation in combination with a therapeutically effective dose of another therapeutic agent, and one or all of these formulations may be administered according to an intermittent dosing strategy. In one embodiment of the present invention, the intermittent dosing strategy comprises administering an initial dose of the AFFIMER formulation to the subject, followed by subsequent doses of the AFFIMER formulation approximately every two weeks. In one embodiment of the present invention, the intermittent dosing strategy comprises administering an initial dose of the AFFIMER formulation to the subject, followed by subsequent doses of the AFFIMER formulation approximately every three weeks. In one embodiment of the present invention, the intermittent dosing strategy includes administering an initial dose of the AFFIMER formulation to the subject, followed by subsequent doses of the AFFIMER formulation approximately every four weeks. In one embodiment of the present invention, the AFFIMER formulation is administered using the intermittent dosing strategy, and the chemotherapeutic agent may be administered weekly.

[0273] Additional embodiments of the present invention are described in the following sections. 1.1x10 -6An engineered modified polypeptide that specifically binds to TNFR2 with a kd value of M or less, characterized in that the engineered modified polypeptide is a variant of the stephin A protein. 2. An engineered polypeptide as described in item 1, comprising an amino acid sequence having at least 80%, at least 90%, 95%, or 98% identity with the following amino acid sequence: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-Xaa2-TNYYIKV RAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa)m-Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4). Here, Xaa is an amino acid residue in each individual case, and n and m are each independent integers between 3 and 20. 3. An engineered polypeptide as described in paragraph 2, wherein the amino acid sequence is 100% identical to the following amino acid sequence: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-Xaa2-TNYYIKV RAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa)m-Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4), Here, Xaa is an amino acid residue in each individual case, and n and m are each independent integers between 3 and 20. 4. An engineered polypeptide as described in item 1, comprising an amino acid sequence having at least 80%, 90%, 95%, or at least 98% identity with the following amino acid sequence: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (Sequence ID 5), Xaa is an amino acid residue in each individual case, and n and m are each independent integers between 3 and 20. 5. An engineered polypeptide as described in Section 4, wherein the amino acid sequence is 100% identical to the following amino acid sequence: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (Sequence ID 5), Xaa is an amino acid residue in each individual case, and n and m are each independent integers between 3 and 20. 6. An engineered polypeptide as described in any one of items 1 to 5, wherein n is an integer between 5 and 18. 7. The engineered polypeptide described in item 6, wherein n is an integer between 7 and 15. 8. The engineered polypeptide described in paragraph 7, wherein n is an integer between 8 and 12. 9. The engineered polypeptide described in any one of the items 1 to 8, wherein m is an integer between 5 and 18. 10. The engineered polypeptide described in paragraph 9, wherein m is an integer between 7 and 15. 11. The engineered polypeptide described in item 10, wherein m is an integer between 8 and 12. 12. The engineered polypeptide has a molecular weight of 1 × 10 ―7 An engineered polypeptide described in any one of items 1 to 11, which binds to TNFR2 at a Kd value of M. 13. The engineered polypeptide has a molecular weight of 1 × 10 ―8 An engineered polypeptide described in section 12 that binds to TNFR2 at a Kd value of M. 14. The (Xaa)n is an engineered polypeptide as described in any one of items 1 to 13, comprising an amino acid sequence selected from the group comprised of SEQ ID NOs: 6-102. 15. The (Xaa)n is an engineered polypeptide as described in item 14, comprising an amino acid sequence selected from the group comprised of Sequence ID Nos. 6-102. 16. The (Xaa)m is an engineered polypeptide as described in any one of items 1 to 15, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-199. 17. The (Xaa)m is an engineered polypeptide as described in section 16, comprising an amino acid sequence selected from the group comprised of sequence numbers 103-199. 18. The (Xaa)n is an engineered polypeptide as described in paragraph 14, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 11, 13, 16, 17, 24, 25, 29, 33, 41, 54, 65, 67, 73, 75, 82, 89, 90, 98, and 99. 19. The (Xaa)n is an engineered polypeptide as described in paragraph 18, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 11, 13, 16, 17, 24, 25, 29, 33, 41, 54, 65, 67, 73, 75, 82, 89, 90, 98, and 99. 20. The (Xaa)m is an engineered polypeptide as described in section 16, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 103, 108, 110, 113, 114, 121, 122, 126, 130, 138, 151, 162, 164, 170, 172, 179, 186, 187, 195, and 196. 21. The (Xaa)m is an engineered polypeptide as described in paragraph 20, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 103, 108, 110, 113, 114, 121, 122, 126, 130, 138, 151, 162, 164, 170, 172, 179, 186, 187, 195, and 196. 22. The engineering modified polypeptide according to item 1, wherein the engineering modified polypeptide comprises an amino acid sequence having at least 75%, at least 85%, or at least 95% identity with the amino acid sequence of SEQ ID NOs: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292, or 293, wherein the sequence can selectively exclude one or more of the 21 carboxy-terminal residues. 23. The engineered polypeptide described in paragraph 22 comprises an amino acid sequence having 100% identity with the amino acid sequence of SEQ ID NOs: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292, or 293, wherein the sequence can selectively exclude one or more of the 21 carboxy-terminal residues. 24. The stephin A polypeptide is an engineered polypeptide as described in any one of the items 1 to 23, which is a mammalian stephin A polypeptide. 25. The stephin A polypeptide is a human stephin A polypeptide, which is an engineered polypeptide as described in any one of items 1 to 24. 26. An engineered polypeptide according to any one of items 1 to 25, further comprising the signal peptide. 27. A fusion protein according to any one of items 1 to 26, comprising a dimer, trimer, or tetramer of the engineeringly modified polypeptide, and selectively comprising one or more linkers. 28. The fusion protein is the fusion protein described in paragraph 27, wherein the linker is a rigid linker. 29. The fusion protein is the fusion protein described in paragraph 28, wherein the rigid linker is a peptide linker containing the (EAAAK)n sequence, and n is 1 to 6. 30. A fusion protein according to any one of items 1 to 29, wherein one or more of the engineeringly modified polypeptides are conjugated with a therapeutic or diagnostic moisture. 31. The therapeutic moiety is a fusion protein as described in paragraph 30, which is a therapeutic protein or peptide. 32. The therapeutic moiety is an antibody, which is a fusion protein as described in paragraph 31. 33. The diagnostic moiety is a fusion protein described in section 30, which is a fluorescent protein. 34. An engineered polypeptide as described in any one of items 1 to 26, or a fusion protein as described in any one of items 27 to 33, further comprising a half-life extension moiety. 35. The half-life extension moiety is an engineered polypeptide or fusion protein as described in paragraph 34, selected from an antibody Fc domain, human serum albumin, and serum-binding protein. 36. The aforementioned half-life extension is 1 × 10⁻⁶ of serum albumin. ―6 An engineered polypeptide or fusion protein described in section 34, which is a variant of the stephin A protein that specifically binds to Kd values ​​below M. 37. The Kd is measured by Biacore kinetic analysis and is an engineered polypeptide or fusion protein as described in any one of items 1 to 36. 38. kd is the engineered lipeptide or fusion protein described in Section 37, measured as described in Example 3, “Biacore assay”. 39. An engineered polypeptide or fusion protein according to any one of items 1 to 38, wherein the engineered polypeptide binds to TNFR2 as a monomer and has an IC50 of 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less in competitive binding analysis with TNFR2. 40. The TNFR2 is a mammalian TNFR2, an engineered polypeptide or fusion protein as described in any one of items 1 to 39. 41. The TNFR2 is human TNFR2, or is an engineered polypeptide or fusion protein as described in paragraph 40. 42. The TNFR2 is mouse TNFR2, or is an engineered polypeptide or fusion protein as described in Section 40. 43. The half-life extension moiety is an engineered polypeptide or fusion protein as described in paragraph 34, selected from the group consisting of Sequence ID No. 471 to Sequence ID No. 477. 44. A polynucleotide or set of polynucleotides described in any one of sections 1 to 43, which encodes the engineered polypeptide or fusion protein. 45. The polynucleotide or set of polynucleotides according to paragraph 44, wherein the polynucleotide or set of polynucleotides includes nucleic acid sequences having at least 80%, at least 90%, at least 95%, or at least 98% identity with sequences selected from the group consisting of SEQ ID NOs 297, 302, 304, 307, 308, 315, 316, 320, 324, 332, 345, 356, 358, 364, 366, 373, 380, 381, 389, and 290, wherein the sequences selectively exclude nucleotides encoding one or more carboxy-terminal residues. 46. ​​A delivery vehicle according to item 44 or 45, comprising the polynucleotide. 47. The carrier described in paragraph 46, wherein the carrier is a viral carrier. 48. The viral carrier according to paragraph 47, wherein the viral carrier is an adenovirus vector, a retrovirus vector, a lentivirus vector, or an adeno-associated virus (AAV) vector. 49. The carrier described in paragraph 46, which is a non-viral carrier. 50. The non-viral mediator is a liposome or lipid nanoparticle, as described in paragraph 49. 51. A plasmid or nicurl described in any one of items 44 to 50, comprising the polynucleotide described above. 52. Messenger RNA (mRNA) as described in any one of paragraphs 1 to 43, including an open reading frame encoding the engineered polypeptide. 53. The mRNA described in paragraph 52, further comprising a read frame encoding an immune cell conjugate. 54. Lipid nanoparticles according to item 52 or 53, which are selectively cationic lipid nanoparticles containing the mRNA. 55. The lipid nanoparticles are cationic lipid nanoparticles, as described in paragraph 54. 56. A pharmaceutical composition according to any one of claims 1 to 50, further comprising a selectively pharmaceutically acceptable carrier comprising the engineered polypeptide, fusion protein, or carrier. 57. A conjugate according to any one of items 1 to 43, wherein the engineered polypeptide or fusion protein is bound to a pharmacologically active moiety. 58. The method according to claim 56, comprising the step of administering the pharmaceutical composition to a subject. 59. The aforementioned subjects include systemic lupus erythematosus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and colitis / ulcerative colitis), graft-versus-host disease (GvHD; associated with hematopoietic stem cell transplantation), also known as allogeneic transplant rejection, transplantation or solid organ transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, The method described in paragraph 58, relating to psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjögren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, glomerulosclerosis, and / or cancer. 60. A polypeptide, fusion protein, or carrier described in any one of the above-mentioned items 1 to 50, as an engineered modification, for use as a pharmaceutical. 61. The aforementioned systemic lupus erythematosus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and colitis / ulcerative colitis), graft-versus-host disease (GvHD) (associated with stem cell transplantation) (also known as allograft rejection), transplantation or solid organ transplantation (SOT), primary biliary cholangitis Engineered polypeptides, fusion proteins, or mediators described in paragraph 60 for use in the treatment of Cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjögren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, glomerulosclerosis, and / or cancer. 62. The aforementioned engineered polypeptides, fusion proteins, or carriers are used to treat systemic lupus erythematosus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and colitis / ulcerative colitis), graft-versus-host disease (GvHD) (associated with stem cell transplantation) (also known as allograft rejection), transplantation or solid organ transplantation (SOT), and primary biliary cholangitis. Uses described in any one of the following paragraphs, 1 to 50, for the manufacture of a pharmaceutical product for the treatment of Primary Biliary Cholangitis (PBC), Psoriasis, Psoriatic Arthritis, Collagen-induced Arthritis, Experimental Allergic Encephalomyelitis (EAE), Oophoritis, Allergic Rhinitis, Asthma, Sjögren's Syndrome, Atopic Eczema, Myasthenia Gravis, Graves' Disease, Glomerulosclerosis, or Cancer. 63. An engineered polypeptide according to any one of the items 1 to 43, which competes with the engineered polypeptide for binding to TNFR2. 64. An engineer-modified polypeptide according to any one of the items 1 to 43, wherein the same epitope as the engineer-modified polypeptide is bound to TNFR2. 65. An engineered polypeptide or fusion protein according to any one of items 1 to 64, comprising the signal sequence and / or transmembrane domain.

[0274] Additional embodiments of the present invention are described in the following sections. 1B.1x10 -6 An engineered modified polypeptide characterized by being a variant of the stephin A protein, wherein the engineered modified polypeptide is an engineered modified polypeptide that specifically binds to TNFR2 with a kd value of M or less. 2B. An engineered polypeptide as described in Section 1B, comprising an amino acid sequence having at least 80%, 90%, 95%, or 98% identity with the following amino acid sequence: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV(Sequence ID 484)Xaa2-(Xaa)n-Xaa3-TNYYIKVRAGDNKYMHLKVF(Sequence ID 485)-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF(Sequence ID 486), Here, Xaa is an amino acid residue in each individual case, and n and m are each independent integers between 3 and 20. 3B. An engineered polypeptide as described in Section 2B, wherein the amino acid sequence is 100% identical to the following amino acid sequence: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV(Sequence ID 484)Xaa2-(Xaa)n-Xaa3-TNYYIKVRAGDNKYMHLKVF(Sequence ID 485)-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF(Sequence ID 486), Here, Xaa is an amino acid residue in each individual case; and n and m are each independent integers between 3 and 20. 4B. An engineered polypeptide as described in Section 1B, comprising an amino acid sequence having at least 80%, 90%, 95%, or 98% identity with the following amino acid sequence: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV(Sequence ID 484) Xaa2-(Xaa)n-Xaa3-TNYYIKVRAGDNKYMHLKVF(Sequence ID 485)-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF(Sequence ID 486), or MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD(Sequence ID 487)-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL(Sequence ID 488)-(Xaa)m-EDLVLTGYQVDKNKDDELTGF(Sequence ID 489), Xaa is any number of amino residues in each case, n and m are each independent integers between 3 and 20. 5B. An engineered polypeptide as described in Section 4B, wherein the amino acid sequence is 100% identical to the following amino acid sequence: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF(Sequence ID 5), Here, Xaa is an amino acid residue in each individual case, and n and m are each independent integers between 3 and 20. 6B. An engineered polypeptide described in any one of the terms 1B to 5B, wherein n is an integer between 5 and 18. 7B. An engineered polypeptide as described in section 6B, wherein n is an integer between 7 and 15. 8B. The engineered polypeptide described in section 7B, wherein n is an integer between 8 and 12. 9B. The above m is an integer between 5 and 18, and is an engineered polypeptide as described in any one of the terms 1B to 8B. 10B. The engineered polypeptide described in section 9B, wherein m is an integer between 7 and 15. 11B. The engineered polypeptide described in section 10B, wherein m is an integer between 8 and 12. 12B. The engineered polypeptide is 1 × 10 ―7 An engineered polypeptide described in any one of sections 1B to 11B that binds to TNFR2 at a Kd value of M. 13B. The engineered polypeptide is 1 × 10 ―8 An engineered polypeptide, as described in Section 12B, that binds to TNFR2 at a Kd value of M. 14B. The (Xaa)n is an engineered polypeptide as described in any one of the items 1B to 13B, comprising an amino acid sequence selected from the group comprised of SEQ ID NOs: 6-102. 15B. The (Xaa)n is an engineered polypeptide as described in section 14B, comprising an amino acid sequence selected from the group comprised of Sequence ID No. 6-102. 16B. The (Xaa)m is an engineered polypeptide described in any one of the items 1B to 15B, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-199. 17B. The (Xaa)m is an engineered polypeptide as described in section 16B, comprising an amino acid sequence selected from the group consisting of sequence numbers 103-199. 18B. The (Xaa)n is an engineered polypeptide as described in section 14B, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 11, 13, 16, 17, 24, 25, 29, 33, 41, 54, 65, 67, 73, 75, 82, 89, 90, 98, and 99. 19B. The (Xaa)n is an engineered polypeptide as described in section 18B, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 6, 11, 13, 16, 17, 24, 25, 29, 33, 41, 54, 65, 67, 73, 75, 82, 89, 90, 98, and 99. 20B. The (Xaa)m is an engineered polypeptide as described in section 16B, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 103, 108, 110, 113, 114, 121, 122, 126, 130, 138, 151, 162, 164, 170, 172, 179, 186, 187, 195, and 196. 21B. The (Xaa)m is an engineered polypeptide as described in section 20B, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 103, 108, 110, 113, 114, 121, 122, 126, 130, 138, 151, 162, 164, 170, 172, 179, 186, 187, 195, and 196. 22B. The engineered polypeptide according to Section 1B, wherein the engineered polypeptide comprises an amino acid sequence having at least 75%, at least 85%, or at least 95% identity with the amino acid sequence of SEQ ID NOs: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292, or 293, wherein the sequence can selectively exclude one or more of the 21 carboxy-terminal residues. 23B. The engineered polypeptide described in section 22B, wherein the engineered polypeptide comprises an amino acid sequence having 100% identity with the amino acid sequence of SEQ ID NOs: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292, or 293, wherein the sequence can selectively exclude one or more of the 21 carboxy-terminal residues. 24B. The stephin A polypeptide is an engineered polypeptide described in any one of the items 1B to 23B, which is a mammalian stephin A polypeptide. 25B. The stephin A polypeptide is a human stephin A polypeptide, which is an engineered modified polypeptide as described in any one of sections 1B to 24B. 26B. An engineered polypeptide according to any one of sections 1B to 25B, further comprising the signal peptide. 27B. A fusion protein according to any one of items 1B to 26B, comprising a dimer, trimer, or tetramer of the engineered polypeptide, and selectively comprising one or more linkers. 28B. The fusion protein is the fusion protein described in section 27B, wherein the linker is a rigid linker. 29B. The fusion protein is the fusion protein described in section 28B, wherein the rigid linker is a peptide linker containing the (EAAAK)n sequence, and n is 1B to 6. 30B. A fusion protein according to any one of sections 1B to 29B, wherein one or more of the engineeringly modified polypeptides are conjugated with a therapeutic or diagnostic moisture. 31B. The therapeutic moiety is a fusion protein as described in section 30B, which is a therapeutic protein or peptide. 32B. The therapeutic moiety is the fusion protein described in section 31B, which is an antibody. 33B. The diagnostic moiety is a fusion protein described in section 30B, which is a fluorescent protein. 34B. An engineered polypeptide as described in any one of sections 1B to 26B, or a fusion protein as described in any one of sections 27B to 33B, further comprising a half-life extension moiety. 35B. The half-life extension moiety is an engineered polypeptide or fusion protein as described in section 34B, selected from an antibody Fc domain, human serum albumin, and serum-binding protein. 36B. The aforementioned half-life extension is 1 × 10⁻⁶ of serum albumin. ―6 An engineered polypeptide or fusion protein described in section 34B, which is a variant of the stephin A protein that specifically binds at kd values ​​of M or less. 37B. kd is an engineered polypeptide or fusion protein as described in any one of sections 1B to 36B, as measured by Biacore kinetic analysis. 38B. kd is an engineered polypeptide or fusion protein as described in Section 37B, measured as described in Example 3, “Biacore assay”. 39B. An engineered polypeptide or fusion protein as described in any one of sections 1B to 38B, wherein the engineered polypeptide binds to TNFR2 as a monomer and has an IC50 of ≤1 μM, ≤100 nM, ≤40 nM, ≤20 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM in competitive binding analysis with TNFR2. 40B. The TNFR2 is a mammalian TNFR2, an engineered polypeptide or fusion protein as described in any one of sections 1B to 39B. 41B. The TNFR2 is human TNFR2, or is an engineered polypeptide or fusion protein as described in Section 40B. 42B. The TNFR2 is mouse TNFR2, an engineered polypeptide or fusion protein as described in section 40B. 43B. The half-life extension molecule is selected from the group consisting of Sequence IDs 471 to 477, and is an engineered polypeptide or fusion protein as described in Section 34B. 44B. A polynucleotide or set of polynucleotides described in any one of sections 1B to 43B, which encodes the engineered polypeptide or fusion protein. 45B. The polynucleotide or set of polynucleotides according to paragraph 44B, wherein the polynucleotide or set of polynucleotides includes nucleic acid sequences having at least 80%, at least 90%, at least 95%, or at least 98% identity with sequences selected from the group consisting of SEQ ID NOs 297, 302, 304, 307, 308, 315, 316, 320, 324, 332, 345, 356, 358, 364, 366, 373, 380, 381, 389, and 290, wherein the sequences selectively exclude nucleotides encoding one or more carboxy-terminal residues. 46B. A delivery vehicle according to section 44B or 45B, comprising the polynucleotide. 47B. The carrier described in section 46B, wherein the carrier is a viral carrier. 48B. The viral carrier according to section 47B, wherein the viral carrier is an adenovirus vector, a retrovirus vector, a lentivirus vector, or an adeno-associated virus (AAV) vector. 49B. The carrier described in section 46B, which is a non-viral carrier. 50B. The non-viral mediator is a liposome or lipid nanoparticle, as described in section 49B. 51B. A plasmid or nicurl described in any one of sections 44B to 50B, comprising the polynucleotide described above. 52B. Messenger RNA (mRNA) as described in any one of sections 1B to 43B, including an open reading frame encoding the engineered polypeptide. 53B. The mRNA described in section 52B, further comprising a read frame encoding an immune cell conjugate. 54B. Lipid nanoparticles according to item 52 or 53, which are selectively cationic lipid nanoparticles containing the mRNA. 55B. The lipid nanoparticles are cationic lipid nanoparticles, as described in section 54B. 56B. A pharmaceutical composition according to any one of sections 1B to 50B, further comprising the engineered polypeptide, fusion protein, or carrier, and further comprising a selectively pharmaceutically acceptable carrier. 57B. A conjugate according to any one of items 1B to 43B, wherein the engineered polypeptide or fusion protein is bound to a pharmacologically active moiety. 58B. The method according to paragraph 56B, comprising the step of administering the pharmaceutical composition to a subject. 59B. The aforementioned subjects include systemic lupus erythematosus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and colitis / ulcerative colitis), graft-versus-host disease (GvHD; associated with hematopoietic stem cell transplantation), also known as allogeneic transplant refusal, transplantation or solid organ transplantation (SOT), primary biliary cholangitis (PBC), and psoriasis. The method described in paragraph 58B, relating to (Psoriasis), Psoriatic Arthritis, Collagen-induced Arthritis, Experimental Allergic Encephalomyelitis (EAE), Oophoritis, Allergic Rhinitis, Asthma, Sjögren's Syndrome, Atopic Eczema, Myasthenia Gravis, Graves' Disease, Glomerulosclerosis, and / or Cancer. 60B. A polypeptide, fusion protein, or carrier described in any one of the above-mentioned engineeringly modified sections 1B to 50B, for use as a pharmaceutical. 61B. The aforementioned systemic lupus erythematosus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease and colitis / ulcerative colitis), graft-versus-host disease (GvHD) (associated with stem cell transplantation) (also known as allograft rejection), transplantation or solid organ transplantation (SOT), primary biliary cholangitis Engineered polypeptides, fusion proteins, or mediators as described in Section 60B, for use in the treatment of Cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjögren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, glomerulosclerosis, and / or cancer. 62B. The aforementioned engineered polypeptides, fusion proteins, or carriers are used in the following conditions: Systemic Lupus Erythematosus (SLE), Lupus nephritis (e.g., drug-induced lupus nephritis), Immune Thrombocytopenia (ITP), Rheumatoid Arthritis (RA), Multiple Sclerosis (MS), Inflammatory Bowel Disease (IBD) (e.g., Crohn's disease and colitis / ulcerative colitis), Graft-versus-Host Disease (GvHD) (associated with stem cell transplantation) (also known as allograft rejection), Transplantation or Solid Organ Transplantation (SOT), and Primary Biliary Cholangitis. Uses described in any one of the following sections, 1B to 50B, for the manufacture of a pharmaceutical product for the treatment of Primary Biliary Cholangitis (PBC), Psoriasis, Psoriatic Arthritis, Collagen-induced Arthritis, Experimental Allergic Encephalomyelitis (EAE), Oophoritis, Allergic Rhinitis, Asthma, Sjögren's Syndrome, Atopic Eczema, Myasthenia Gravis, Graves' Disease, Glomerulosclerosis, or Cancer. 63B. An engineer-modified polypeptide according to any one of clauses 1B to 43B, which competes with the engineer-modified polypeptide for binding to TNFR2. 64B. An engineer-modified polypeptide described in any one of the items 1B to 43B, which binds to TNFR2 at the same epitope as the engineer-modified polypeptide. 65B. An engineered polypeptide or fusion protein according to any one of items 1B to 64B, comprising the signal sequence and / or transmembrane domain. [Examples]

[0275] To identify AFFIMER polypeptides with specific activity in human and mouse TNFR2, the following experiments were conducted.

[0276] Example 1: Selection of TNFR2 AFFIMER polypeptides from a phage display library Candidate clone identification The TNFR2 AFFIMER polypeptide of the present invention was identified by sorting from a library of stephin A protein variants having two random loop sequences, with each loop being displayed on a consistent stephin A protein variant framework skeleton based on the stephin A amino acid sequence and having a length of approximately 9 amino acids. Such sorting procedures are well known in the art (see Tiede et al. Protein Eng Des Sel. 2014.27(5):145-155 and Hughes et al. Sci Signal. 2018.10(505):eaaj2005). Following such a procedure, phage suspensions expressing the AFFIMER protein were cultured with human or mouse TNFR2 as needed, and their sequences are shown in Table 7 below.

[0277] [Table 7] JPEG2026520086000050.jpg129153 (Table 7) TNFR protein sequences used in the selection process (provided by Sino Biologicals).

[0278] [Table 8] JPEG2026520086000052.jpg187153 (Table 8) These are the commercial antigens used during selection. TNFR2 antigens with sequences shown in Table 7, along with other proteins listed below, were tested in various forms for use in biotinylation, selection of steffin A protein variants that specifically bind to TNFR2, and other analyses.

[0279] In some cases, TNFR2 was biotinylated and alternately captured on streptavidin and neutravidin beads; otherwise, TNFR2 was passively adsorbed onto the surface. Next, unbound phage particles were washed, and after washing, bound phages were eluted. Elution of bound phages was performed by exposure to trypsin. The eluted phage particles were then used to infect E. coli, and the infected bacteria were cultured under conditions suitable for bacteriophage replication. After bacteriophage particles were released from these infected bacteria, the phage particles were bound to target antigens, and the bound phage particles were eluted. The cycle of growing the eluted phage particles in bacteria and separating the phage particles released from the infected bacteria was repeated to enrich the bacteriophage population against phage particles displaying proteins that bind to target antigens. In this cycle, specific conditions were modified, such as increasing the number of washing steps, reducing the amount of available antigen, or adding blockers, to select phage particle display proteins that bind more strongly or specifically to the target antigen.

[0280] After multiple rounds of phage display library selection and amplification, the proteins expressed by the phages were expressed and screened via enzyme-linked immunosorbent assay (ELISA). Specifically, AFFIMER polypeptides were overexpressed from phagemide vectors, bacterial cells were lysed, and the lysates were used as substrates in ELISA. In ELISA, human, mouse, and cynomolgus monkey TNFR2 were immobilized on plates, the lysates were added, and the amount of TNFR2 AFFIMER polypeptide was measured in each plate using a detection antibody specific to the Myc tag expressed in candidate StefinA protein variants. Phagemide vectors encoding the AFFIMER polypeptide with the best human TNFR2 binding activity were sequenced to confirm the DNA sequences of candidate clones for further development. Specificity was sometimes confirmed using TNFR1 polypeptides. The amino acid sequences of loop 2 and loop 4 of each of these candidate clones are shown in Table 1.

[0281] Example 2: Production and screening of AFFIMER as a water-soluble protein in mammalian cells Cloning to interest vectors To evaluate the ability of selected AFFIMER proteins to bind to TNFR2 when expressed on the surface of mammalian cells, a cell pool transformed with 30 clones 'fast-tracked' based on the most repeatedly found sequences during the manual selection process was constructed. These clones were also used for initial characterization analysis and validation optimization. The TNFR2 and TNFR2 AFFIMER sequences were identified using Expi29. TM The phagemide from Example 1 was cloned into the mammalian expression vector pD609KanR (ATUM custom vector) for expression in cells (ThermoFisher). The average concentration obtained through expression was 2.8 mg / mL, and 19 clones were selected based on purity.

[0282] The aforementioned 19 clones contained glycine only in Loop2 and Loop4, and compared to the SQT-GLY and 3T0-GLY negative control group, which did not use a specific binder, Expi293F was expressed at 1 μM and 0.1 μM by flow cytometry. TM Screening was performed using hTNFR2 cells. From the signals detected in transduced cells, untransduced Expi293F was identified. TM Signals obtained from cells were excluded. Of these, 16 clones showed more than 10% hTNFR2-Expi293F at 1 μM after binding to the parent cell. TM Cellular binding was confirmed, while clones 15, 22, and 23 did not show such binding (Figure 2).

[0283] AFFIMER protein purified from mammalian cells is processed according to the manufacturer's protocol using HEK Blue. TM The clones were screened using a TNFα SEAP reporter assay (Invivogen) and clustered with an anti-his antibody coated on the plate. Their ability to bind to TNFR2 and induce the NFκB pathway and SEAP production was then evaluated. In this evaluation, five of the fast-tracked clones (clones 01, 09, 12, 21, and 26) induced SEAP release, with clone 12 showing the most consistent agonist activity.

[0284] An additional 90 AFFIMER polypeptides were cloned from phagemids and characterized using the same method as described above. 55 clones were selected, and their binding to TNFR2 was confirmed via Mirrorball analysis. A total of 75 Stephin A protein variants (20 fast-track selections and 55 additional selections) were further characterized using competitive ELISA to evaluate their ability to block the interaction between hTNFR2-hFc-his-avi and TNF-alpha. A series of experiments were performed to determine the EC80 of hTNFR2-hFc-his-avi when bound to coated hTNF-alpha (593 pM). AFFIMER proteins were tested at three concentrations (10 μM, 1 μM, and 0.1 μM) where the hTNFR2 antigen was present at the determined EC80. Detection of bound hTNFR2-hFc-his-avi was performed using an anti-hFc-HRP antibody. Of the 75 clones tested, 32 showed more than 65% inhibition of the interaction between hTNFR2 and TNF-alpha at 10 μM, and additional analyses were performed to determine the IC50 for these 32 clones (Figure 3). Clones 06, 21, 26, 27, 44, 108, and 115 were confirmed to completely block the interaction between TNFα and hTNFR2 at the test concentrations.

[0285] At this point, 55 clones and 14 clones from the fast-track group (13 agonist clones and 1 negative clone, clone 06) were cloned into the pDisplay vector (Thermo Fisher). This vector contains a PDGFR protein transmembrane domain at the C-terminus to ensure that AFFIMER is expressed on the surface of expressing cells and not on water-soluble proteins. Additionally, an HA tag is encoded at the N-terminus, allowing for easy verification of AFFIMER expression. After cloning, these AFFIMERs were subjected to SEAP analysis using Expi293F, as previously described. TM The cells were temporarily transformed. Based on the combined data, 43 clones were selected for further characterization.

[0286] Example 3: Characterization of 43 selected clones Biacore assay The selected 43 clones were tested as water-soluble proteins via Biacore kinetic analysis. Briefly, multicycle kinetics were performed using a CM5 chip immobilized with the dimeric antigen hTNFR2-hFc-His-avi at 500 RU. The AFFIMER protein was titrated in a 1:2 dilution system starting from 1 μM with HBS-EP+ buffer. The association and disassociation times were 200 seconds and 400 seconds, respectively, and the flow rate was measured at 20 μl / min. Regeneration was performed with 10 mM glycine (pH 1.5) at 30 μl / min for 30 seconds.

[0287] Twelve clones showed extremely low response, making it impossible to estimate the KD value. Clone 09 was fitted to a 1:1 binding model, but the reported KD value was too high to be reliable within the concentration range used (>E-05). Sixteen clones yielded sufficient signal and were successfully fitted to the 1:1 binding model; their curves and fitting results are presented together. Two clones (21 and 69) were dimers, and were fitted to both the 1:1 binding model and the steady-state model to estimate their KD. On the other hand, for the twelve clones, the 1:1 binding model was not applicable due to low Rmax values, so the steady-state model was used; in this case, the KD value was not reported, and only an approximate magnitude was presented instead. The results are shown in Figure 4.

[0288] Binding ELISA cross-reactivity The specificity of TNFR2 AFFIMER clones and others to hTNFR2 was evaluated by ELISA comparing the binding of these clones to TNFR2 with their binding to TNFR1. None of the clones showed binding to TNFR1 as a recombinant antigen. Furthermore, hTNFR1 was found to be Expi293F TMAlthough it was expressed in cells, no binding was observed to this cell-expressed hTNFR1, confirming that there is no cross-reactivity between the hTNFR2 AFFIMER polypeptide conjugate and hTNFR1.

[0289] In addition to binding to human TNFR2, the ability of clones to bind to mouse TNFR2 was also tested to verify cross-reactivity. Since the extracellular domain sequences of the two proteins share only about 68% homology, the possibility of cross-reactivity is extremely low, and in fact, all clones express mTNFR2. TM It did not show binding to cells.

[0290] HEK293-HEK Blue™ TNFα SEAP Reporter Assay Forty-three clones were transiently transduced into HEK293 cells, along with four control groups including SQT-Gly, 3t0-Gly, an empty vector, and clone 06, which was confirmed to be negative. Approximately 29 hours after transduction, cell viability was confirmed using a cell counter, and AFFIMER expression was evaluated by flow cytometry using an anti-HA antibody. Forty-eight hours after transduction, AFFIMER-expressing HEK293 cells were transduced into HEK Blue TM Reporter cells were fixed at 50,000 cells / well and co-cultured at four different cell densities (40,000, 20,000, 10,000, and 5,000). In addition, the positive control MR2-1 was cultured with HEK Blue cells for 24 hours at various concentration ranges.

[0291] After 24 hours, SEAP release in the supernatant was quantified by absorbance measurements using Quanti-Blue and at 640 nm. Staining with monoclonal anti-HA antibody showed that AFFIMER expression levels exceeded 90% in both experiments. Co-culture reporter analysis confirmed the effect of TNFR2 AFFIMER protein displayed on HEK293 cells on HEK Blue cells, and the effect of the MR2-1 clone was also observed. Sixteen clones that consistently ranked in the top 20 across both experiments were ultimately selected, and as shown in Figure 5, the selected clones were: clones 001, 007, 009, 012, 013, 026, 027, 037, 044, 059, 069, 079, 100, 103, 112, and 115.

[0292] Analysis of binding to human and cynomolgus monkey TNFR2-expressing cells. Sixteen selected agonist clones were analyzed in aqueous form to confirm their binding to human TNFR2-overexpressing Expi293FTM and cynomolgus monkey TNFR2-overexpressing Expi293FTM. The experiment was repeated three times, using two other batches of plasma-infected cells. All clones consistently showed specific binding to hTNFR2-Expi293FTM cells, with clones 26 and 69 showing the best binding affinity to both human and cynomolgus monkey TNFR2-expressing cells. Of the 16 clones tested, 13 showed cross-reactivity with cynomolgus monkey TNFR2, while clones 001, 007, and 009 did not. However, while it is not necessary to be the strongest conjugate, clones DAW02-013, DAW02-044, and DAW02-112 were confirmed to be the clones with the least binding difference between human TNFR2 and cynomolgus monkey TNFR2-overexpressing cells when the results of three experiments were combined (Figure 5).

[0293] Example 4. In-line fusion of the same type of dimer Clones 001, 009, and 012 were selected to be formatted into in-line fusion (ILF) allodimers to investigate whether this format offers advantages in inducing agonist activity as a water-soluble protein or when displayed in cells, compared to monomeric forms. Two constructs were used to express the ILF allodimers using a flexible linker; one was codon-optimized for mammalian expression, and the other was not. Both constructs were cloned into a mammalian secretion vector (pD609_KanR), and the recombinant proteins were purified and evaluated for expression levels, protein quality, binding kinetics with hTNFR2-hFc-his-avi by Biacore, and agonist activity using reporter assays with HEK Blue™ TNFα SEAP reporter cells. Furthermore, both structures were expressed on the surface of Expi293FTM cells and cloned into pDisplay vectors to assess their expression levels. Agonist activity was evaluated by binding to hTNFR2-hFc-his-avi and by co-culture reporter testing with HEK Blue™ TNFα SEAP reporter cells. Codon optimization did not improve the yield or purity of the water-soluble ILF allodimer, as confirmed by HPLC.

[0294] Biacore analysis revealed that clones 001 and 009 showed increased affinity in the water-soluble ILF form compared to the water-soluble monomer form, but clone 12 did not show an increase in affinity. Agonist activity was confirmed via SEAP analysis in HEK Blue™ TNFα SEAP reporter cells, and only clone 12 showed increased activity in the water-soluble ILF form. However, when expressed on the cell surface, no difference was observed between the ILF form and the monomer form in SEAP analysis and flow cytometry (flow cell analysis).

[0295] Summary of selection and characterization of StefinA protein variants that specifically bind to hTNFR2 Over 2500 single-clonal phages were screened using phage ELISA, with a hit rate of over 60% and good diversity (20-40%). ELISA screening using crude cell extracts confirmed that over 400 clones bound to the hTNFR2 antigen. To reduce the number of clones to be cloned into mammalian expression vectors at this stage, an additional step was introduced to the selective screening campaign. In parallel, 30 fast-track clones directly expressed as water-soluble AFFIMER protein were selected via passive selection to facilitate assay development and the construction of screening steps for the remaining clones. Of these clones, 20 passed the protein production quality standard of over 80% purity by SEC-HPLC and were used for material characterization through various assays. Five of the 20 clones showed agonist activity in preliminary agonist analysis; at this point, the monomeric water-soluble AFFIMER protein was clustered with an anti-his antibody via a histidine tag and then incubated with a HEK reporter cell line.

[0296] In parallel with the analytical development using fast-track clones, a phage screening campaign was completed, and a total of 364 human TNFR2-binding AFFIMER proteins were purified from phagemids and tested for binding to TNFR2-overexpressing cell lines. Following this analysis, 90 human TNFR2-binding AFFIMER proteins were reformatted into mammalian expression vectors. Of these clones, 55 passed the protein production quality standard of over 80% purity by SEC-HPLC and were used for subsequent analysis.

[0297] Whole clones (fast-track and non-fast-track clones) were subjected to binding of AFFIMER water-soluble protein to hTNFR2 overexpressing cells, agonist analysis using HEK reporter cells, and Expi293F TMThe number of clones was reduced from 75 to 43 through a series of experiments, including preliminary data from HEK reporter cell agonist analysis performed in co-culture with AFFIMER proteins expressed on the cell surface. These 43 superior clones were evaluated as water-soluble proteins by binding characterization analysis to human TNFR2 using Biacore, and KD was successfully calculated for 16 of the 43 clones using a 1:1 binding model. None of the clones showed binding to human TNFR1 by ELISA and not to mouse TNFR2 by cell binding analysis. As AFFIMER proteins displayed on the HEK293 cell surface, these 43 clones were tested for their ability to induce TNFR2 activity in co-culture with HEK reporter cell lines. The majority of the tested clones (80%) showed dose-dependent effects in HEK reporter cells. Sixteen AFFIMER proteins were selected as optimal agonists and used for additional characterization to evaluate cynomorgus TNFR2 binding and human TNFR1 binding on cells. The 16 selected hTNFR2 AFFIMER polypeptides are clones 001, 007, 009, 012, 013, 026, 027, 037, 044, 059, 069, 079, 100, 103, 112, and 115.

[0298] As a water-soluble protein, the inline fusion allodimer showed advantages over the monomer in binding to recombinant TNFR2 in Biacore analysis and also showed potential advantages in the analysis of anti-His clustering agonists using HEK reporter cells. However, when the inline fusion AFFIMER protein was displayed on the cell surface and induced the active pathway of the reporter cell line via TNFR2, no advantages were observed compared to the monomer. The reason why the inline fusion allodimer did not show superior performance compared to the monomer in this final analysis may be that the inline fusion format was not well displayed on the cell surface, allowing only one of the two AFFIMER proteins to bind, or that the limitations of reporter analysis were reached and the differences between the two formats could not be distinguished.

[0299] Example 5. StefinA protein mutant that specifically binds to mouse TNFR2. Phage selection and screening The mTNFR2 antigens, having the sequences shown in Table 7 and provided in three formats (mFc, hFc, and His tag), were tested to evaluate their suitability for use in analyses for selecting mTNFR2 AFFIMER polypeptides. Of the three formats, mTNFR2-mFc was selected for use in biotinylation and AFFIMER selection due to its superior performance in the analysis.

[0300] choice Similar to the hTNFR2 AFFIMER selection, passive and solution selections were performed on two AFFIMER phage libraries. Enrichment was observed when the mTNFR2 concentration was maintained at 10 nM in round 3. More than 2000 phage-displayed AFFIMER polypeptides from rounds 2 and 3 were screened by single-clone phage ELISA. More than 900 of these clones showed mTNFR2 binding, and no nonspecific binding to mouse Fc, plastic, streptavidin, or neutraavidin was detected. Each clone was defined as a "hit" by an absorbance ≥0.5 on an mTNFR2-coated plate and <0.05 on a control antigen (e.g., mouse Fc, plastic, neutraavidin)-coated plate (450 nm to 630 nm). Sequencing of phage ELISA hits identified 281 unique clone sequences, of which 109 showed mTNFR2 binding after ELISA on crude cell extracts. Further binding analysis using mTNFR2-Expi293FTM cells selected 58 clones, which are shown as clones 122-179 in Table 2.

[0301] Water-soluble protein characterization Similar to the process performed with the hTNFR2 AFFIMER polypeptide, the mTNFR2 AFFIMER polypeptide was cloned from a phagemide into a mammalian vector and expressed in Expi293FTM cells. The expressed protein was evaluated for purity by SDS-PAGE and HPLC, affinity was evaluated with Biacore, and cell-based binding of mTNFR2 to mTNFR2 in Expi293FTM cells was assessed. Specificity for mTNFR2 was tested using ELISA and cell-based assays targeting mTNFR1 and mTNFR2 to confirm the specificity of the identified clones. For the characterization of the water-soluble protein, the AFFIMER polypeptide was evaluated for its ability to inhibit the binding of mTNFR2-mFc to mTNFα in ELISA, and four clones (157, 160, 171, and 172) showed clear competitive responses within the test range.

[0302] T cell stimulation analysis Since activated T cells express high levels of TNFR2 and CD25 expression increases upon stimulation with HM102 (a TNFR2 agonist), T cell stimulation assays were used as a screening tool.

[0303] The mTNFR2 AFFIMER polypeptide was first expressed on the surface of HEK293 cells. Subsequently, these HEK293 cells were co-cultured with BALB / c splenocytes for 48 hours, and the increase in CD25 expression in CD4-positive T cells and CD8-positive T cells was evaluated. AFFIMER protein expression on the surface of HEK293 cells was confirmed by flow cytometry staining of the HA tag located at the start site of the open reading frame, preceding the AFFIMER protein and cell anchoring proteins. To evaluate the effects of AFFIMER protein-HEK293 cells on CD4+ and CD8+ cells, after 48 hours of incubation, the cells were stained with Live / Dead staining and a marker panel including CD90.2, CD4, CD8, and CD25.

[0304] In response to HEK293 cells expressing the mTNFR2 AFFIMER protein, CD25 expression in CD4+ and CD8+ T cells increased in a dose-dependent manner, similar to the response observed with the TNFR2 agonist HM102. The response obtained in CD8+ T cells was stronger than in CD4+ T cells, but when focusing on the two highest cell counts used, similar clonal rankings were derived between the CD8+ and CD4+ T cell subsets.

[0305] The six AFFIMER polypeptides that consistently ranked highly in both main analysis experiments were clones 174, 160, 175, 125, 157, and 179.

[0306] Summary of Selection and Characterization Analysis of mTNFR2 AFFIMER Polypeptides Over 2000 single-clonal phages were screened using phage ELISA, exhibiting moderate hit rates of 35–60% and good diversity of 25–35%. ELISA screening using crude cell extracts confirmed that 109 clones bound to the mTNFR2 antigen. Using the same screening procedure as the human program, the phagemide-identified binders were purified and tested for binding to TNFR2-overexpressing cell lines. Based on this analysis, 60 AFFIMER proteins that specifically bind to mTNFR2 were reformatted into mammalian expression vectors for production, and their biophysical properties as water-soluble proteins were evaluated. Of these, 22 AFFIMER proteins exceeded 80% purity by SEC-HPLC analysis, meeting protein production quality standards. These 22 clones were characterized as water-soluble proteins using various assays and then expressed on the surface of HEK293 cells for co-culture assays with splenocytes.

[0307] When only 10 of the 22 selected AFFIMER proteins were tested with Biacore, they showed nanomolar binding affinity (KD) in the triple digits. However, AFFIMER proteins with even higher KD values ​​were found to exhibit good agonist activity when expressed on the cell surface. Furthermore, the performance of the clones as water-soluble proteins differed depending on whether they were native dimers or trimers. For these reasons, Biacore data were considered in the context of mTNFR2 agonist activity but were not used for clone selection. When tested in cells expressing mTNFR2, 20 of the 22 clones showed specific binding across a wide range of EC50 values. Similar to the Biacore data, cell binding results were also considered only in the context of mTNFR2 agonist activity and were not used for clone selection. None of the clones bound to recombinant protein or TNFR1 expressed on the cell surface. Five of the 22 clones were confirmed to completely inhibit the interaction between TNF-α and TNFR2 by competitive ELISA.

[0308] Twenty-two mouse clones were expressed on the surface of HEK293 cells for co-culture testing with splenocytes. The majority of the tested clones showed a dose-dependent increase in CD25 expression in CD4+ T cells and CD8+ T cells (≥70%). Clones were ranked by CD25 expression in both CD4+ and CD8+ T cells, and based on the results of this crucial test, six AFFIMER proteins (clones 174, 160, 175, 125, 157, and 179) that specifically bind to mTNFR2 were identified.

Claims

1. 1 x 10 －６ An engineered modified polypeptide that specifically binds to TNFR2 with a Kd value of M or less, wherein the engineered modified polypeptide is a variant of the stephin A protein.

2. An engineered polypeptide according to claim 1, characterized by comprising an amino acid sequence having at least 80%, 90%, 95%, or 98% identity with the following amino acid sequence: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-Xaa2-TNYYIKV RAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa)m-Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4). Here, Xaa is an amino acid residue in each individual case, n and m are each independent integers between 3 and 20.

3. An engineered polypeptide according to claim 1, characterized by comprising an amino acid sequence having at least 80%, 90%, 95%, or 98% identity with the following amino acid sequence: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (Sequence ID 5). Xaa is an amino acid residue in each individual case. n and m are each independent integers between 3 and 20.

4. An engineered polypeptide according to claim 1, characterized by comprising an amino acid sequence having at least 80%, 90%, 95%, or 98% identity with the following amino acid sequence: MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEEKTGETYGKLEAVQYKTQV (Sequence ID 484)Xaa2-(Xaa)n-Xaa3-TNYYIKVRAGDNKYMHLKVF (Sequence ID 485)-Xaa4-Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF (Sequence ID 486). Here, Xaa is an amino acid residue in each individual case, n and m are each independent integers between 3 and 20.

5. 1 x 10 ―７ M's Kd, preferably 1 × 10 －８ An engineered polypeptide according to any one of claims 1 to 4, characterized by binding to TNFR2 with M's Kd.

6. The (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 102, and / or The engineered polypeptide according to claim 2 or 3, characterized in that (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103 to 199.

7. The (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 11, 13, 16, 17, 24, 25, 29, 33, 41, 54, 65, 67, 73, 75, 82, 89, 90, 98, and 99, and / or The engineered polypeptide according to claim 6, characterized in that (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103, 108, 110, 113, 114, 121, 122, 126, 130, 138, 151, 162, 164, 170, 172, 179, 186, 187, 195, and 196.

8. The engineering-modified polypeptide according to claim 1, wherein the engineering-modified polypeptide comprises an amino acid sequence having at least 75%, 85%, or 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292, and 293, wherein the sequence is characterized by selectively excluding one or more of the 21 carboxy-terminal residues.

9. An engineered polypeptide according to any one of claims 1 to 8, further comprising the signal peptide.

10. An engineered polypeptide according to any one of claims 1 to 9, further comprising the transmembrane domain.

11. A fusion protein according to any one of claims 1 to 8, comprising a dimer, trimer, or tetramer of the engineeringly modified polypeptide, and selectively comprising a linker.

12. The fusion protein according to claim 11, characterized in that the linker is a rigid linker.

13. The fusion protein according to claim 12, characterized in that the rigid linker is a peptide linker containing the sequence (EAAAK)n, and n is selectively 1 to 6.

14. A fusion protein comprising one or more engineered polypeptides according to any one of claims 1 to 13, linked to a therapeutic moiety or a diagnostic moiety.

15. The fusion protein according to claim 14, characterized in that the therapeutic moiety is a therapeutic protein or peptide and selectively contains an antibody.

16. The fusion protein according to claim 15, characterized in that the diagnostic moiety is a fluorescent protein.

17. The engineered polypeptide or fusion protein according to any one of claims 1 to 10 or any one of claims 11 to 16 further comprises a half-life extension moiety.

18. The engineered polypeptide or fusion protein according to claim 17, characterized in that the half-life extension moiety is selected from the group consisting of an antibody Fc domain, human serum albumin, and serum-binding proteins.

19. The aforementioned half-life extension moiety is 1 × 10⁻⁶ compared to human serum albumin. ―６ The engineered polypeptide or fusion protein according to claim 18, characterized in that it is an engineered polypeptide that is a variant of the stephin A protein that specifically binds at Kd values ​​of M or less.

20. The engineered polypeptide or fusion protein according to claim 19, characterized in that the half-life extension moiety is selected from the group consisting of Sequence ID No. 471 to Sequence ID No.

477.

21. A polynucleotide or set of polynucleotides characterized by encoding an engineered polypeptide according to any one of claims 1 to 20 or a fusion protein according to any one of claims 1 to 20.

22. A polynucleotide or set of polynucleotides comprising a nucleic acid sequence having at least 80%, 90%, 95%, or 98% identity with a sequence selected from the group consisting of SEQ ID NOs: 297, 302, 304, 307, 308, 315, 316, 320, 324, 332, 345, 356, 358, 364, 366, 373, 380, 381, 389, and 290, wherein the sequence selectively excludes nucleotides encoding 20 carboxyl-terminal residues.

23. A delivery vehicle characterized by containing a polynucleotide as described in any one of claims 1 to 22.

24. The carrier according to claim 23, characterized in that the carrier is a viral delivery vehicle.

25. The carrier according to claim 24, characterized in that the virus carrier is an adenovirus vector, a retrovirus vector, a lentivirus vector, or an adeno-associated virus (AAV) vector.

26. The carrier according to claim 24, characterized in that the delivery vehicle is a non-viral carrier.

27. The carrier according to claim 26, characterized in that the non-viral carrier is a liposome or a lipid nanoparticle.

28. A plasmid or minicircle characterized by comprising a polynucleotide as described in any one of claims 1 to 27.

29. Messenger RNA (mRNA) comprising an open reading frame encoding an engineered polypeptide as described in any one of claims 1 to 28.

30. The mRNA according to claim 29, further comprising a reading frame encoding the immune cell conjugate.

31. Lipid nanoparticles characterized by being selectively cationic lipid nanoparticles containing the mRNA described in claim 29 or 30.

32. A pharmaceutical composition comprising an engineered polypeptide, fusion protein, or carrier as described in any one of claims 1 to 31, further comprising a selectively pharmaceutically acceptable carrier (excipient).

33. A conjugate characterized by comprising an engineered polypeptide or fusion protein according to any one of claims 1 to 32, linked to a pharmacologically active moiety.

34. A method characterized by comprising the step of administering the pharmaceutical composition described in claim 32 to a subject.

35. The aforementioned subjects (Subjects) include systemic lupus erythematosus (SLE), lupus nephritis (e.g., drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD) (e.g., Crohn's disease), and colitis / ulcerative colitis. Colitis), Graft-versus-Host Disease (GvHD) (associated with stem cell transplantation) (also known as allograft rejection), Transplant or Solid Organ Transplantation (SOT), Primary Biliary Cholangitis (PBC), Psoriasis, Psoriatic Arthritis, Collagen-Induced Arthritis, Experimental Allergy The method according to claim 34, characterized in that the patient has Encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjögren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, glomerulosclerosis, and / or cancer.

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