FR3147278A1 - TNFR2-BINDING POLYPEPTIDES AND METHODS OF USE - Google Patents

Abstract

The present invention provides engineered variants of the TNFR2-binding Stefin A polypeptide, polynucleotides encoding the engineered variants of the TNFR2-binding Stefin A polypeptide, cells expressing the polypeptide variants, pharmaceutical preparations of the polypeptide variants, and uses of the polypeptide variants in the treatment of various human conditions, including inflammatory, autoimmunity, and cancers.

Description

TNFR2-BINDING POLYPEPTIDES AND METHODS OF USE

Maintaining control of cell-mediated and humoral immune responses is an important dimension of healthy immune system activity. Abnormal regulation of T-cell and B-cell-mediated immune responses has been associated with a wide range of human diseases, as inappropriate augmentation of an immune response against various self antigens and foreign antigens plays a causal role in conditions such as autoimmune disorders, asthma, allergic reactions, graft-versus-host disease (GvHD), transplant rejection, and a variety of other immunological disorders including cancer.

These diseases are mediated by T and B lymphocytes that exhibit reactivity against autoantigens and those derived from nonthreatening sources, such as allergens or transplant allografts. Regulatory T cells (Tregs) have evolved to inhibit the activity of immune cells that cross-react with self major histocompatibility complex (MHC) proteins and other benign antigens. The best-known Treg populations are CD4+, CD25+, FoxP3+, and CD17+ Tregs.

Tumor necrosis factor receptor (TNFR) subtypes 1 and 2 have been identified specifically on the surface of Tregs. Activation of TNFR1 by soluble TNF-alpha amplifies the caspase signaling cascade leading to Treg apoptosis. On the other hand, activation of TNFR2 by predominantly membrane-bound TNF-alpha induces signaling through the mitogen-activated protein kinase (MAPK) signaling pathway, which orchestrates TRAF2/3- and NF-κB-mediated transcription of genes that promote cell proliferation and escape apoptosis.

The importance of TNFR2 in Treg proliferation and function has been demonstrated in various studies. TNFR2-deficient mice showed reduced numbers of thymic and peripheral Tregs (2013_Chen X, et al. PMID: 23277487), and TNFR2 -/- Tregs were unable to control the inflammatory response in vivo (2008_Van Mierlo GJ, et al. PMID: 18292492). In humans, Tregs have been shown to express higher levels of TNFR2 than effector T cells (2013_Okubo Y, et al. PMID: 24193319 & 2002_Annunziato F, et al. PMID: 12163566) and TNFR2+ Tregs exhibited the most potent suppression of proliferation and cytokine production of co-cultured T-responder cells (2010_Chen X, et al. PMID: 20127680). Furthermore, TNFR2 has been shown to play a role in carcinogenesis and tumor growth by mediating TNF responses in immunosuppressive cells, enabling immune evasion and tumor development (Sheng Y., et al. doi: 10.3389/fimmu.2018.01170).

Moreover, in autoimmune diseases affecting the central nervous system (CNS), particularly multiple sclerosis (MS), pathogenic lymphocytes are generated peripherally to infiltrate the CNS and cause local inflammation and demyelination. Demyelination is the damage of the protective layer (myelin sheath) that surrounds nerve fibers in the brain. Protecting against or reversing demyelination are strategies explored to combat MS. Interestingly, several studies have reported the involvement of TNFR2 in neuroprotection. For example, in a mouse model of cuprizone-induced demyelination, TNFR2 was shown to be essential for the regeneration of oligodendrocytes, the cells primarily responsible for maintaining and generating the myelin sheath that surrounds axons, whereas TNF signaling via TNFR1 promoted nerve demyelination (2001_HA Arnett, et al. PMID: 11600888).

Due to its role in directing cell survival and growth, TNFR2 represents an attractive target for the treatment of diseases such as autoimmune diseases, GvHD, allograft rejection, allergic reactions, asthma, and cancer.

AFFIMER®, developed by Avacta Life Sciences Limited, is a stable engineered small molecule protein based on stefin A protein, which is an in vivo protein. AFFIMER® comprises two short peptide sequences having a random sequence and an N-terminal sequence, and is capable of binding to a target material with high affinity and specificity in a manner similar to a monoclonal antibody. AFFIMER® shows remarkably improved binding affinity and specificity compared with the free peptide library, and has a very small size and high stability compared with antibodies, and thus has received great attention as a next-generation alternative pharmaceutical platform to replace antibodies (US Patent Nos. 9,447,170, 8,853,131, etc.).

The present invention relates to polypeptides that specifically bind to TNFR2 by engineering the natural protein stefin A, resulting in the development of AFFIMER® polypeptides capable of binding to TNFR2 with excellent affinity and specificity.

Summary

In one aspect, there is provided an engineered polypeptide that specifically binds to TNFR2 with a Kd of 1×10 ^-6 M or less, the engineered polypeptide being a variant of a Stefin A protein.

In another aspect, there is provided a fusion protein comprising a dimer, trimer or tetramer of the engineered polypeptides of the invention, optionally comprising one or more linkers.

In another aspect, there is provided a fusion protein comprising one or more of the engineered polypeptides of the invention linked to a therapeutic or diagnostic moiety.

In another aspect, there is provided a polynucleotide or set of polynucleotides encoding the engineered polypeptide or fusion protein of the invention.

In one aspect, there is provided a delivery vehicle comprising the polynucleotide of the invention.

In another aspect, there is provided a plasmid or minicircle comprising the polynucleotide of the invention.

In another aspect, there is provided a messenger RNA (mRNA) comprising an open reading frame encoding the engineered polypeptide of the invention.

In one aspect, there is provided a lipid nanoparticle, optionally a cationic lipid nanoparticle, comprising the mRNA of the invention.

In another aspect there is provided a pharmaceutical composition comprising the engineered polypeptide, fusion protein or delivery vehicle according to the invention, and optionally a pharmaceutically acceptable excipient.

In one aspect, there is provided a conjugate comprising the engineered polypeptide or fusion protein of the invention linked to a pharmacologically active moiety.

In another aspect, there is provided a method comprising administering to a subject the pharmaceutical composition of the invention.

In one aspect there is provided an engineered polypeptide, fusion protein or delivery vehicle of the invention for use as a medicament.

In another aspect, there is provided an engineered polypeptide that competes for binding to TNFR2 with an engineered polypeptide according to the invention.

In another aspect, there is provided an engineered polypeptide that binds to the same epitope on TNFR2 as an engineered polypeptide according to the invention.

Fig. 1

Schematic of the selection strategy for the human TNFR2 selection campaign. Both libraries underwent solution and passive selections. Stringency was introduced by decreasing the hTNFR2 concentration as the rounds progressed and increasing the number of wash steps between round 1 (5x PBS/0.1% Tween 20, 2x PBS) and round 2 (15x PBS/0.1% Tween 20, 2x PBS). (PBS, from “Phosphate-buffered saline”) For passive selections using Avacta’s proprietary AFFIMER® Type 3 phage library (US Patent No. 9,932,575), both deselection and competition approaches were explored simultaneously. For the deselection approach, the Fc concentration at round n+1 was the hTNFR2 concentration at round n. For the competition approach, the Fc concentration was 10 times the molar excess of the hTNFR2 concentration specified in the given round. For passive selections using Avacta's proprietary AFFIMER® Type 1 phage library (U.S. Patent No. 8,841,491), the deselection approach was performed exclusively and a round 4 was introduced since early screening of round 3 results indicated high diversity and hit rates. For solution selections, both libraries underwent the competition approach to reduce enrichment of AFFIMER® polypeptides displayed on Fc-binding phage. Streptavidin- and neutravidin-coated beads were alternated between rounds to reduce enrichment of peptides presented on streptavidin- and/or neutravidin-binding phages;

Fig. 2

Binding of AFFIMER® polypeptide to Expi293F™ cells overexpressing human TNFR2;

Fig. 3

Compilation of data from titration experiments. % inhibition at 3 µM and IC50 for the best blockers;

Fig. 4

SEAP duplicate results, ranking of 20 clones;

Fig. 5

Cross-reactivity of hTNFR2 AFFIMER® clones with cynomolgus TNFR2, in triplicate.

Detailed description

AFFIMER®

Stefin polypeptides comprise a subgroup of proteins within the cystatin superfamily, a family that encompasses proteins that contain multiple cystatin-like sequences. The Stefin subgroup of the cystatin family includes relatively small (approximately 100 amino acids), single-domain proteins. They undergo no known post-translational modifications and lack disulfide bonds, suggesting that they will be able to fold identically in a wide range of extracellular and intracellular environments. Stefin A itself is a monomeric, single-chain, single-domain protein of 98 amino acids. The structure of Stefin A has been determined, facilitating the rational mutation of Stefin A into the AFFIMER® polypeptide. The only known biological activity of cystatins is inhibition of cathepsin activity, which allowed exhaustive testing of the residual biological activity of the engineered proteins.

An “AFFIMER® polypeptide” (also referred to herein as an “AFFIMER® protein”) refers to a small, highly stable protein that is an engineered variant of a Stefin polypeptide. AFFIMER® proteins feature two peptide loops and an N-terminal sequence that can all be randomized to bind to desired target proteins with high affinity and specificity, in a manner similar to monoclonal antibodies. The stabilization of the two peptides by the Stefin A protein scaffold limits the possible conformations that the peptides can assume, increasing binding affinity and specificity compared to free peptide libraries. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in a variety of applications. Variations of other portions of the Stefin A polypeptide sequence may be made, such variations improving the properties of these affinity reagents, such as increasing stability, making them robust over a range of temperatures and pH, etc. In some embodiments, an AFFIMER® polypeptide includes a sequence derived from Stefin A, sharing substantial identity with a wild-type Stefin A sequence, such as human Stefin A. It will be apparent to one skilled in the art that modifications may be made to the scaffold sequence without departing from the disclosure. In particular, an AFFIMER® polypeptide may have an amino acid sequence that is at least 25%, 35%, 45%, 55% or 60% identical to the sequences corresponding to human Stefin A, e.g. at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95% identical, e.g. where the sequence variations do not adversely affect the ability of the scaffold to bind to the desired target (such as TFNR2), and e.g. which do not restore or generate biological functions such as those possessed by wild-type Stefin A but which are abolished in the mutational changes described herein.

An “AFFIMER® agent” refers to a polypeptide that includes an AFFIMER® polypeptide sequence and any other modification(s) (e.g., conjugation, post-translational modifications, etc.) so as to represent a therapeutically active protein for delivery to an individual.

An “AFFIMER®-linked conjugate” refers to an AFFIMER® agent having at least one moiety conjugated thereto via chemical conjugation other than by formation of a contiguous peptide bond via the C-terminus or N-terminus of the polypeptide portion of the AFFIMER® agent containing the AFFIMER® polypeptide sequence. An AFFIMER®-linked conjugate may be an “AFFIMER®-drug conjugate,” which refers to an AFFIMER® agent including at least one pharmacologically active moiety conjugated thereto. An AFFIMER®-linked conjugate may also be an “AFFIMER®-label conjugate,” which refers to an AFFIMER® agent including at least one detectable moiety (e.g., a detectable label) conjugated thereto.

An “AFFIMER®-encoded polynucleotide” refers to a nucleic acid construct that, when introduced into and expressed by cells, produces an intended AFFIMER® agent.

TNFR2

Tumor necrosis factor receptor 2 (“TNFR2”), also known as tumor necrosis factor receptor 1 (TNFR1), is a Type 1 membrane-bound receptor that binds to tumor necrosis factor alpha (“TNFα”). TNFR2, also known as p75, TNF Receptor Superfamily Member 1B or CD120b, is encoded in humans as TNFRSF1B which expresses a 48 kDa protein of 461 amino acids in length (UniProt P20333). In mice, the TNFR2 protein is 474 amino acids in length and has a mass of 50 kDa.

A “TFNR2 AFFIMER® agent” refers to an AFFIMER® agent that comprises at least one AFFIMER® polypeptide that binds to TFNR2, particularly human TFNR2 and optionally cynomolgus TNFR2, with a dissociation constant (Kd) of at least 10 ^-6 M. In some embodiments, the TFNR2 AFFIMER® agent binds TFNR2 at a Kd of 1×10−7M or less, a Kd of 1×10−8M or less, a Kd of 1×10−9M or less, or a Kd of 1×10−10M or less. It is to be understood that the terms “TFNR2 AFFIMER® polypeptide,” “TFNR2 AFFIMER® protein,” and “engineered variant of TFNR2-binding Stefin A polypeptide” are used interchangeably herein. Thus, a “TFNR2 AFFIMER® polypeptide” is an engineered polypeptide that specifically binds to TFNR2 with a Kd of 1 x 10 ^-6 M or less, wherein the engineered polypeptide is a variant of a Stefin A protein.

Polypeptides

Polypeptides (which include peptides and proteins) are polymers of amino acids of any length. The polymer may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing at least one analogue of an amino acid (including, for example, non-naturally occurring amino acids), as well as other modifications known in the art.

Amino acids (also referred to herein as amino acid residues) participate in one or more peptide bonds of a polypeptide. In general, the abbreviations used herein to designate amino acids 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 "residues" of methionine, isoleucine, leucine, alanine, and glycine, respectively. A residue is defined as a radical derived from the corresponding α-amino acid by removing the OH portion of the carboxyl group and the H portion of the α-amino group. The term "amino acid side chain" is that portion of an amino acid excluding the -CH(NH2)COOH portion, as defined by K. D. Kopple, "Peptides and Amino Acids", W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33.

For the most part, the amino acids used in the application of this disclosure are the naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids that contain amino and carboxyl groups. Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine and tryptophan, and amino acids and amino acid analogs that have been identified as constituents of bacterial cell wall peptidylglycans.

Amino acid residues having "basic side chains" include Arg, Lys, and His. Amino acid residues having "acidic side chains" include Glu and Asp. Amino acid residues having "neutral polar side chains" include Ser, Thr, Asn, Gln, Cys, and Tyr. Amino acid residues having "neutral nonpolar side chains" include Gly, Ala, Val, Ile, Leu, Met, Pro, Trp, and Phe. Amino acid residues having "nonpolar aliphatic side chains" include Gly, Ala, Val, Ile, and Leu. Amino acid residues having "hydrophobic side chains" include Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp. Amino acid residues having "small hydrophobic side chains" include Ala and Val. Amino acid residues with "aromatic side chains" include Tyr, Trp, and Phe.

Amino acid residues further include analogs, derivatives, and congeners of any specific amino acid referred to herein, such as, for example, the subject AFFIMER® polypeptide (particularly if generated by chemical synthesis) may include an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxyphenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminiopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring amino acid metabolites or precursors having side chains suitable herein will be recognized by those skilled in the art and are included within the scope of this disclosure.

Also included are the (D) and (L) stereoisomers of these amino acids where the structure of the amino acid admits of stereoisomeric forms. The configuration of amino acids and amino acid residues herein is designated by the appropriate symbols (D), (L) or (DL), further where the configuration is not designated, the amino acid or residue may have the (D), (L) or (DL) configuration. It will be noted that the structure of some of the compounds of this disclosure includes asymmetric carbon atoms. It is to be understood accordingly that isomers resulting from such asymmetry are included within the scope of this disclosure. Such isomers can be obtained in substantially pure form by conventional separation techniques and by sterically controlled synthesis. For the purposes of this application, unless expressly indicated otherwise, a named amino acid is to be construed as including both the (D) and (L) stereoisomers.

The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum match, without regard to conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megaalign, BestFit, GCG Wisconsin Package and variations thereof. In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% identity to amino acid or nucleotide residues, when compared and aligned for maximum match, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues long, or any integer value therebetween. In some embodiments, identity exists over a region longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical throughout the length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases long, or any integer therebetween. In some embodiments, identity exists over a region longer than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical throughout the length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

When calculating percent identity, all residues defined as "Xaa" or "X" in a reference sequence are included in the percent identity calculation, i.e., any amino acid at that position in a comparison sequence matches the reference sequence.

A conservative amino acid substitution is a substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have generally been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of a phenylalanine for a tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins and/or antibodies of the disclosure do not abrogate binding of the polypeptide, soluble protein or antibody containing the amino acid sequence to the target binding site. Methods for identifying conservative amino acid substitutions that do not eliminate binding are well known in the art.

An "isolated" polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is isolated is substantially pure.

A material is considered substantially pure if it is at least 50% pure (e.g. free of contaminants), at least 60%, at least 70%, at least 80%, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

A fusion polypeptide (e.g., a fusion protein) is a hybrid polypeptide expressed by a nucleic acid molecule comprising at least two open reading frames (e.g., from two individual molecules, e.g., two individual genes).

A linker (also referred to as a linker region) may be inserted between a first polypeptide (e.g., copies of a TFNR2 AFFIMER® polypeptide) and a second polypeptide (e.g., another AFFIMER® polypeptide, an Fc domain, a ligand binding domain, etc.). In some embodiments, a linker is a peptide linker. The linkers must not adversely affect the expression, secretion, or bioactivity of the polypeptides. In some embodiments, the linkers are non-antigenic and do not elicit an immune response.

A transmembrane domain (also referred to as a TM domain) is a sequence that can be added to an AFFIMER® polypeptide, such as by adding a polynucleotide sequence encoding the TM domain to the polynucleotide sequence encoding the AFFIMER® polypeptide, to ensure that the AFFIMER® is retained on the surface of the cell expressing it. A variety of TM domains are available and readily usable with the AFFIMER® polypeptide.

An “AFFIMER®-antibody fusion” is a fusion protein that comprises a portion of an AFFIMER® polypeptide and a variable region of an antibody. AFFIMER®-antibody fusions may include full-length antibodies having, for example, at least one AFFIMER® polypeptide sequence attached to the C-terminus or N-terminus of at least one of its VH and/or VL chains, e.g. at least one chain of the assembled antibody is a fusion protein with an AFFIMER® polypeptide. AFFIMER®-antibody fusions may also comprise at least one AFFIMER® polypeptide sequence as part of a fusion protein with an antigen binding site or a variable region of an antibody fragment.

An antibody is an immunoglobulin molecule that specifically recognizes and binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination of any of the above, through at least one antigen-binding site, wherein the antigen-binding site is typically within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” includes intact (whole) polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies provided that such fragments have been formatted to include an Fc or other FcγRIII binding domain, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen binding site of an antibody (formatted to include an Fc or other FcγRIII binding domain), antibody mimetics, and any other modified immunoglobulin molecule comprising an antigen binding site provided that the antibodies exhibit the desired biological activity.

The antibody may belong to one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains called alpha, delta, epsilon, gamma, and mu. In some embodiments, the Fc is null binding to FcyR2b. In some embodiments, the antibody is not an IgG1 Fc.

A variable region of an antibody may be a variable region of an antibody light chain or a variable region of an antibody heavy chain, either alone or in combination. Typically, the variable region of both heavy and light chains includes four framework regions (FRs) and three complementarity determining regions (CDRs), also called hypervariable regions. The CDRs of each chain are held together in close proximity by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antibody's antigen-binding sites. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 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). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

A humanized antibody is a form of non-human (e.g., murine) antibody that is: specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which CDR residues are replaced with CDR residues from a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding capacity. In some cases, residues in the Fv framework region of a human immunoglobulin are replaced with corresponding residues in an antibody from a non-human species. The humanized antibody may be further modified by substituting additional residues either in the Fv framework region and/or among the replaced non-human residues to refine and optimize the specificity, affinity, and/or binding capacity of the antibody. The humanized antibody may include variable domains containing all or substantially all of the CDRs that correspond to the non-human immunoglobulin while all or substantially all of the framework regions are those of a human immunoglobulin sequence. In some embodiments, the variable domains include the framework regions of a human immunoglobulin sequence. In some embodiments, the variable domains include the framework regions of a human immunoglobulin consensus sequence. The humanized antibody may also include at least a portion of an immunoglobulin constant (Fc) region or domain, typically that of a human immunoglobulin. A humanized antibody is generally considered distinct from a chimeric antibody.

An epitope (also referred to herein as an antigenic determinant) is that portion of an antigen capable of being recognized and specifically bound by a particular antibody, a particular AFFIMER® polypeptide, or other particular binding domain. When the antigen is a polypeptide, epitopes may be formed from both contiguous amino acids and non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturation, while epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturation. An epitope typically comprises at least 3, and more typically, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

“Binds specifically to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target (e.g., TFNR2) and an AFFIMER® polypeptide, antibody, or other binding partner, that allow the presence of the target to be determined in the presence of a heterogeneous population of molecules including biological molecules. For example, an AFFIMER® polypeptide that binds specifically to TFNR2 is an AFFIMER® polypeptide that binds to TFNR2 with greater affinity, avidity (if formatted multimeric), more readily, and/or with greater duration than it binds to other targets.

"Conjugate", "conjugation" and their grammatical variations refer to the joining or linking together of two or more compounds resulting in the formation of another compound, by any joining or linking process known in the art. They may also refer to a compound that is generated by the joining or linking of two or more compounds. For example, a TFNR2 AFFIMER® polypeptide linked directly or indirectly to at least one chemical moiety or polypeptide is an example of a conjugate. Such conjugates include fusion proteins, those produced by chemical conjugates, and those produced by any other process.

Polynucleotides

A polynucleotide (also referred to herein as a nucleic acid or nucleic acid molecule) is a polymer of nucleotides of any length and may include DNA, RNA (e.g., messenger RNA (mRNA)), or a combination of DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogues, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

A polynucleotide encoding a polypeptide refers to the order or sequence of nucleotides along a strand of deoxyribonucleotides of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (e.g. protein) chain. Thus, a nucleic acid sequence encodes the sequence of amino acids.

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

Nucleic acid sequences may be altered, e.g. mutated, compared to naturally occurring nucleic acid sequences, for example.

The nucleic acid sequence may be of any length, for example from 2 to 000,000 nucleotides or more (or any integer value above or between) a nucleic acid, for example from about 100 to about 10,000, or from about 200 nucleotides to about 500 nucleotides.

Transfection is the process of introducing an exogenous nucleic acid into a eukaryotic cell. Transfection can be accomplished by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistic technology (biolistics).

A vector is a construct that is capable of delivering, and typically expressing, at least one gene or sequence of interest into a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes. A vector may, in some embodiments, be an isolated nucleic acid that can be used to deliver a composition into the cell. A number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, a vector may be a plasmid or a virus, replicating autonomously. The term should also be interpreted to include facilitating the transfer of nucleic acid into cells of non-plasmid and non-viral compounds, e.g., polylysine compounds, liposomes, and the like. Non-limiting examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, and retroviral vectors.

An expression vector is a vector comprising a recombinant polynucleotide comprising an expression control sequence and a nucleotide sequence to be expressed operably linked. The expression vector comprises sufficient cis-acting elements (cis-acting elements) used for expression; other expression elements may be provided by the host cell or the in vitro expression system. Expression vectors include, for example, cosmids, plasmids (e.g. naked or contained in liposomes) and viruses (e.g. lentiviruses, retroviruses, adenoviruses and adeno-associated viruses).

“Operably linked” refers to a functional coupling between the regulatory sequence and a heterologous nucleic acid sequence that drives expression of the latter. For example, if the promoter affects transcription or expression of the coding sequence, the promoter is operably linked to a coding sequence. Typically, operably linked DNA sequences are contiguous and can join two protein-coding regions in the same reading frame.

A promoter is a DNA sequence recognized by the synthesis machinery required for the synthesis machinery of cell-specific transcription of a polynucleotide or introduced sequence.

“Inducible expression” refers to expression under certain conditions, such as activation (or inactivation) of an intracellular signaling pathway or contacting the cells harboring the expression construct with a small molecule that regulates the expression (or level of expression) of a gene operably linked to an inducible promoter that is sensitive to the concentration of the small molecule. This is in contrast to constitutive expression, which refers to expression under physiological conditions (not limited by certain conditions).

"Electroporation" refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a biomembrane; their presence allows biomolecules such as plasmids or other oligonucleotides to pass from one side of the cell membrane to the other.

Treatments

“Treatment” refers to biomembrane both (1) therapeutic measures that cure, slow, alleviate symptoms, and/or stop the progression of a diagnosed disease or disorder and (2) prophylactic or preventive measures that prevent or slow the development of a targeted disease or disorder. Thus, those in need of treatment include those who already have the disorder; those who are likely to have the disorder; and those in whom the disorder is to be prevented.

“Subject,” “individual,” and “patient,” used interchangeably herein, refer to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, and rodents.

“Sustained response” refers to the sustained effect on reducing inflammation after stopping a treatment. In some embodiments, the sustained response has a duration that is at least the same as the duration of the treatment, at least 1.5x, 2.0x, 2.5x or 3.0x the extent of the duration of the treatment.

A “complete response” or “CR” refers to the disappearance of all target lesions; “partial response” or “PR” refers to a decrease of at least 30% in the sum of the longest diameters (SLD) of target lesions, taking the baseline SLD as a reference; and “stable disease” or “SD” refers to neither sufficient shrinkage of target lesions to qualify for PR nor sufficient increase to qualify for PD, taking the smallest SLD since treatment initiation as a reference.

Progression-free survival (PFS) refers to the length of time during and after treatment that the disease being treated (e.g., an inflammatory or autoimmune disease) does not get worse. Progression-free survival can include the length of time that patients have had a complete response or partial response, as well as the length of time that patients have had stable disease.

The "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

"Overall survival" refers to the percentage of individuals in a group who are likely to be alive after a given period of time.

“Agonist” and “agonist” refer to agents that are capable of, directly or indirectly, inducing, activating, promoting, increasing or substantially amplifying the biological activity of a target or target pathway. “Agonist” is used herein to include any agent that partially or completely induces, activates, promotes, increases or amplifies the activity of a protein or other target of interest.

“Antagonist” and “antagonist” refer to or describe an agent that is capable, directly or indirectly, partially or completely, of blocking, inhibiting, reducing or neutralizing a biological activity of a target and/or pathway. The term “antagonist” is used herein to include any agent that partially or completely blocks, inhibits, reduces or neutralizes the activity of a protein or other target of interest.

“Modulation” and “modulate” refer to a change or alteration of a biological activity. Modulation includes, but is not limited to, stimulation of an activity or inhibition of an activity. Modulation may be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in biological, functional, or immunological properties associated with the activity of a protein, pathway, system, or other biological target of interest.

An immune response includes responses from both the innate immune system and the adaptive immune system. It includes both cell-mediated and/or humoral immune responses. It includes both T-cell and B-cell responses, as well as responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, etc.

“Pharmaceutically acceptable” means a substance that is approved or approvable by a federal or state government regulatory agency or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, including humans.

"Pharmaceutically acceptable excipient" is an excipient, carrier or adjuvant that can be administered to a subject, together with at least one agent of the present disclosure, and that does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to provide a therapeutic effect. In general, those skilled in the art and the United States Food and Drug Administration (FDA) consider a pharmaceutically acceptable excipient, carrier or adjuvant to be an inactive ingredient in any formulation.

An “effective amount” (also referred to herein as a “therapeutically effective amount”) is an amount of an agent, such as a TFNR2 AFFIMER® agent, effective to treat a disease or disorder in a subject such as a mammal. In the case of an inflammatory or autoimmune disease, the therapeutically effective amount of a TFNR2 AFFIMER® agent has a therapeutic effect and may thereby reduce inflammation; alleviate to some extent at least one of the symptoms associated with the inflammatory or autoimmune disease; reduce morbidity and mortality; improve quality of life; or a combination of these effects.

Miscellaneous

It is understood that wherever embodiments are described herein with the term "comprising," otherwise analogous embodiments described with the terms "consisting of" and/or "consisting essentially of" are also provided. It is further understood that wherever embodiments are described herein with the language "consisting essentially of," otherwise analogous embodiments described with the terms "consisting of" are also provided.

As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments that relate to that value or parameter. For example, the description referring to "about X" includes the description of "X".

The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: 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 (alone); B (alone); and C (alone).

The phrase "at least one" can be used interchangeably with "one or more." It should be understood that "one" is not limited to one but rather means "at least one."

Polypeptide TFNR2 AFFIMER®

An AFFIMER® polypeptide is a scaffold based on a Stefin A polypeptide, meaning that it has a sequence that is derived from a Stefin A polypeptide, e.g., a mammalian Stefin A polypeptide, e.g., a human Stefin A polypeptide. Certain aspects of the application relate to AFFIMER® polypeptides that bind to TFNR2 (also referred to as “TFNR2 AFFIMER® polypeptides”) wherein at least one of the solvent accessible loops of the wild-type Stefin A protein has the ability to bind to TFNR2, preferably selectively, and preferably with a Kd of 10 ^-6 M or less.

In some embodiments, a TFNR2 AFFIMER® polypeptide is derived from wild-type human Stefin A polypeptide having a backbone sequence and wherein one or both of loop 2 [designated (Xaa)n] and loop 4 [designated (Xaa)m] are replaced with alternative loop sequences (Xaa)n and (Xaa)m, to have the general formula (I)

FR1-(Xaa)n-FR2-(Xaa)m-FR3 (I)

Or

FR1 is a polypeptide sequence represented by MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TGETYGKLEA VQYKTQVX (SEQ ID NO: 1) or 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 to the amino acid sequence of SEQ ID NO: 1, wherein X is any number of independently selected amino acids, more suitably three or less independently selected amino acids, or more suitably X is V; and/or

FR2 is a polypeptide sequence comprising the amino acid sequence of GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2) or 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 amino acid sequence of SEQ ID NO: 2;

FR3 is a polypeptide sequence comprising the amino acid sequence of EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3) or 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 amino acid sequence of SEQ ID NO: 3; and

Xaa, individually for each occurrence, is an amino acid residue; and

n and m are each, independently, an integer from 3 to 20.

In some embodiments, FR1 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology to SEQ ID NO: 1. In some embodiments, FR1 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% identity to SEQ ID NO: 1; in some embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology to SEQ ID NO: 2. In some embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% identity to SEQ ID NO: 2; in some embodiments, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% homology to SEQ ID NO: 3. In some embodiments, FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95%, or even 98% identity to SEQ ID NO: 3.

In some embodiments, the TNFR2 AFFIMER® polypeptide has an amino acid sequence represented in General Formula (II):

(SEQ ID NO: 4)

Or

Xaa, individually for each occurrence, is an amino acid residue, and

n and m are each, independently, an integer from 3 to 20.

In some embodiments, the TNFR2 AFFIMER® polypeptide comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence of:

MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5), where

Xaa, individually for each occurrence, is an amino acid residue, and

n and m are each, independently, an integer from 3 to 20.

In some embodiments, 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 Gly, Ala, Val, Ser or Thr, more preferably Gly or Ser; Xaa3 is Arg, Lys, Asn, Gln, Ser, Thr, more preferably Arg, Lys, Asn or Gln, and even more preferably Lys or Asn; Xaa4 is Gly, Ala, Val, Ser or Thr, more preferably Gly or Ser; Xaa5 is Ala, Val, Ile, Leu, Gly or Pro, more preferably Ile, Leu or Pro, and even more preferably Leu or Pro; Xaa6 is Gly, Ala, Val, Asp or Glu, more preferably Ala, Val, Asp or Glu, and even more preferably Ala or Glu; and Xaa7 is Ala, Val, Ile, Leu, Arg or Lys, more preferably Ile, Leu or Arg, and even more preferably Leu or Arg.

In some embodiments, n is 3 to 15, 3 to 12, 3 to 9, 3 to 7, 5 to 7, 5 to 9, 5 to 12, 5 to 15, 7 to 12, or 7 to 9.

In some embodiments, m is 3 to 15, 3 to 12, 3 to 9, 3 to 7, 5 to 7, 5 to 9, 5 to 12, 5 to 15, 7 to 12, or 7 to 9.

In some embodiments, Xaa, independently for each occurrence, is an amino acid that can be added to a polypeptide by recombinant expression in a prokaryotic or eukaryotic cell, and even more preferably one of the 20 naturally occurring amino acids.

In some embodiments of the above sequences and formulas, (Xaa)n is an amino acid sequence selected from SEQ ID NOs: 6 to 102, or is an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% homology to a sequence selected from SEQ ID NOs: 6 to 102. In some embodiments, (Xaa)n is an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% identity to a sequence selected from SEQ ID NOs: 6 to 102.

In some embodiments of the above sequences and formulas, (Xaa)m is an amino acid sequence selected from SEQ ID NOs: 103 to 199, or is an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% homology to a sequence selected from SEQ ID NOs: 103 to 199. In some embodiments, (Xaa)m is an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% identity to a sequence selected from SEQ ID NOs: 103 to 199.

In some embodiments, the TNFR2 AFFIMER® polypeptide has an amino acid sequence selected from SEQ ID NO: 200 to 296, wherein the sequence optionally excludes one or more of the twenty-one carboxy terminal residues. In some embodiments, the TNFR2 AFFIMER® polypeptide has an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or even 98% identity to a sequence selected from SEQ ID NO: 200 to 296, wherein the sequence optionally excludes one or more of the twenty-one carboxy terminal residues.

Table 1. Polypeptide sequences of TNFR2 AFFIMER®. The C-terminal 21 amino acids are additional sequences added for cloning and testing purposes: 3xAla linker, ten amino acid Myc tag, 2xAla linker, and 6xHis tag, all of which are optional and not necessary for the present invention. Clone Name Loop 2 SEQ ID NO of Loop 2: Loop 4 SEQ ID NO of Loop 4: Protein sequence Protein SEQ ID NO: CLONE 001 GQDQWGNYA 6 PDIPHNHWH 103 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVGQDQWGNYAGTNYYIKVRAGDNKYMHLKVFKSLPDIPHNHWHEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 200 CLONE 003 GEDKWLNYA 7 ESEDHGQNT 104 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVGEDKWLNYAGTNYYIKVRAGDNKYMHLKVFKSLESEDHGQNTEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 201 CLONE 004 RNLGNIYQW 8 SLNARYALD 105 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVRNLGNIYQWGTNYYIKVRAGDNKYMHLKVFKSLSLNARYALDELVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 202 CLONE 005 KLSYDLPPV 9 SLNKESIIV 106 MIPGGLSEAKPATPEIQEI VDKVKPQLEEKTGETYGKLEAVQYKTQVVKLSYDLPPVGTNYYIKVRAGDNKYMHLKVFKSLSLNKESIIVEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 203 CLONE 006 VYWDILVEL 10 ISGGYSAQT 107 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVVYWDILVELGTNYYIKVRAGDNKYMHLKVFKSLISGGYSAQTEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 204 CLONE 007 GLDIWGNNA 11 ADTARTEEA 108 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVGLDIWGNNAGTNYYIKVRAGDNKYMHLKVFKSLADTARTEEAEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 205 CLONE 008 FVLDTNLWQ 12 RGFGNVKKV 109 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVFVLDTNLWQGTNYYIKVRAGDNKYMHLKVFKSLRGFGNVKKVEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 206 CLONE 009 RLFHIWRWW 13 IHIYTDSIQ 110 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVRLFHIWRWWGTNYYIKVRAGDNKYMHLKVFKSLIHIYTDSIQEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 207 CLONE 010 QEPGVWWWW 14 REGDFDYDI 111 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVQEPGVWWWWGTNYYIKVRAGDNKYMHLKVFKSLREGDFDYDIEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 208 CLONE 011 LNWNDIYEH 15 SLTDQYKIS 112 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVLNWNDIYEHGTNYYIKVRAGDNKYMHLKVFKSLSLTDQYKISEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 209 CLONE 012 YWGVQGALD 16 NWNHEFDLV 113 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVYWGVQGALDGTNYYIKVRAGDNKYMHLKVFKSLNWNHEFDLVEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 210 CLONE 013 AHDYDGYKW 17 LLSNRASEH 114 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVAHDYDGYKWGTNYYIKVRAGDNKYMHLKVFKSLLLSNRASEHEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 211 CLONE 014 WQDYQWRGQ 18 YQHIDPPYS 115 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVWQDYQWRGQGTNYYIKVRAGDNKYMHLKVFKSLYQHIDPPYSEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 212 CLONE 015 EWEVGDVWG 19 WWVRGFFEP 116 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVEWEVGDVWGGTNYYIKVRAGDNKYMHLKVFKSLWWVRGFFEPEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 213 CLONE 016 AETAKWFFY 20 VSYIEGAWK 117 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAAETAKWFFYSTNYYIKVRAGDNKYMHLKVFNGPVSYIEGAWKADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 214 CLONE 021 GELGEWQWY 21 QPPDWGWTF 118 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAGELGEWQWYSTNYYIKVRAGDNKYMHLKVFNGPQPPDWGWTFADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 215 CLONE 022 GHTDHYWYL 22 THERETGFT 119 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAGHTDHYWYLSTNYYIKVRAGDNKYMHLKVFNGPTHERETGFTADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 216 CLONE 023 GHTDQFWLT 23 IWQGDYDRY 120 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAGHTDQFWLTSTNYYIKVRAGDNKYMHLKVFNGPIWQGDYDRYADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 217 CLONE 026 LEIPSAIIY 24 YFWGDQEHW 121 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLALEIPSAIIYSTNYYIKVRAGDNKYMHLKVFNGPYFWGDQEHWADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 218 CLONE 027 NQLGNGWDA 25 YQAKPNWRH 122 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLANQLGNGWDASTNYYIKVRAGDNKYMHLKVFNGPYQAKPNWRHADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 219 CLONE 032 DSNGVSAVI 26 TWNIKPQWP 123 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLADSNGVSAVISTNYYIKVRAGDNKYMHLKVFNGPTWNIKPQWPADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 220 CLONE 035 KWHQKQLPL 27 SLNNNFKIQ 124 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVKWHQKQLPLGTNYYIKVRAGDNKYMHLKVFKSLSLNNNFKIQEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 221 CLONE 036 HDEHISHPV 28 DWWANINGT 125 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVHDEHISHPVGTNYYIKVRAGDNKYMHLKVFKSLDWWANINGTEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 222 CLONE 037 AVNDFEPSL 29 FQLPDYSVN 126 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVAVNDFEPSLGTNYYIKVRAGDNKYMHLKVFKSLFQLPDYSVNEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 223 CLONE 038 QHAFDGYAW 30 LQSAIKGIF 127 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVQHAFDGYAWGTNYYIKVRAGDNKYMHLKVFKSLLQSAIKGIFEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 224 CLONE 042 VGDETFDAQ 31 AIQVIGYGT 128 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVVGDETFDAQGTNYYIKVRAGDNKYMHLKVFKSLAIQVIGYGTEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 225 CLONE 043 HYHERHRIQ 32 SLDKQYNII 129 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVHYHERHRIQGTNYYIKVRAGDNKYMHLKVFKSLSLDKQYNIIEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 226 CLONE 044 LFPEAVYWV 33 IWNDNAAQH 130 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVLFPEAVYWVGTNYYIKVRAGDNKYMHLKVFKSLIWNDNAAQHEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 227 CLONE 045 VNDEGDHIA 34 YNVTGYVHW 131 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVVNDEGDHIAGTNYYIKVRAGDNKYMHLKVFKSLYNVTGYVHWEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 228 CLONE 049 YDTDWQQDA 35 AYWYDVQGW 132 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVYDTDWQQDAGTNYYIKVRAGDNKYMHLKVFKSLAYWYDVQGWEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 229 CLONE 051 WLVHAKTWL 36 LEPQYNVAN 133 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVWLVHAKTWLGTNYYIKVRAGDNKYMHLKVFKSLLEPQYNVANEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 230 CLONE 052 KRELVHYWW 37 DEDVSYAYE 134 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVKRELVHYWWGTNYYIKVRAGDNKYMHLKVFKSLDEDVSYAYEEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 231 CLONE 056 EWNQHAVWL 38 LEHPYDQYG 135 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVEWNQHAVWLGTNYYIKVRAGDNKYMHLKVFKSLLEHPYDQYGEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 232 CLONE 057 KGWGEKTWE 39 QLDGWAEYFG 136 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVKGWGEKTWEGTNYYIKVRAGDNKYMHLKVFKSLQLDGWAEYFGEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 233 CLONE 058 EWGFGLSIS 40 EYQRYWFEF 137 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVEWGFGLSISGTNYYIKVRAGDNKYMHLKVFKSLEYQRYWFEFEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 234 CLONE 059 FNVQDIWNH 41 ALNHQYTIQ 138 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVFNVQDIWNHGTNYYIKVRAGDNKYMHLKVFKSLALNHQYTIQEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 235 CLONE 060 TESFEQLGY 42 STGGFSWKV 139 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLATESFEQLGYSTNYYIKVRAGDNKYMHLKVFNGPSTGGFSWKVADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 236 CLONE 063 EETGYHVSY 43 SLNRQYAVI 140 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVEETGYHVSYGTNYYIKVRAGDNKYMHLKVFKSLSLNRQYAVIEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 237 CLONE 064 HQPEVKHAW 44 PINPWNLLP 141 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVHQPEVKHAWGTNYYIKVRAGDNKYMHLKVFKSLPINPWNLLPEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 238 CLONE 065 EQWDKSVDV 45 RQYYDEET 142 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVEQWDKSVDVGTNYYIKVRAGDNKYMHLKVFKSLRQYYDEETEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 239 CLONE 067 VEGHEHWDW 46 DDWQQQTEV 143 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAVEGHEHWDWSTNYYIKVRAGDNKYMHLKVFNGPDDWQQQTEVADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 240 CLONE 069 GPFQLQLED 47 WELVNAYFP 144 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAGPFQLQLEDSTNYYIKVRAGDNKYMHLKVFNGPWELVNAYFPADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 241 CLONE 071 EPYNRLSVH 48 SLNKDYFIE 145 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVEPYNRLSVHGTNYYIKVRAGDNKYMHLKVFKSLNKDYFIEEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 242 CLONE 072 KSVHDVQQH 49 TLTRGYAVE 146 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLKSVHDVQQHGTNYYIKVRAGDNKYMHLKVFKSLTLTRGYAVEEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 243 CLONE 073 DDYDDLHWW 50 TSVEYWWDA 147 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVDDYDDLHWWGTNYYIKVRAGDNKYMHLKVFKSLTSVEYWWDAEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 244 CLONE 076 AWHDDDSLQ 51 NWPEVDIRW 148 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAAWHDDDSLQSTNYYIKVRAGDNKYMHLKVFNGPNWPEVDIRWADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 245 CLONE 077 TEEQVLGIS 52 TLTRHYLIH 149 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVTEEQVLGISGTNYYIKVRAGDNKYMHLKVFKSLTLTRHYLIHEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 246 CLONE 078 SIYDDEVND 53 FLNNAYQIF 150 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVSIYDDEVNDGTNYYIKVRADDNKYMHLKVFKSLFLNNAYQIFEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 247 CLONE 079 RWWDEEHPI 54 SLDSLYHKV 151 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVRWWDEEHPIGTNYYIKVRAGDNKYMHLKVFKSLSLDSLYHKVEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 248 CLONE 080 WWVEWEPEI 55 FQDNDYHIF 152 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVWWVEWEPEIGTNYYIKVRAGDNKYMHLKVFKSLFQDNDYHIFEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 249 CLONE 081 FWEHTWVEQ 56 SLSEEYTII 153 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVFWEHTWVEQGTNYYIKVRAGDNKYMHLKVFKSLSLSEEYTIIEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 250 CLONE 085 HVWSEKHHV 57 SLTAKYEIL 154 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVHVWSEKHHVGTNYYIKVRAGDNKYMHLKVFKSLSLTAKYEILEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 251 CLONE 087 QHQKSLHAI 58 NLNRDYVII 155 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVQHQKSLHAIGTNYYIKVRAGDNKYMHLKVFKSLNLNRDYVIIEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 252 CLONE 088 YFHYLGYEW 59 GPPWQYQKG 156 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVYFHYLGYEWGTNYYIKVRAGDNKYMHLKVFKSLGPPWQYQKGEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 253 CLONE 089 YHDVDPTTT 60 NKLPTGYGV 157 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVYHDVDPTTTGTNYYIKVRAGDNKYMHLKVFKSLNKLPTGYGVEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 254 CLONE 093 WIDDEQFPV 61 TLDFDYVIQ 158 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVWIDDEQFPVGTNYYIKVRAGDNKYMHLKVFKSLTLDFDYVIQEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 255 CLONE 095 IKQYPAELAWW 62 FTDKNPASV 159 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVIKQYPAELAWWGTNYYIKVRAGDNKYMHLKVFKSLFTDKNPASVEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 256 CLONE 096 DAHLDIHWY 63 DRIEYWFGN 160 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVDAHLDIHWYGTNYYIKVRAGDNKYMHLKVFKSLDRIEYWFGNEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 257 CLONE 097 WDNPKELWE 64 KEHPYDEKF 161 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVWDNPKELWEGTNYYIKVRAGDNKYMHLKVFKSLKEHPYDEKFEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 258 CLONE 100 QSWDKHYQLRE 65 EVPHHAHGV 162 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAQSWDKHYQLRESTNYYIKVRAGDNKYMHLKVFNGPEVPHHAHGVADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 259 CLONE 102 WAEVAWVED 66 WETYDSYGF 163 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVWAEVAWVEDGTNYYIKVRAGDNKYMHLKVFKSLWETYDSYGFEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 260 CLONE 103 DYHWLWVVG 67 SLTREYTTQ 164 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVDYHWLWVVGGTNYYIKVRAGDNKYMHLKVFKSLSLTREYTTQEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 261 CLONE 105 KWSQNSVTQ 68 TLERTFRVV 165 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVKWSQNSVTQGTNYYIKVRAGDNKYMHLKVFKSLTLERTFRVVEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 262 CLONE 108 YWSDLWFVY 69 WKINLFAED 166 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAYWSDLWFVYSTNYYIKVRAGDNKYMHLKVFNGPWKINLFAEDADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHHHAAAEQKLISEEDLAAHHHHHH 263 CLONE 109 DQIRFQAHH 70 SLDKLYQVI 167 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVDQIRFQAHHGTNYYIKVRAGDNKYMHLKVFKSLSLDKLYQVIEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 264 CLONE 110 YPGEGDFAV 71 ALNRDYYII 168 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVYPGEGDFAVGTNYYIKVRAGDNKYMHLKVFKSLALNRDYYIIEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 265 CLONE 111 LWLESLINQ 72 NLSRNYQLW 169 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVLWLESLINQGTNYYIKVRAGNNKYMHLKVFKSLNLSRNYQLWEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 266 CLONE 112 YFRKHVHPV 73 SIDKYYQII 170 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVYFRKHVHPVGTNYYIKVRAGDNKYMHLKVFKSLSIDKYYQIIEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 267 CLONE 113 DEDEYLLVS 74 FLDYFYNIG 171 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVDEDEYLLVSGTNYYIKVRAGDNKYMHLKVFKSLFLDYFYNIGEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 268 CLONE 115 DHYAEHWDD 75 YKADIQWKH 172 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLADHYAEHWDDSTNYYIKVRAGDNKYMHLKVFNGPYKADIQWKHADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 269 CLONE 116 WPQGQDKTH 76 DDGSLEWLHG 173 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAWPQGQDKTHSTNYYIKVRAGDNKYMHLKVFNGPDDGSLEWLHGADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 270 CLONE 117 WLLDGLLWK 77 LEHQYDEFL 174 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVWLLDGLLWKGTNYYIKVRAGDNKYMHLKVFKSLLEHQYDEFLEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 271 CLONE 118 KWQQLGPIQ 78 ALAKDYTLI 175 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVKWQQLGPIQGTNYYIKVRAGDNKYMHLKVFKSLALAKDYTLIEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 272 CLONE 119 KDHQASHTL 79 SLSGEYIVI 176 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVKDHQASHTLGTNYYIKVRAGDNKYMHLKVFKSLSLSGEYIVIEDDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 273 CLONE 120 HYLYSLPVL 80 QDVKYWVIE 177 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVHYLYSLPVLGTNYYIKVRAGDNKYMHLKVFKSLQDVKYWVIEEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 274 CLONE 124 YWAGKQLVL 81 NLALQFDTV 178 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAYWAGKQLVLSTNYYIKVRAGDNKYMHLKVFNGPNLALQFDTVADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 275 CLONE 125 FWIEPGYIY 82 IHLDPELTW 179 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAFWIEPGYIYSTNYYIKVRAGDNKYMHLKVFNGPIHLDPELTWADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 276 CLONE 129 LEGAQYYIS 83 FEQLYFSPK 180 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLALEGAQYYISSTNYYIKVRAGDNKYMHLKVFNGPFEQLYFSPKADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 277 CLONE 130 DWAGLSFNE 84 IFPDVWEWG 181 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLADWAGLSFNESTNYYIKVRAGDNKYMHLKVFNGPIFPDVWEWGADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 278 CLONE 133 ADHGQGIIW 85 AQLWVSPDW 182 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAADHGQGIIWSTNYYIKVRAGDNKYMHLKVFNGPAQLWVSPDWADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 279 CLONE 139 HQLGDYVRI 86 WLIILINND 183 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLAHQLGDYVRISTNYYIKVRAGDNKYMHLKVFNGPWLIILINNDADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 280 CLONE 141 NEEPWGEWY 87 WDPYTQLIS 184 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLANEEPWGEWYSTNYYIKVRAGDNKYMHLKVFNGPWDPYTQLISADRVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 281 CLONE 145 VYEKWLAVV 88 IPNNDIILA 185 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVVYEKWLAVVGTNYYIKVRAGDNKYMHLKVFKSLIPNNDIILAEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 282 CLONE 157 DHRVPDIGI 89 GPSLYFNHF 186 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVDHRVPDIGIGTNYYIKVRAGDNKYMHLKVFKSLGPSLYFNHFEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 283 CLONE 160 VSLHYVSAV 90 SEWAFFFND 187 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVVSLHYVSAVGTNYYIKVRAGDNKYMHLKVFKSLSEWAFFFNDELVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 284 CLONE 162 VWRVSWAVE 91 WGVYEESKW 188 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVVWRVSWAVEGTNYYIKVRAGDNKYMHLKVFKSLWGVYEESKWEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 285 CLONE 163 PRVEYVNNE 92 GGPNGWLYA 189 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVPRVEYVNNEGTNYYIKVRAGDNKYMHLKVFKSLGGPNGWLYAEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 286 CLONE 164 IDRQSFSVV 93 WFFPPKYHP 190 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVIDRQSFSVVGTNYYIKVRAGDNKYMHLKVFKSLWFFPPKYHPEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 287 CLONE 165 VDLQYVQVI 94 FNDANQIHW 191 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVVDLQYVQVIGTNYYIKVRAGDNKYMHLKVFKSLFNDANQIHWEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 288 CLONE 166 VGWEGDIEE 95 SDQWPYVAI 192 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVVGWEGDIEEGTNYYIKVRAGDNKYMHLKVFKSLSDQWPYVAIEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 289 CLONE 171 LQWHGYLNG 96 FPIQEGIVH 193 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVLQWHGYLNGGTNYYIKVRAGDNKYMHLKVFKSLFPIQEGIVHEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 290 CLONE 172 WAEQAPYVHHF 97 LTFPVREAW 194 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVWAEQAPYVHHFGTNYYIKVRAGDNKYMHLKVFKSLLTPFPVREAWEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 291 CLONE 174 AHGQYLIVW 98 SNEVHAGDG 195 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVAHGQYLIVWGTNYYIKVRAGDNKYMHLKVFKSLSNEVHAGDGEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 292 CLONE 175 IDPIPFAVY 99 DYFKFNHEH 196 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVIDPIPFAVYGTNYYIKVRAGDNKYMHLKVFKSLDYFKFNHEHEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 293 CLONE 176 WDYWRELHA 100 RYLGQAADD 197 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVWDYWRELHAGTNYYIKVRAGDNKYMHLKVFKSLRYLGQAADDELVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 294 CLONE 177 YHLEWLQQW 101 YFDSLGWDP 198 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVYHLEWLQQWGTNYYIKVRAGDNKYMHLKVFKSLYFDSLGWDPEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 295 CLONE 179 PYERWLVST 102 VDIANLFEY 199 MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVVPYERWLVSTGTNYYIKVRAGDNKYMHLKVFKSLVDIANLFEYEDLVLTGYQVDKNKDDELTGF AAAEQKLISEEDLAAHHHHHH 296

In some embodiments, the TNFR2 AFFIMER® polypeptide has an amino acid sequence that is encoded by a nucleic acid having a coding sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or even 98% identical to a sequence selected from SEQ ID NO: 297 to 393, optionally excluding the nucleotides encoding the twenty-one carboxy-terminal amino acids. In some embodiments, the TNFR2 AFFIMER® polypeptide has an amino acid sequence that is encoded by a nucleic acid that has a coding sequence that hybridizes to a sequence selected from SEQ ID NO: 297 to 393 optionally excluding nucleotides encoding the twenty-one carboxy-terminal amino acids under stringent conditions (such as in the presence of 6X sodium chloride/sodium citrate (SSC) at 45°C followed by washing in 0.2X SSC at 65°C.

Table 2: Nucleic Acid Sequences of the Present Invention. The 5' 63 nucleotides encoding the C-terminal 21 amino acids are additional sequences added for cloning and testing purposes: 3xAla linker, ten amino acid Myc tag, 2xAla linker, and 6xHis tag, all of which are optional and not required for the present invention. Clone Name DNA sequence DNA SEQ ID NO: CLONE 001 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGGTCAGGATCAGTGGGGTAACTACGCAGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGCCAGATATCCCACATAACCATTGGCATGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 297 CLONE 003 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGGTGAAGATAAATGGCTGAACTACGCAGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGAATCTGAAGATCATGGTCAGAACACCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 298 CLONE 004 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCGTAACCTGGGTAACATCTACCAGTGGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGAACGCAAGATACGCACTGGATGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 299 CLONE 005 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCAAACTGTCTTACGATCTGCCACCAGTTGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGAACAAAGAATCTATCATCGTTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 300 CLONE 006 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGTTTACTGGGATATCCTGGTTGAACTGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGATCTCTGGTGGTTACTCTGCACAGACCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 301 CLONE 007 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGGTCTGGATATCTGGGGTAACAACGCAGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGCAGATACCGCAAGAACCGAAGAAGCAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 302 CLONE 008 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTTCGTTCTGGATACCAACCTGTGGCAGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGAGAGGTTTCGGTAACGTTAAAAAAGTTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 303 CLONE 009 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCGTCTGTTCCATATCTGGCGTTGGTGGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGATCCATATCTACACCGATTCTATCCAGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 304 CLONE 010 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCAGGAACCAGGTGTTTGGTGGTGGTGGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGAGAGAAGGTGATTTCGATTACGATATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 305 CLONE 011 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCTGAACTGGAACGATATCTACGAACATGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGACCGATCAGTACAAAATCTCTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 306 CLONE 012 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTACTGGGGTGTTCAGGGTGCACTGGATGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGAACTGGAACCATGAATTCGATCTGGTTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 307 CLONE 013 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGCACATGATTACGATGGTTACAAATGGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGCTGCTGTCTAACAGAGCATCTGAACATGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 308 CLONE 014 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTGGCAGGATTACCAGTGGCGTGGTCAGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTACCAGCATATCGATCCACCATACTCTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 309 CLONE 015 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGAATGGGAAGTTGGTGATGTTTGGGGTGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTGGTGGGTTAGAGGTTTCTTCGAACCAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 310 CLONE 016 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGCAGAAACCGCAAAATGGTTTTTTTTACTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGGTTTCTTACATCGAAGGTGCATGGAAAGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 311 CLONE 021 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGGTGAACTGGGTGAATGGCAGTGGTACTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGCAGCCACCAGATTGGGGTTGGACCTTTGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 312 CLONE 022 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGGTCATACCGATCATTACTGGTACCTGTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGACCCATGAACGTGAAACCGGTTTTACCGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 313 CLONE 023 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGGTCATACCGATCAGTTTTGGCTGACCTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGATCTGGCAGGGTGATTACGATCGTTACGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 314 CLONE 026 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCACTGGAAATCCCATCTGCAATCATCTACTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGTACTTTTGGGGTGATCAGGAACATTGGGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 315 CLONE 027 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAAACCAGCTGGGTAACGGTTGGGATGCATCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGTACCAGGCAAAACCAAAACTGGCGTCATGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 316 CLONE 032 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGATTCTAACGGTTTCTGCAGTTATCTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGACCTGGAACATCAAACCACAGTGGCCAGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 317 CLONE 035 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCAAATGGCATCAGAAACAGCTGCCACTGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGAACAACAACAACTTCAAAATCCAGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 318 CLONE 036 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCATGATGAACATATCTCTCATCCAGTTGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGATTGGTGGGCAAACATCAACGGTACCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 319 CLONE 037 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGCAGTTAACGATTTCGAACCATCTCTGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTTCCAGCTGCCAGATTACTCTGTTAACGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 320 CLONE 038 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCAGCATGCATTCGATGGTTACGCATGGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGCTGCAGTCTGCAATCAAAGGTATCTTCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 321 CLONE 042 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGTTGGTGATGAAACCTTCGATGCACAGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGCAATCCAGGTTATCGGTTACGGTACCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 322 CLONE 043 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCATTACCATGAACGTCATCGTATCCAGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGGATAAACAGTACAACATCATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 323 CLONE 044 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCTGTTCCCAGAAGCAGTTTACTGGGTTGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGATCTGGAACGATAACGCAGCACAGCATGAAGATTTGGTGCTGACGGGCTACCAAGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 324 CLONE 045 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGTTAACGATGAAGGTGATCATATCGCAGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTACAACGTTACCGGTTACGTTCATTGGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 325 CLONE 049 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTACGATACCGATTGGCAGCAGGATGCAGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGCATACTGGTACGATGTTCAGGGTTGGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 326 CLONE 051 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTGGCTGGTTCATGCAAAAACCTGGCTGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGCTGGAACCACAGTACAACGTTGCAAACGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 327 CLONE 052 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCAAACGTGAACTGGTTCATTACTGGTGGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGATGAAGATGTTTCTTACGCATACGAAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 328 CLONE 056 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGAATGGAACCAGCATGCAGTTTGGCTGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGCTGGAACATCCATACGATCAGTACGGTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 329 CLONE 057 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCAAAGGTTGGGGTGAAAAAACCTGGGAAGGTACGAACTACTACTACTACATCAAGGTTCGTG CGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGCAGCTGGATGGTTGGGCAGAATACTTCGGTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 330 CLONE 058 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGAATGGGGTTTCGGTCTGTCTATCTCTGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGAATACCAGAGATACTGGTTCGAATTCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 331 CLONE 059 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTTCAACGTTCAGGATATCTGGAACCATGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGCACTGAACCATCAGTACACCATCCAGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 332 CLONE 060 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAACCGAATCTTTTGAACAGCTGGGTTACTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGTCTACCGGTGGTTTTCTTGGAAAGTTGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 333 CLONE 063 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGAAGAAACCGGTTACCATGTTTCTTACGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGAACAGACAGTACGCAGTTATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 334 CLONE 064 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCATCAGCCAGAAGTTAAACATGCATGGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGCCAATCAACCCATGGAACCTGCTGCCAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 335 CLONE 065 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGAACAGTGGGATAAATCTGTTGATGTTGGTACGAACTACTACATCAAGGTTC GTGCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGAGACAGTACTACGATGAAGAAACCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 336 CLONE 067 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGTTGAAGGTCATGAACATTGGGATTGGTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGGATGATTGGCAGCAGACCGAAGTTGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 337 CLONE 069 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGGTCCATTTCAGCTGCAGCTGGAAGATTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGTGGGAACTGGTTAACGCATACTTTCCAGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 338 CLONE 071 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGAACCATACAACCGTCTGTCTGTTCATGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGAACAAAGATTACTTCATCGAAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 339 CLONE 072 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCCTCAAATCTGTTCATGATGTTCAGCAGCATGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGACCCTGACCAGGTTACGCAGTTGAAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 340 CLONE 073 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGATGATTACGATGATCTGCATTGGTGGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGACCTCTGTTGAATACTGGTGGGATGCAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 341 CLONE 076 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGCATGGCATGATGATTCTCTGCAGTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGAACTGGCCAGAAGTTGATATCCGTTGGGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 342 CLONE 077 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCACCGAAGAACAGGTTCTGGGTATCTCTGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGACCCTGACCAGACATTACCTGATCCATGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 343 CLONE 078 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTCTATCTACGATGATGAAGTTAACGATGGTACGAACTACTACTACATCAAGGTTCGT GCGGATGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTTCCTGAACAACGCATACCAGATCTTCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 344 CLONE 079 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCGTCGGTGGATGAAGAAACATCCAATCGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGGATTCTCTGTACCATAAAGTTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 345 CLONE 080 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTGGTGGGTTGAATGGGAACCAGAAATCGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTTCCAGGATAACGATTACCATATCTTCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 346 CLONE 081 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTTCTGGGAACATACCTGGGTTGAACAGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGTCTGAAGAATACACCATCATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 347 CLONE 085 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCATGTTTGGTCTGAAAACATCATGTTGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGACCGCAAAATACGAAATCCTGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 348 CLONE 087 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCAGCATCAGAAATCTCTGCATGCAATCGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGAACCTGAACAGAGATTACGTTATCATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 349 CLONE 088 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTACTTCCATTACCTGGGTTACGAATGGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGGTCCACCATGGCAGTACCAGAAAGGTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 350 CLONE 089 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTACCATGATGTTGATCCAACCACCACCGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGAACAAACTGCCAACCGGTTACGGTGTTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 351 CLONE 093 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTGGATCGATGATGAACAGTTCCCAGTTGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGACCCTGGATTTCGATTACGTTATCCAGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 352 CLONE 095 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCATCAAACAGTACCCAGCTGAACTGGCATGGTGGGGTACGAACTACTACTACATCAAGGTT CGTCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTTCACCGATAAAAACCCAGCATCTGTTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 353 CLONE 096 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGATGCACATCTGGATATCCATTGGTACGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGATAGAATCGAATACTGGTTCGGTAACGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 354 CLONE 097 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTGGGATAACCCAAAAGAACTGTGGGAAGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGAAAGAACATCCATACGATGAAAAATTCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 355 CLONE 100 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCACAGTCTTGGGATAAACATTACCAGCTGCGTGAATCCACCAACTATTACATTAAGG TTCGTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGGAAGTTCCACATCATGCACATGGTGTTGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 356 CLONE 102 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTGGGCAGAAGTTGCATGGGTTGAAGATGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTGGGAAACCTACGATTCTTACGGTTTCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 357 CLONE 103 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGATTACCATTGGCTGTGGGTTGTTGGTGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGACCAGAGAATACACCACCACCAGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 358 CLONE 105 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCAAATGGTCTCAGAACTCTGTTACCCAGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGACCCTGGAAAGAACCTTCAGAGTTGTTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 359 CLONE 108 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCATACTGGTCTGATCTGTGGTTTGTTTACTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGTGGAAAATCAACCTGTTTGCAGAAGATGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 360 CLONE 109 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGATCAGATCCGTTTCCAGGCACATCATGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGGATAAACTGTACCAGGTTATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 361 CLONE 110 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTACCCAGGTGAAGGTGATTTCGCAGTTGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGCACTGAACAGAGATTACTACATCATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 362 CLONE 111 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCTGTGGCTGGAATCTCTGATCAACCAGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTAACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGAACCTGTCTAGAAACTACCAGCTGTGGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 363 CLONE 112 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTACTTCCGTAAACATGTTCATCCAGTTGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTATCGATAAATACTACCAGATCATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 364 CLONE 113 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGATGAAGATGAATACCTGCTGGTTTCTGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTTCCTGGATTACTTCTACAACATCGGTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 365 CLONE 115 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGATCATTACGCAGAACATTGGGATGATTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGTACAAAGCAGATATCCAGTGGAAACATGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 366 CLONE 116 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCATGGCCACAGGGTCAGGATAAAACCCATTCCACCAACTATTACATTAAGGTTCGT GCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGGATGATGGTTCTCTGGAATGGCTGCATGGTGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 367 CLONE 117 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTGGCTGCTGGATGGTCTGCTGTGGAAAGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGCTGGAACATCAGTACGATGAATTCCTGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 368 CLONE 118 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCAAATGGCAGCAGCTGGGTCCAATCCAGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGCACTGGCAAAAGATTACACCCTGATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 369 CLONE 119 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCAAAGATCATCAGGCATCTCATACCCTGGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTCTGTCTGGTGAATACATCGTTATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 370 CLONE 120 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCATTACCTGTACTCTTCGCCAGTTCTGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGCAGGATGTTAAATACTGGGTTATCGAAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 371 CLONE 124 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCATACTGGGCAGGTAAACAGCTGGTTCTGTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGAACCTGGCACTGCAGTTTGATACCGTTGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 372 CLONE 125 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCATTTTGGATCGAACCAGGTTACATCTACTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGATCCATCTGGATCCAGAACTGACCTGGGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 373 CLONE 129 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCACTGGAAGGTGCACAGTACTACATCTCTTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGTTTGAACAGCTGTACTTTTCTCCAAAAGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 374 CLONE 130 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGATTGGGCAGGTCTGTCTTTTAACGAATCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGATCTTTCCAGATGTTTGGGAATGGGGTGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 375 CLONE 133 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAGCAGATCATGGTCAGGGTATCATCTGGTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGGCACAGCTGTGGGTTTCTCCAGATTGGGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 376 CLONE 139 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCACATCAGCTGGGTGATTACGTTCGTATCTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGTGGCTGATCATCCTGATCAACAACGATGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 377 CLONE 141 ATGATCCCGAGGGGACTGTTCCGAGGCCAAGCCTGCCACCCCTGAGATCCAGGAAATTGTCGACAAAGTCAAGCCGCAGCTGGAAGAGAAAACGGGCGAAACCTACGGTAAGCTGGAAGCGGTCCAGTACAAAACCCAAGTGCTAGCAAACGAAGAACCATGGGGTGAATGGTACTCCACCAACTATTACATTAAGGTTC GTGCCGGTGACAATAAGTATATGCACCTGAAAGTGTTCAACGGCCCGTGGGATCCATACACCCAGCTGATCTCTGCGGACCGTGTTCTGACCGGTTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 378 CLONE 145 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGTTTACGAAAAAATGGCTGGCAGTTGTTGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGATCCCAAACAACGATATCATCCTGGCAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 379 CLONE 157 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGATCATCGTGTTCCAGATATCGGTATCGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGGTCCATCTCTGTACTTCAACCATTTCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 380 CLONE 160 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGTTTCTCTGCATTACGTTTCTGCAGTTGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTGAATGGGCATTCTTCTTCAACGATGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 381 CLONE 162 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGTTTGGCGTGTTTCTTGGGCAGTTGAAGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTGGGGTGTTTACGAAGAATCTAAATGGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 382 CLONE 163 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCCACGTGTTGAATACGTTAACAACGAAGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGGTGGTCCAAACGGTTGGCTGTACGCAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 383 CLONE 164 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCATCGATCGTCAGTCTTTCTCTGTTGTTGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTGGTTCTTCCCACCAAAATACCATCCAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 384 CLONE 165 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGTTGATCTGCAGTACGTTCAGGTTATCGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTTCAACGATGCAAACCAGATCCATTGGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 385 CLONE 166 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGTTGGTTGGGAAGGTGATATCGAAGAAGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTGATCAGTGGCCATACGTTGCAATCGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 386 CLONE 171 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCTGCAGTGGCATGGTTACCTGAACGGTGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTTCCCAATCCAGGAAGGTATCGTTCATGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 387 CLONE 172 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTGGGCAGAACAGGCACCATACGTTCATCATTTCGGTACGAACTACTACTACATCAAGGTT CGTGCGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGCTGACCTTCCCAGTTAGAGAAGCATGGGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 388 CLONE 174 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCGCACATGGTCAGTACCTGATCGTTTGGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTCTAACGAAGTTCATGCAGGTGATGGTGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 389 CLONE 175 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCATCGATCCAATCCCATTCGCAGTTTACGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGATTACTTCAAATTCAACCATGAACATGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 390 CLONE 176 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTGGGATTACTGGCGGGAACTGCATGCAGGTACGAACTACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGAGATACCTGGGTCAGGCAGCAGATGATGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 391 CLONE 177 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCTACCATCTGGAATGGCTGCAGCAGTGGGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGTACTTCGATTCTCTGGTTGGGATCCAGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 392 CLONE 179 ATGATTCCCGGCGGACTTTCGGAAGCCAAGCCAGCCACTCCGGAGATTCAGGAAATCGTGGACAAGGTCAAACCGCAGCTGGAAGAGAAAACCGGCGAAACCTACGGCAAACTGGAAGCCGTCCAGTATAAGACTCAAGTCGTCCCATACGAACGTTGGCTGGTTTCTACCGGTACGAACTACTACATCAAGGTTCGT GCGGGTGACAACAAGTATATGCACCTGAAAGTGTTTAAGAGCCTGGTTGATATCGCAAACCTGTTCGAATACGAAGATTTGGTGCTGACGGGCTACCAGGTTGACAAGAACAAAGATGACGAGCTGACGGGTTTCGCGGCCGCTGAACAGAAGCTGATCTCCGAAGAAGATCTGGCCGCGCATCACCATCACCACCAC 393

Additionally, minor modifications may also include small deletions or additions - beyond the loop 2 and loop 4 inserts described above - to the Stefin A or Stefin A-derived sequences described herein, such as the addition or deletion of up to 10 amino acids relative to the Stefin A or Stefin A-derived AFFIMER® polypeptide.

In some embodiments, the AFFIMER® agent is a TNFR2 AFFIMER® agent comprising an AFFIMER® polypeptide portion that binds to human TNFR2 as a monomer with a dissociation constant (KD) of about 1 uM 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 some embodiments, the AFFIMER® agent is a TNFR2 AFFIMER® agent comprising an AFFIMER® polypeptide portion that binds to human TNFR2 as a monomer with an off-rate constant (Koff), as measured by the BIACORE™ assay, of about 10 ^-3 s ^-1 (e.g., units in 1/second) or slower; about 10 ^-4 s ^-1 or less; or even about 10 ^-5 s ^-1 or less.

In some embodiments, the AFFIMER® agent is a TNFR2 AFFIMER® agent comprising an AFFIMER® polypeptide portion that binds to human TNFR2 as a monomer with an association constant (Kon), as measured by the BIACORE™ assay, of at least about 103 M-1s-1 or faster; at least about 104 M-1s-1 or faster; at least about 105 M-1s-1 or faster; or even at least about 106 M-1s-1 or faster.

In some embodiments, the AFFIMER® agent is a TNFR2 AFFIMER® agent comprising an AFFIMER® polypeptide portion that binds to human TNFR2 as a monomer with an IC50 in a competitive binding assay with human TNFR2 of 1 uM 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 some embodiments, the AFFIMER® agent has a melting temperature (Tm, e.g., the temperature at which the folded and unfolded states are equally populated) of 65°C or greater, and preferably at least 70°C, 75°C, 80°C, or even 85°C or greater. The melting temperature is a particularly useful indicator of protein stability. The relative proportions of folded and unfolded proteins can be determined by many techniques known to those skilled in the art, including differential scanning calorimetry, UV difference spectroscopy, fluorescence, circular dichroism (CD), and NMR (Pace et al. (1997) "Measuring the conformational stability of a protein" in Protein structure: A practical approach 2: 299-321).

Fusion Proteins - General

In some embodiments, the AFFIMER® polypeptides may further comprise an additional insertion, substitution, and/or deletion that modulates the biological activity of the AFFIMER® polypeptide. For example, the additions, substitutions, and/or deletions may modulate at least one property or activity of the modified AFFIMER® polypeptides. For example, the additions, substitutions, or deletions may modulate affinity for the AFFIMER® polypeptide, e.g., for binding to, and inhibition of, TNFR2, modulate circulating half-life, modulate therapeutic half-life, modulate stability of the AFFIMER® polypeptide, modulate protease cleavage, modulate dose, modulate release or bioavailability, facilitate purification, decrease deamidation, improve shelf life, or improve or alter a particular route of administration. Similarly, AFFIMER® polypeptides may include protease cleavage sequences, reactive groups, antibody binding domains (including, but not limited to, FLAG or poly-His) or other affinity-based sequences (including, but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including, but not limited to, biotin) that enhance the detection, purification or other characteristics of the polypeptide.

In some cases, these additional sequences are added to one end and/or the other of the AFFIMER® polypeptide in the form of a fusion protein. Accordingly, in some aspects of the disclosure, the AFFIMER® agent is a fusion protein having at least one AFFIMER® polypeptide sequence and at least one heterologous polypeptide sequence ("fusion domain" herein). A fusion domain may be selected to confer a desired property, such as secretion from a cell or retention on the cell surface (e.g., for an encoded AFFIMER® polynucleotide), serve as a substrate or other recognition sequence for post-translational modifications, create multimeric structures that aggregate through protein-protein interactions, alter (often to extend) serum half-life, or alter tissue localization or tissue exclusion and other ADME (Absorption, Distribution, Metabolism, and Excretion) properties - by way of example only.

For example, certain fusion domains are particularly useful for the isolation and/or purification of fusion proteins, such as by affinity chromatography. Well-known examples of such fusion domains that facilitate expression or purification include, by way of illustration only, affinity tags such as polyhistidine (e.g., a His6 tag), Strep II tag, streptavidin-binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA-tag, c-Myc-tag, thioredoxin, protein A, and protein G.

In order for the AFFIMER® agent to be secreted, it will typically contain a signal sequence that directs the protein to be transported to the lumen of the endoplasmic reticulum and ultimately to be secreted (or retained on the cell surface if a transmembrane domain or other retention signal on the cell surface). Signal sequences (also called signal peptides or leader sequences) are located at the N-terminus of nascent polypeptides. They target the polypeptide to the endoplasmic reticulum and the proteins are sorted to their destinations, such as to the internal space of an organelle, to an inner membrane, to the outer membrane of the cell, or to the outside of the cell by secretion. Many signal sequences are cleaved from the protein by a signal peptidase after the proteins have been transported to the endoplasmic reticulum. Cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is dependent on the amino acid residues in the signal sequence.

In some embodiments, the signal peptide is about 5 to about 40 amino acids in length (such as about 5 to about 7, about 7 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids in length).

In some embodiments, the signal peptide is a native signal peptide of a human protein. In other embodiments, the signal peptide is a non-native signal peptide. For example, in some embodiments, the non-native signal peptide is a mutant native signal peptide from the corresponding native secreted human protein, and may include at least one (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitutions, insertions, and/or deletions.

In some embodiments, the signal peptide is a signal peptide or mutant thereof from a non-IgSF protein family, such as a signal peptide from an immunoglobulin (such as an IgG heavy chain or an IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), or CD33), a serum albumin protein (e.g., HSA or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g., chymotrypsinogen or trypsinogen), or another signal peptide capable of efficiently secreting a protein from a cell. Examples of signal peptides include, but are not limited to:

Table 3. Signal peptide sequences Native protein Signal sequence SEQ ID NO: Human serum albumin (HSA) MKWVTFISLLFLFSSAYS 483 Ig kappa light chain MDMRAPAGIFGFLLVLFPGYRS 394 Human azurocidin preprotein MTRLTVLALLAGLLASSRA 395 IgG heavy chain MELGLSWIFLLAILKGVQC 396 IgG heavy chain MELGLRWVFLVAILEGVQC 397 IgG heavy chain MKHLWFFLLLVAAPRWVLS 398 IgG heavy chain MDWTWRILFLVAAATGAHS 399 IgG heavy chain MDWTWRFLFVVAAATGVQS 400 IgG heavy chain MEFGLSWLFLVAILKGVQC 401 IgG heavy chain MEFGLSWVFLVALFRGVQC 402 IgG heavy chain MDLLHKNMKHLWFFLLLVAAPRWVLS 403 Ig kappa light MDMRVPAQLLGLLLLWLSGARC 404 Ig kappa light MKYLLPTAAAGLLLLAAQPAMA 405 Gaussia luciferase MGVKVLFALICIAVAEA 406 Human chymotrypsinogen MAFLWLLSCWALLGTTFG 407 human interleukin-2 MQLLSCIALILALV 408 Human trypsinogen-2 MNLLLILTFVAAAVA 409 Human CD33 MPLLLLLPLLWAGALA 410 Prolactin MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS 411 human tPA MDAMKRGLCCVLLLCGAVFVSPS 412 Synthetic/Consensus MLLLLLLLLLLALALA 413 Synthetic/Consensus MWWRLWWLLLLLLLLWPMVWA 414

Many natural linkers exhibited α-helical structures. The α-helical structure was rigid and stable, with intrasegment hydrogen bonds and a tightly packed backbone. Therefore, stiff α-helical linkers can act 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, stiff linkers exhibit relatively stiff structures by adopting α-helical structures or by containing multiple Pro residues. In many circumstances, they separate functional domains more efficiently than flexible linkers. The length of the linkers can be easily adjusted by changing the copy number to achieve an optimal distance between domains. Accordingly, stiff linkers are chosen when spatial separation of domains is essential to preserve the stability or bioactivity of fusion proteins. In this regard, alpha-helix linkers with the sequence of (EAAAK)n (SEQ ID NO: 421) have been applied to the construction of many recombinant fusion proteins. Another type of rigid linkers has a Pro-rich sequence, (XP)n, with X denoting any amino acid, preferably Ala, Lys or Glu.

For illustration purposes only, examples of linkers include GRA, poly(Gly), poly(Ala) and those provided in Table 4.

Table 4. Linker sequences. Sequence SEQ ID NO: (GGGGS) _n (eg, n = 1-6) 415 (Gly) ₈ 416 (Gly) ₆ 417 KESGSVSSEQLAQFRSLD 418 EGKSSGSGSESKST 419 GSAGSAAGSGEF 420 (EAAAK) _n (eg, n = 1-6) 421 A(EAAAK) ₄ ALEA(EAAAK) ₄ A 422 DADDY 423 AEAAAKEAAAKA 424 (Ala-Pro)n (10 to 34 aa) 425 GGSG 426 GSGS 427 GGGS 428 S(GGS)n (n=1-7) 429 GGGSGGG 430 ESGGGGVT 431 LESGGGGVT 432 GRAQVT 433 WRAQVT 434 ARGRAQVT 435 SGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT 436 SGTSPGLSAGATVGIMIGVLVGVALI 437 SAPVLSAVATVGITIGVLARVALI 438 SSPDLSAGTAVSIMIGVLAGMALI 439 TLGGNSASYTFVSLLFSAVTLLLLC 440 SGTSPGLSAGATVGIMIGVLVGVALI 441

The AFFIMER® polypeptide may further comprise, for example be fused to, a transmembrane domain (TM domain). Such TM domains ensure that the AFFIMER® polypeptide remains on the surface of the cell expressing the AFFIMER®. Such TM domains are readily available and include CD3, CD8, CD28, PDGFR TMD and others.

Still further modifications that may be made to the AFFIMER® polypeptide sequence or a flanking polypeptide moiety provided as part of a fusion protein are at least one sequence that is a site of post-translational modification by an enzyme. These may include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid linkage modification, etc.

Multispecific fusion proteins

In some embodiments, an AFFIMER® agent is a multispecific polypeptide including, for example, a first AFFIMER® TNFR2 polypeptide and at least one additional binding domain. The additional binding domain may be a polypeptide sequence selected from, for example, a second AFFIMER® polypeptide (which may be the same as or different from the first AFFIMER® polypeptide), an antibody or fragment thereof or another antigen-binding polypeptide, a ligand-binding portion of a receptor (such as a receptor trap polypeptide), a receptor-binding ligand (such as a cytokine, growth factor, or the like), an engineered T cell receptor, an enzyme or catalytic fragment thereof.

In some embodiments, an AFFIMER® agent includes at least one additional AFFIMER® polypeptide sequence that is also directed against TNFR2. The additional AFFIMER® TNFR2 polypeptide(s) may be the same or different (or a mixture thereof) from the first AFFIMER® TNFR2 polypeptide to create a multispecific AFFIMER® fusion protein. The AFFIMER® agents may bind to the same or overlapping sites on TNFR2 or may bind to two different sites such that the AFFIMER® TNFR2 agent may bind simultaneously to two sites on the same TNFR2 protein (biparatopic) or to more than two sites (multiparatopic).

In some embodiments, an AFFIMER® agent includes at least one antigen binding site from an antibody. The resulting AFFIMER® agent may be a single chain comprising both the AFFIMER® TNFR2 polypeptide and the antigen binding site (as in the case of a scFv) or may be a multimeric protein complex such as in an assembled antibody with heavy and/or light chains to which the anti-TNFR2 antibody sequence has also been fused.

In some embodiments, relative to a multispecific AFFIMER® agent comprising a full-length immunoglobulin, fusion of the AFFIMER® polypeptide sequence to the antibody will preserve the Fc function of the Fc region of the immunoglobulin. For example, the AFFIMER® agent may be capable of binding, via its Fc portion, to the Fc receptor of Fc receptor-positive cells. In some other embodiments, the AFFIMER® agent may activate the Fc receptor-positive cell by binding to the Fc receptor-positive cell, thereby initiating or increasing the expression of costimulatory cytokines and/or antigens. Additionally, the AFFIMER® agent may transfer at least one second activation signal necessary for physiological activation of the T cell to the T cell via the costimulatory antigens and/or cytokines.

In some embodiments, resulting from the binding of its Fc portion to other cells that express Fc receptors present on the surface of immune system effector cells, such as immune cells, hepatocytes and endothelial cells, the AFFIMER® agent can possess antibody-dependent cellular cytotoxicity (ADCC) function, a cell-mediated immune defense mechanism by which an immune system effector cell actively lyses a target cell, whose membrane surface antigen has been bound by an antibody, and thereby triggers tumor cell death via ADCC. In some other embodiments, the AFFIMER® agent is capable of demonstrating ADCC function.

As described above, in addition to Fc-mediated cytotoxicity, the Fc moiety may contribute to maintaining serum levels of the AFFIMER® agent, which are critical for its stability and persistence in the body. For example, when the Fc moiety binds to Fc receptors on endothelial cells and phagocytes, the AFFIMER® agent can be internalized and recycled into the bloodstream, amplifying its half-life in the body.

Examples of targets of the additional AFFIMER® polypeptides include, but are not limited to, another immune checkpoint protein and an immune costimulatory receptor (particularly if the additional AFFIMER®(s) can agonize the costimulatory receptor), a receptor, a cytokine, a growth factor, or a tumor-associated antigen, by way of illustration only. In some embodiments, the immunoglobulin portion may be a monoclonal antibody directed against at least one autoimmune target (e.g., TNFR2 or IL6-R). In some embodiments, the AFFIMER® TNFR2 polypeptide is part of an AFFIMER® agent that includes one or more binding domains that bind to a protein upregulated in autoimmune conditions (e.g., TNFR2 or IL6-R).

See William BA et al. J. Clin. Med. 2019;8(8):1261 for a review of the TNFR2 AFFIMER® agent formats encompassed by this disclosure.

In some embodiments, a multispecific TNFR2 AFFIMER® agent may further comprise a half-life extending moiety, such as any of those described herein. For example, a TNFR2 AFFIMER® agent may comprise at least one TNFR2 AFFIMER® polypeptide linked by a peptide linker to a specific binding domain of at least one binding domain (e.g., CD3ε chain or CD16) of an immune cell (e.g., T cell and/or NK cell) further linked to a half-life extending moiety, such as a fragment crystallizable (Fc) domain (e.g., a non-Fc-gammaR binding Fc), human serum albumin (HSA), or an AFFIMER® HSA polypeptide. In some embodiments, the half-life extending moiety is a fragment crystallizable (Fc) domain. In some embodiments, the half-life extending moiety is a human serum albumin (HSA). In some embodiments, the half-life extending moiety is an AFFIMER® HSA polypeptide.

PK and ADME property engineering

In some embodiments, the AFFIMER® agent may not have an optimal half-life and/or PK profile for the route of administration, such as parenteral therapeutic dosing. A "half-life" is the amount of time it takes for a substance, such as an AFFIMER® agent of the present invention, to lose half of its pharmacological or physiological activity or concentration. The biological half-life may be affected by elimination, excretion, degradation (e.g., enzymatic) of the substance, or absorption and concentration in certain organs or tissues of the body. In some embodiments, the biological half-life can be assessed by determining the time taken for the plasma concentration of the substance to reach half of its steady-state level ("plasma half-life"). To address this shortcoming, a variety of general strategies to extend half-life have been used in the case of other therapeutic proteins, including the incorporation of half-life extending moieties as parts of the AFFIMER® agent.

The term “half-life extending moiety” refers to a pharmaceutically acceptable moiety, domain or molecule covalently linked (chemically conjugated or fused) to an AFFIMER® polypeptide to form an AFFIMER® agent described herein, optionally via a non-naturally encoded amino acid, directly or via a linker, that prevents or attenuates in vivo proteolytic degradation or other modification that decreases the activity of the AFFIMER® polypeptide, increases the half-life, and/or improves or alters other pharmacokinetic or biophysical properties, including, but not limited to, increasing the rate of absorption, reducing toxicity, improving solubility, reducing protein aggregation, increasing the biological activity and/or target selectivity of the modified AFFIMER® polypeptide, increasing manufacturability and/or reducing the immunogenicity of the modified AFFIMER® polypeptide, relative to a comparator such as an unconjugated form of the modified AFFIMER® polypeptide. The term “half-life extending moiety” includes non-proteinaceous half-life extending moieties, such as a water-soluble polymer such as polyethylene glycol (PEG) or discrete PEG, hydroxyethyl starch (HEA), a lipid, a branched or unbranched acyl group, a branched or unbranched C8-C30 acyl group, a branched or unbranched alkyl group, and a branched or unbranched C8-C30 alkyl group; and protein half-life extending moieties, such as serum albumin, transferrin, adnectins (e.g., albumin-binding or pharmacokinetics extending (PKE) adnectins), the Fc domain, and an unstructured polypeptide, such as XTEN and PAS polypeptides (e.g., conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala and/or Ser), and a fragment of any of such elements.

In some embodiments, the half-life extending moiety extends the half-life of the resulting AFFIMER® agent circulating in mammalian blood serum relative to the half-life of the protein that is not thereby conjugated to the moiety (e.g., relative to the AFFIMER® polypeptide alone). In some embodiments, the half-life is extended by more than or equal to about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold. In some embodiments, the half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours, or more than 1 week following in vivo administration relative to the protein without the half-life extending moiety.

As additional exemplary means, half-life extending moieties that may be used in the generation of AFFIMER® agents of the disclosure include:

• Genetic fusion of the AFFIMER® pharmacological sequence to a protein or protein domain with a naturally long half-life (e.g., Fc fusion, transferrin [Tf] fusion, or albumin fusion. See, 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.

• Genetic fusion of the AFFIMER® pharmacological sequence to an inert polypeptide, e.g. XTEN (also known as recombinant PEG or ''rPEG''), a homoamino acid polymer (HAP; HAPylation), a proline-alanine-serine polymer (PAS; PASylation), or an elastin-like peptide (ELP; ELPylation). See, 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 halflife of pharmaceutically active proteins. Protein Eng Des Salt. 26:489–501. Floss et al. (2012) “Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends Biotechnology. 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.

• Increasing the hydrodynamic radius by chemical conjugation of the pharmacologically active peptide or protein to repeating chemical groups, e.g. to PEG (PEGylation) or hyaluronic acid. See, 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.

• Significantly increasing the negative fusion charge of the pharmacologically active peptide or protein by polysialylation; or, alternatively, (b) fusion of a negatively charged, highly sialylated peptide (e.g., carboxy-terminal peptide [CTP; of human chorionic gonadotropin (GC) b-chain]), known to prolong the half-life of natural proteins such as human GC, b-subunit, to the biologic drug candidate. See, 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 long-acting 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.

• Noncovalent binding, via attachment of a peptide or protein-binding domain to the bioactive protein, to proteins with normally long half-lives such as HSA, human IgG, transferrin, or fibronectin. See, 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.

Classical gene fusions with long-lived serum proteins offer an alternative method of half-life extension distinct from chemical conjugation to PEG or lipids. Two major proteins have traditionally been used as fusion partners: antibody Fc domains and human serum albumin (HSA). Fc fusions involve the fusion of receptor peptides, proteins, or exodomains to the Fc portion of an antibody. Fc and albumin fusions achieve extended half-lives not only by increasing the size of the peptide drug, but both also take advantage of the body's natural recycling mechanism: the neonatal Fc receptor, FcRn. The pH-dependent binding of these proteins to FcRn prevents degradation of the fusion protein in the endosome. Fusions based on these proteins can have half-lives in the range of 3 to 16 days, much longer than typical PEGylated or lipidated peptides. Fusion to antibody Fc domains can improve the solubility and stability of the peptide or protein drug. An example of a Fc peptide fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin, the same protein exploited by acylated fatty peptides, is the other popular fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. A major difference between Fc and albumin is the dimeric nature of Fc compared to the monomeric structure of HSA leading to the presentation of a fused peptide as a dimer or monomer depending on the choice of fusion partner. The dimeric nature of an AFFIMER®-Fc fusion can produce an avidity effect if the AFFIMER® targets are close enough together or are themselves dimers. This may or may not be desirable depending on the target.

Fc Mergers

In some embodiments, the AFFIMER® polypeptide may be part of a fusion protein with an immunoglobulin Fc domain ("Fc domain"), or a fragment or variant thereof, such as a functional Fc region. In some embodiments, the Fc region is a non-Fc-gammaR binding Fc region. In this context, an Fc fusion ("Fc-fusion"), such as an AFFIMER® TNFR2 agent created as an Fc-AFFIMER® fusion protein, is a polypeptide comprising at least one AFFIMER® TNFR2 sequence covalently linked via a peptide backbone (directly or indirectly) to an Fc region of an immunoglobulin. An Fc-fusion may comprise, for example, the Fc region of an antibody (which facilitates effector functions and pharmacokinetics) and an AFFIMER® TNFR2 sequence as part of the same polypeptide. An immunoglobulin Fc region may also be indirectly linked to at least one TNFR2 AFFIMER® polypeptide. Various linkers are known in the art and may optionally be used to link an Fc to a polypeptide including a TNFR2 AFFIMER® sequence to generate an Fc-fusion. In some embodiments, the Fc-fusions may be dimerized to form Fc-fusion homodimers, or using non-identical Fc domains, to form Fc-fusion heterodimers.

In some embodiments, a fusion-Fc homodimer comprises a dimer of a TNFR2 AFFIMER® agent that comprises a TNFR2 AFFIMER® polypeptide linked to an Fc domain linked to another TNFR2 AFFIMER® polypeptide (TNFR2 AFFIMER® polypeptide-Fc domain-TNFR2 AFFIMER® polypeptide).

There are several reasons to choose the Fc region of human antibodies for use in the generation of TNFR2 AFFIMER® agents as TNFR2 AFFIMER® fusion proteins. The primary reason is to produce a stable protein, large enough to demonstrate a pharmacokinetic profile similar to those of antibodies, and to take advantage of the properties conferred by the Fc region; this includes the neonatal FcRn receptor retrieval pathway involving FcRn-mediated recycling of the fusion protein to the cell surface after endocytosis, avoiding lysosomal degradation and resulting in re-release into the bloodstream, thus contributing to an extended serum half-life. Another obvious advantage is the linkage of the Fc domain to Protein A, which can simplify downstream processing during AFFIMER® agent production and allow the generation of a highly pure preparation of AFFIMER® agent.

In general, an Fc domain will comprise the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, the Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible N-terminal hinge of these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises the Cγ2 and Cγ3 immunoglobulin domains and the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc domain may vary, the Fc region of human IgG heavy chain is generally defined as consisting of residues C226 or P230 to its carboxyl terminus, with numbering according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). Fc may refer to this region in isolation, or to this region in the context of a whole antibody, an antibody fragment, or an Fc fusion protein. Polymorphisms have been observed at a number of different Fc positions and are also included as Fc domains as used herein.

In some embodiments, the Fc As used herein, a “functional Fc region” refers to an Fc domain or fragment thereof that retains the ability to bind to FcRn. A functional Fc region binds to FcRn but does not possess an effector function. The ability of the Fc region or fragment thereof to bind to FcRn can be determined by standard binding assays known in the art. Examples of “effector functions” include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions can be assessed using various assays known in the art for evaluating such antibody effector functions.

In an exemplary embodiment, the Fc domain is derived from a subclass of IgG1, however, other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used. An exemplary sequence of a human IgG1 immunoglobulin Fc domain that may be used is: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 442)

In some embodiments, the Fc region used in the fusion protein may comprise the hinge region of an Fc molecule. An exemplary hinge region includes the central hinge residues spanning positions 1-16 (e.g., DKTHTCPPCPAPELLG ((SEQ ID NO: 484)) of the exemplary human immunoglobulin IgG1 Fc domain sequence provided above. In some embodiments, the AFFIMER®-containing fusion protein may adopt a multimeric structure (e.g., a dimer) due, in part, to the cysteine residues at positions 6 and 9 within the hinge region of the exemplary human immunoglobulin IgG1 Fc domain sequence provided above. In other embodiments, the hinge region as used herein may further include residues derived from the CH1 and CH2 regions that flank the central hinge sequence of the exemplary human immunoglobulin IgG1 Fc domain sequence provided above. In still other embodiments, the hinge sequence may comprise or consist of GSTHTCPPCPAPELLG (SEQ ID NO: 443) or EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 444).

In some embodiments, the hinge sequence may include at least one substitution that confers desirable pharmacokinetic, biophysical and/or biological properties. Examples of hinge sequences include:

EPKSCDKTHTCPPCPAPELLGGPS (SEQ ID NO: 445);

EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 446);

EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 447);

EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 448);

DKTHTCPPCPAPELLGGPS (SEQ ID NO: 449); And

DKTHTCPPCPAPELLGGSS (SEQ ID NO: 450).

In some embodiments, the P residue at position 18 of the exemplary human IgG1 immunoglobulin Fc domain sequence provided above may be replaced with S to remove the Fc effector function; this replacement is exemplified in hinges having the sequences

EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 451), EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 452), and DKTHTCPPCPAPELLGGSS (SEQ ID NO: 453).

In another embodiment, the DK residues at positions 1-2 of the exemplary human immunoglobulin IgG1 Fc domain sequence provided above may be replaced with GS to eliminate a potential clip site; this replacement is exemplified in the sequence EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 448). In another embodiment, the C at position 103 of the human IgG1 heavy chain constant region (e.g., CH1-CH3 domains) may be replaced with S to prevent improper formation of a cysteine bond in the absence of a light chain; This replacement is illustrated by way of example by EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 446), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 451), and EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 452).

In some embodiments, the Fc is a mammalian Fc such as a human Fc, comprising Fc domains derived from IgG1, IgG2, IgG3 or IgG4. The Fc region may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a native Fc region and/or to an Fc region of a parent polypeptide. In some embodiments, the Fc region may have at least about 90% sequence identity to a native Fc region and/or to an Fc region of a parent polypeptide.

In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NO: 454-467 or an Fc sequence exemplified by SEQ ID NO: 454-467. It is to be understood that the C-terminal lysine of an Fc domain is an optional component of a fusion protein comprising an Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence selected from SEQ ID NO: 454-467, except that the C-terminal lysine thereof is omitted. In some embodiments, the Fc domain comprises the amino acid sequence selected from SEQ ID NO: 454-467. In some embodiments, the Fc domain comprises the amino acid sequence selected from SEQ ID NO: 454-467 except that the C-terminal lysine thereof is omitted.

Table 5. Fc domain sequences. Name Sequence SEQ ID NO: hIgG1a_191
[Subtype A]
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 454 hIgG1a_189 [hIgG1a_191 without "GK" on C-terminus; Subtype A]
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 455 hIgG1a_191b
[Subtype A/F]
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 456 hIgG1f_1.1_191
[Contains five point mutations to impair ADCC function, subtype F] DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 457 hIgG1f_1.1_186
[Contains five point mutations to impair ADCC function and C225S (Edlemen numbering); subtype F]

EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSS IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 458 hIgG1a_(N297G)_191
[Subtype A]
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 459 hIgG1a_190
[hIgG1a_190 without "K" on C terminus; Subtype A]
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 460 hIgG1a_(N297Q)_191
[Subtype A]
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 461 hIgG1a_(N297S)_191
[Subtype A]
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 462 hIgG1a_(N297A)_191
[Subtype A]
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 463 hIgG1a_(N297H)_191
[Subtype A]
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYHSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 464 hIgG4
DKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 465 hIgG4_(S241P)
DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 466 hIgG1 (contains two point mutations to impair ADCC function L20A, L21A) SEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 467

Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins.

In some embodiments, the fusion protein comprises an Fc domain sequence for which the resulting AFFIMER® agent has no (or reduced) ADCC and/or complement activation or effector functionality. For example, the Fc domain may comprise a naturally deactivated constant region of IgG2 or IgG4 isotype or a mutated IgG1 constant region. Examples of suitable modifications are described in EP0307434. An example includes substitutions of alanine residues at positions 235 and 237 (EU index numbering).

In other embodiments, the fusion protein comprises an Fc domain sequence for which the resulting AFFIMER® agent will retain some or all of the Fc functionality, e.g. will be capable of one or both of ADCC and CDC activities, such as for example if the fusion protein comprises the Fc domain of human IgG1 or IgG3. The levels of effector function may be varied according to known techniques, e.g. by mutations in the CH2 domain, e.g. where the IgG1 CH2 domain has at least one mutation at positions selected from 239 and 332 and 330, e.g. the mutations being selected from S239D and I332E and A330L such that the antibody has enhanced effector function, and/or e.g. by altering the glycosylation profile of the antigen binding protein of the disclosure such that there is reduced fucosylation of the Fc region.

Albumin fusions

In some embodiments, the AFFIMER® agent is a fusion protein comprising, in addition to at least one AFFIMER® sequence, an albumin sequence or albumin fragment. In other embodiments, the AFFIMER® agent is conjugated to the albumin sequence or albumin fragment via a chemical bond other than incorporation into the polypeptide sequence including the AFFIMER® polypeptide. In some embodiments, the albumin, albumin variant, or albumin fragment is human serum albumin (HSA), a human serum albumin variant, or a human serum albumin fragment. Serum albumin proteins comparable to HSA are found, e.g., in cynomolgus monkeys, cows, dogs, rabbits, and rats. Among non-human species, bovine serum albumin (BSA) is structurally most similar to HSA. See, e.g., Kosa et al., (2007) J Pharm Sci. 96(11):3117-24. The present disclosure contemplates the use of albumin from non-human species, including, but not limited to, an albumin sequence derived from cyno serum albumin or bovine serum albumin.

Mature HSA, a 585 amino acid (approx. 67 kDa) polypeptide with a serum half-life of approximately 20 days, is primarily responsible for maintaining colloidal osmotic blood pressure, blood pH, and the transport and distribution of numerous endogenous and exogenous ligands. The protein has three structurally homologous domains (domains I, II, and III), is almost entirely alpha-helical in conformation, and is highly stabilized by 17 disulfide bridges. In some embodiments, the AFFIMER® agent may be an albumin fusion protein comprising at least one AFFIMER® polypeptide sequence and the sequence for mature human serum albumin (SEQ ID NO: 468) or a variant or fragment thereof that maintains the PK and/or biodistribution properties of mature albumin to the extent desired in the fusion protein.

The albumin sequence can be separated from the AFFIMER® polypeptide sequence or other flanking sequences in the AFFIMER® agent by use of linker sequences as described above.

Unless otherwise indicated, reference herein to “albumin” or “mature albumin” is intended to refer to HSA. However, it is noted that full-length HSA has an 18 amino acid signal peptide (MKWVTFISLLFLFSSAYS (SEQ ID NO: 483) followed by a 6 amino acid prodomain (RGVFRR) (SEQ ID NO: 469); this 24 amino acid residue peptide may be referred to as the preprodomain. The AFFIMER®-HSA fusion proteins may be expressed and secreted by utilizing the HSA preprodomain in the coding sequence of the recombinant proteins. Alternatively, the AFFIMER®-HSA fusion protein may be expressed and secreted by including other secretory signal sequences, as described above.

In alternative embodiments, rather than being provided as part of a fusion protein with the AFFIMER® polypeptide, the serum albumin polypeptide may be covalently coupled to the AFFIMER®-containing polypeptide via a linkage other than a backbone amide linkage, such as cross-linked via chemical conjugation between amino acid side chains on each of the albumin polypeptide and the AFFIMER®-containing polypeptide.

In some embodiments, one chemical modification method that can be applied in the generation of the subject AFFIMER® agents to increase protein half-life is lipidation, which involves the covalent attachment of fatty acids to peptide side chains. Originally conceived and developed as a method to extend the half-life of insulin, lipidation shares the same basic mechanism of half-life extension as PEGylation, namely increasing the hydrodynamic radius to reduce renal filtration. However, the lipid moiety itself is relatively small and the effect is mediated indirectly by non-covalent attachment of the lipid moiety to circulating albumin. A consequence of lipidation is that it reduces the water solubility of the peptide, but engineering of the linker between the peptide and the fatty acid can modulate this, for example by using glutamate or mini PEGs within the linker. Linker engineering and lipid moiety variation can affect self-aggregation, which may contribute to increased half-life by slowing biodistribution, independent of albumin. See, for example, Jonassen et al. (2012) Pharm Res. 29(8):2104–14.

Other examples of albumin-binding moieties for use in the generation of certain AFFIMER® agents include albumin-binding adnectins (PKE2) (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 Streptococcus G148 strain G protein, and the albumin-binding domain antibody GSK2374697 ("AlbudAb") or the albumin-binding nanobody portion of ATN-103 (Ozoralizumab).

AFFIMER® XT

In some embodiments, the molecule that binds to a serum protein such as HSA comprises an AFFIMER® HSA polypeptide. Examples of such AFFIMER® HSA polypeptides can be found in WO2022/023540. An AFFIMER® HSA polypeptide provided herein, in some embodiments, is linked to another molecule and extends the half-life of that molecule (e.g., a therapeutic polypeptide). These AFFIMER® HSA polypeptides have been shown in in vivo pharmacokinetic (PK) studies to controllably extend the serum half-life of any other therapeutic AFFIMER® polypeptide to which it is conjugated in a single genetic fusion, e.g., which may be made in E. Coli. AFFIMER® XT™ polypeptides may also be used to extend the half-life of other peptide or protein therapeutic agents, such as the TNFR2 AFFIMER® of the present invention.

In some embodiments, an AFFIMER® HSA polypeptide prolongs the serum half-life of the AFFIMER® TNFR2 polypeptide in vivo. For example, an AFFIMER® HSA polypeptide can extend the half-life of the AFFIMER® TNFR2 polypeptide by at least 2-fold, relative to the half-life of the molecule unbound to an AFFIMER® HSA polypeptide. In some embodiments, an AFFIMER® HSA polypeptide prolongs the half-life of the AFFIMER® TNFR2 polypeptide by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, or at least 30-fold, relative to the half-life of the AFFIMER® TNFR2 polypeptide unbound to an AFFIMER® HSA polypeptide. In some embodiments, an AFFIMER® HSA polypeptide extends the half-life of the AFFIMER® TNFR2 polypeptide by 2-fold to 5-fold, 2-fold to 10-fold, 3-fold to 5-fold, 3-fold to 10-fold, 15-fold to 5-fold, 4-fold to 10-fold, or 5-fold to 10-fold, relative to the half-life of the AFFIMER® TNFR2 polypeptide unbound to an AFFIMER® HSA polypeptide. In some embodiments, an AFFIMER® HSA polypeptide extends the half-life of the AFFIMER® TNFR2 polypeptide by at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, e.g., at least 1 week after in vivo administration, relative to the half-life of the molecule unbound to an AFFIMER® HSA polypeptide.

An AFFIMER® HSA polypeptide comprises an AFFIMER® polypeptide wherein at least one of the solvent accessible loops is derived from wild-type Stefin A protein having amino acid sequences to enable an AFFIMER® polypeptide to bind to HSA, selectively, and in some embodiments, with a Kd of 10 ^-6 M or less.

In some embodiments, the HSA AFFIMER® polypeptide is derived from wild-type human Stefin A protein having a backbone sequence and wherein one or both of loop 2 (designated (Xaa)n) and loop 4 (designated (Xaa)m) are replaced with alternative loop sequences (Xaa)n and (Xaa)m, to have the general Formula (I):

FR1-(Xaa)n-FR2-(Xaa)m-FR3 (I),

where FR1 is an amino acid 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 MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 470); FR2 is an amino acid 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 GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2); FR3 is an amino acid 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 to EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3); Xaa, individually for each occurrence, is an amino acid; and n is an integer from 3 to 20, and m is an integer from 3 to 20. Additional variations of this general structure can be found in WO2022/023540.

In all embodiments, each amino acid of (Xaa)n may be the same or selected from any amino acid. The same is true for (Xaa)m.

In some embodiments, the TNFR2 AFFIMER® polypeptide comprises an HSA AFFIMER® polypeptide comprising an amino acid sequence selected from any of SEQ ID NOs: 471-477 (Table 6). In some embodiments, the TNFR2 AFFIMER® polypeptide has an extended serum half-life and comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or even 98% identical to a sequence selected from SEQ ID NOs: 471-477 (Table 6). Additional HSA AFFIMER® polypeptide sequences for use with the present invention can be found in WO2022/023540.

Table 6. Examples of HSA AFFIMER® polypeptide sequences Sequence SEQ ID NO: MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLADWWQAKWPHSTNYYIKVRAGDNKYMHLKVFNGPYKVHQSSGGADRVLTGYQVDKNKDDELTGF 471 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLAGYWAAKWPGSTNYYIKVRAGDNKYMHLKVFNGPFPNTSYDLQADRVLTGYQVDKNKDDELTGF 472 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLANWYQQRWPGSTNYYIKVRAGDNKYMHLKVFNGPWHNYGESSGADRVLTGYQVDKNKDDELTGF 473 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLAVWGPEYQHQSTNYYIKVRAGDNKYMHLKVFNGPNAGWPLVPEADRVLTGYQVDKNKDDELTGF 474 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLAGHYARYWPGSTNYYIKVRAGDNKYMHLKVFNGPWAQKSKVHQADRVLTGYQVDKNKDDELTGF 475 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLAGFWASKWPGSTNYYIKVRAGDNKYMHLKVFNGPFTAVSKKDAADRVLTGYQVDKNKDDELTGF 476 MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVLANFFQRRWPGSTNYYIKVRAGDNKYMHLKVFNGPWKFRNTDRGADRVLTGYQVDKNKDDELTGF 477

Conjugated

The subject AFFIMER® agents may also include at least one functional group intended to provide additional detectability or pharmacological activity to the AFFIMER® agent. Functional groups for detection are those that can be used to detect the association of the AFFIMER® agent with a cell or tissue in vivo. Functional groups having pharmacological activity are those agents that are intended to be delivered to the tissue expressing the target of the AFFIMER® agent (TNFR2 in the case of the TNFR2 AFFIMER® agents of the present disclosure) and, in doing so, have a pharmacological consequence for the targeted tissues or cells.

The present disclosure provides AFFIMER® agents including conjugates of substances having a wide variety of groups, substituents or functional moieties, such Functional Moieties including, but not limited to, a label; a dye; an immunoadhesion molecule; a radionuclide; a cytotoxic compound; a drug; an affinity tag; a photoaffinity tag; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin; an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing group; a radioactive group; a novel functional group; a group that interacts covalently or non-covalently with other molecules; a photocaged group; a group excitable by actinic radiation; a photoisomerizable group; biotin; a biotin derivative; a biotin analogue; a group incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a sugar linked to a carbon; a redox-active agent; an aminothioacid; a toxic group; an isotopically labeled group; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron-dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent; a detectable label; a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; a radio emitter; a neutron capture agent; or any combination of the foregoing, or any other desirable compound or substance.

Detectable markers and groups

When the moiety is a detectable label, it may be a fluorescent label, a radioactive label, an enzymatic label or any other label known to those skilled in the art. In some embodiments, the functional group is a detectable label that may be included as part of a conjugate to form certain AFFIMER® agents suitable for medical imaging. By "medical imaging" is meant any technique for visualizing an internal region of the human or animal body, for diagnostic, research or therapeutic treatment purposes. For example, the AFFIMER® agent may be detected (and quantified) by X-ray scanning, magnetic resonance imaging (MRI), computed tomography (CT) scan, nuclear imaging, positron emission tomography (PET) contrast agent, optical imaging (such as fluorescence imaging including near-infrared fluorescence (NIRF) imaging), bioluminescence imaging, or combinations thereof. The Functional Moiety is optionally a contrast agent for X-ray imaging. Agents useful for enhancing these techniques are materials that allow visualization of a particular locus, organ, or disease site in the body, and/or that lead to some improvement in the quality of images generated by the imaging techniques, providing improved or easier interpretation of those images. Such agents are herein referred to as contrast agents, the use of which facilitates the differentiation of different parts of the image, by increasing the "contrast" between these different regions of the image. The term "contrast agents" thus encompasses agents which serve to amplify the quality of an image which can nevertheless be generated in the absence of such an agent (as is the case, for example, in MRI), as well as agents which are prior to the generation of an image (as is the case, for example, in nuclear imaging).

In some embodiments, the detectable label comprises a chelate moiety for chelating a metal, e.g., a chelator for a radiometal or a paramagnetic ion. In some embodiments, the detectable label is a chelator for a radionuclide useful for radiation therapy or imaging procedures. Radionuclides useful in the present disclosure include gamma emitters, positron emitters, Auger electron emitters, X-ray emitters, and fluorescence emitters, with beta or alpha emitters for therapeutic use. Examples of radionuclides useful as toxins in radiotherapy include: 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 chelator will coordinate a metal are described, for example, by Gansow et al., US Patent Nos.: 4,831,175, 4,454,106 and 4,472,509. Examples of chelators include, by way of illustration only, 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA).

Other detectable isotopes that can be incorporated directly into the amino acid residues of the AFFIMER® polypeptide or that otherwise do not require a chelator, include 3H, 14C, 32P, 35S and 36Cl.

Paramagnetic ions, useful for diagnostic procedures, may also be administered. Examples of paramagnetic ions include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), erbium(III), or combinations of these paramagnetic ions.

Examples of fluorescent labels 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-Phycoerythrin, etc.), and quantum dots.

Non-limiting fluorescent compounds that may be used in the present disclosure 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), rhodamine 6G (R6G), rhodamine derivative JA133, Alexa Fluorescent Dyes (such as Alexa Fluorescent Dyes), and the like. Fluorine 488, Alexa Fluorine 546, Alexa Fluorine 633, Alexa Fluorine 555, and Alexa Fluorine 647), 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine, and fluorescent transition metal complexes, such as europium. Fluorescent compounds that can be used also include fluorescent proteins, such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CyPet), and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet). WO2008142571, WO2009056282, WO9922026.

Examples of enzyme markers include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, and ß-galactosidase.

Another well-known label is biotin. Biotin labels are usually composed of the biotinyl group, a spacer arm, and a reactive group that is responsible for attachment to target functional groups on proteins. Biotin can be useful for attaching the labeled protein to other moieties that include an avidin group.

AFFIMER® polypeptide-drug conjugates

In some embodiments, the AFFIMER® agent comprises at least one therapeutic agent, e.g., to form an AFFIMER® polypeptide-drug conjugate. As used herein, the term “therapeutic agent” refers to a substance that can be used in the cure, mitigation, treatment, or prevention of disease in a human or other animal. Such therapeutic agents include substances recognized in the Official United States Pharmacopeia, the Official Homeopathic Pharmacopeia of the United States, the Official National Formulary, or any combination thereof, and include, but are not limited to, small molecules, nucleotides, oligopeptides, polypeptides, and the like. Therapeutic agents that may be attached to AFFIMER®-containing polypeptides include, but are not limited to, cytotoxic agent(s), anti-metabolites, alkylating agents, antibiotics, growth factors, cytokines, anti-angiogenic agents, anti-mitotic agents, toxins, apoptotic agents or the like, such as DNA alkylating agents, topoisomerase inhibitors, microtubule inhibitors (e.g., DM1, DM4, MMAF and MMAE), endoplasmic reticulum stress inducing agents, platinum compounds, antimetabolites, vincalcaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, radiosensitizers and chemotherapeutic combination therapies, as illustrated.

Any method known in the art for conjugation to antibodies and other proteins can be used to generate the conjugates of the present disclosure, including the methods described by Hunter, et al., (1962) Nature 144:945; David et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating peptides, polypeptides, and organic and inorganic moieties to antibodies and other proteins are conventional and very well known in the art and readily adapted to the generation of these versions of the subject AFFIMER® agents.

Where the conjugated moiety is a peptide or polypeptide, such moiety may be chemically cross-linked to the AFFIMER®-containing polypeptide or may be included as part of a fusion protein with the AFFIMER®-containing polypeptide. And an illustrative example would be a diphtheria toxin-AFFIMER® fusion protein. In the case of non-peptide entities, addition to the AFFIMER®-containing polypeptide will generally be by way of chemical conjugation to the AFFIMER®-containing polypeptide - such as via a functional group on an amino acid side chain or the carboxyl group at the C-terminal or amino group at the N-terminus of the polypeptide. In some embodiments, whether a fusion protein or a chemically cross-linked moiety, the conjugated moiety will include at least one site that is responsive to an environmental condition (such as pH) that allows the conjugated moiety to be released from the AFFIMER®-containing polypeptide, such as in diseased tissue (or tissue to be protected if the conjugated moiety functions to protect healthy tissue).

Spacers

In some embodiments, an AFFIMER® polypeptide-drug conjugate comprises a spacer or linkage (L1) between the half-life extension moiety and the substrate recognition sequence (SRS) cleavable by the enzyme, e.g., present in an inflammatory microenvironment.

The spacer may be any molecule, e.g., one or more nucleotides, amino acids, chemical functional groups. In some embodiments, the spacer is a peptide linker (e.g., two or more amino acids). The spacers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. In some embodiments, the spacers are non-antigenic and do not elicit an immune response. An immune response includes a response from the innate immune system and/or the adaptive immune system. Thus, an immune response may be a cell-mediated response and/or a humoral immune response. The immune response may be, for example, a T cell response, a B cell response, a natural killer (NK) cell response, a monocyte response, and/or a macrophage response. Other cellular responses are contemplated herein. In some embodiments, the linkers do not encode proteins.

In some embodiments, L1 is a hydrocarbon (straight chain or cyclic) such as 6-maleimidocaproyl, maleimidopropanoyl and maleimidom ethyl cyclohexane-1-carboxylate, or L1 is N-succinimidyl 4-(2-pyridylthio) pentanoate, N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate, N-succinimidyl (4-iodo-acetyl) aminobenzoate.

In some embodiments, L1 is a polyether such as a poly(ethylene glycol) or other hydrophilic linker. For example, when the CBM (carbohydrate-binding module) comprises a thiol (such as a cysteine residue), L1 may be a polyethylene glycol) coupled to the thiol group through a maleimide moiety.

Non-limiting examples of linkers for use in accordance with the present disclosure are described in International Publication No. WO 2019/236567, published on December 12, 2019.

AFFIMER® polynucleotide encoded for in vivo administration

An alternative approach to the delivery of AFFIMER® therapeutic agents, such as an AFFIMER® TNF2 agent, would be to have the body itself produce the therapeutic polypeptide. A multitude of clinical studies have illustrated the utility of in vivo gene transfer into cells using a variety of different delivery systems. In vivo gene transfer seeks to deliver the encoded AFFIMER® polynucleotide, rather than the AFFIMER® agent, to patients. This allows the patient's body to produce the AFFIMER® therapeutic agent of interest for an extended period of time, and to secrete it either systemically or locally, depending on the site of production. The gene-encoded AFFIMER® polynucleotide may present a time- and cost-effective alternative to conventional production, purification, and administration of the polypeptide version of the AFFIMER® agent. A number of antibody expression platforms, to which delivery of the AFFIMER®-encoded polynucleotide can be adapted, have been monitored in vivo, including viral vectors, naked DNA and RNA.

The success of gene therapy has been driven in large part by improvements in nonviral and viral gene transfer vectors. A variety of nonviral physical and chemical methods have been used to transfer DNA and mRNA to mammalian cells and a substantial number of these have been developed as clinical stage technologies for gene therapy, both ex vivo and in vivo, and are readily adapted to deliver the encoded TNFR2 AFFIMER® polynucleotide of the present disclosure.

An efficient approach to encoded AFFIMER® gene transfer results in expression that must be at a level appropriate to the specific application. Promoters are a major cis-acting element within the design of the vector genome that can dictate the overall strength of expression as well as cellular specificity. Similarly, polyadenylation of an encoded AFFIMER® transcript can also be important for nuclear export, translation, and mRNA stability. Therefore, in some embodiments, the encoded AFFIMER® polynucleotide will include a promoter and/or a polyadenylation signal sequence, as well as other regulatory elements well known in the art, such as enhancers, intronic sequences, post-transcriptional regulatory agents, and the like.

Viral vectors

Examples of viral gene therapy systems that are readily adapted for use in the present invention include plasmids, adenoviruses, adeno-associated viruses (AAVs), retroviruses, lentiviruses, herpes simplex viruses, vaccinia virus, poxvirus, reoviruses, measles viruses, Semliki Forest virus, etc. Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced by the nucleic acid construct carrying the nucleic acid sequences encoding the epitopes and targeting sequences of interest.

To further illustrate, the encoded AFFIMER® polynucleotides may be delivered in vivo using adenoviruses and adeno-associated viruses (AAVs), which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The AFFIMER® TNFR2 polypeptides may be encoded and delivered in vivo using retroviral vectors, such as gammaretroviruses or lentiviruses. In some embodiments, the viral vector is pseudotyped with an envelope selected from one of the envelopes of amphotropic, polytropic, xenotropic, 10A1, GALV, VSV-G, endogenous baboon virus, RD114, rhabdoviruses, alphaviruses, measles or influenza viruses.

Non-viral vectors

Examples of nucleic acids or polynucleotides for the encoded TNFR2 AFFIMER® agents of the present disclosure include, but are not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA; including LNA having a β-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA) "), cyclohexenyl nucleic acids (CeNA), or hybrids or combinations thereof. Accordingly, the encoded TNFR2 AFFIMER® polynucleotide may be delivered by plasmid DNA, minicircle DNA, mRNA, RNA replicons, and the like. Means of delivery include transfection using electroporation, use of lipofection and other transfection reagents, delivery of naked DNA, delivery of gold particles, and other means known in the art.

Expression methods and systems for protein production

The AFFIMER® TNFR2 agent proteins described herein may be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing these sequences in a suitable host. For recombinant AFFIMER® agent proteins comprising other modifications, such as chemical modifications or conjugation, the recombinant AFFIMER® agent protein may be further chemically or enzymatically manipulated after isolation from the host cell or chemical synthesis. The AFFIMER® polypeptide may be secreted or associated with the membrane of the expressing cell, for example by tethering, such as by inclusion of a transmembrane domain.

The present disclosure includes recombinant methods and nucleic acids for recombinantly expressing the recombinant AFFIMER® agent proteins of the present disclosure comprising (i) introducing into a host cell a polynucleotide encoding the amino acid sequence of said AFFIMER® agent, for example, wherein the polynucleotide is in a vector and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., eukaryotic or prokaryotic) under conditions favorable to expression of the polynucleotide; and (iii) optionally, isolating the AFFIMER® agent from the host cell and/or the medium in which the host cell is cultured. See e.g. WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

In some embodiments, a DNA sequence encoding a recombinant AFFIMER® agent protein of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Additionally, a DNA oligomer containing a nucleotide sequence encoding the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' extensions for complementary assembly.

Once a nucleic acid sequence encoding a recombinant AFFIMER® agent protein of the disclosure has been obtained, the vector for producing the recombinant AFFIMER® agent protein can be produced by recombinant DNA technology using techniques well known in the art. Methods well known to those skilled in the art can be used to construct expression vectors containing the sequences encoding the recombinant AFFIMER® agent and appropriate transcriptional and translational control signals. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., techniques described in Sambrook et al, 1990, MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al. eds., 1998, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of a recombinant AFFIMER® agent protein can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the recombinant AFFIMER® agent protein of the disclosure. In specific embodiments, expression of the recombinant AFFIMER® agent protein is regulated by a tissue-specific, constitutive, or inducible promoter.

The expression vector may include an origin of replication, such as may be selected based on the type of host cell used for expression. For example, the origin of replication of plasmid pBR322 (Product No. 303-3s, New England Biolabs, Beverly, Mass.) is useful for most Gram-negative bacteria while various origins from SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomaviruses (such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not required for mammalian expression vectors (e.g., the SV40 origin is often used because it contains the early promoter).

The vector may comprise at least one selectable marker gene, e.g., genetic elements that encode a protein required for the survival and growth of a host cell cultured in a selective culture medium. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) provide critical nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. A neomycin resistance gene may also be used for selection in prokaryotic and eukaryotic host cells. Other selectable genes may be used to amplify the gene to be expressed. Amplification is a process where genes that are in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. Transformants of mammalian cells are placed under a selection pressure where only transformants are uniquely adapted to survive by virtue of the marker present in the vector. The selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively varied, thereby leading to amplification of both the selection gene and the DNA that encodes the recombinant AFFIMER®-containing protein. As a result, increased amounts of the recombinant AFFIMER®-containing protein are synthesized from the amplified DNA.

The vector may also include at least one ribosome binding site, which will be transcribed into the mRNA comprising the coding sequence for the recombinant AFFIMER® agent protein. For example, such a site is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed. The Shine-Dalgarno sequence is varied but is typically a polypurine (having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using the methods set forth above and used in a prokaryotic vector.

Expression vectors will typically contain a promoter that is recognized by the host organism and operably linked to a nucleic acid molecule encoding the recombinant AFFIMER® agent protein. A native or heterologous promoter may be used depending on the host cell used for expression and the desired yield.

Promoters for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems; the alkaline phosphatase, tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences have been published, and they can be ligated to one or more desired nucleic acid sequences, using linkers or adaptors as desired to provide restriction sites.

Promoters for use with yeast hosts are also known in the art. Yeast enhancers are advantageously used with yeast promoters. Promoters suitable for use with mammalian host cells are well known and include those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and most preferably Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, e.g., heat shock promoters and the actin promoter.

Additional promoters that may be used to express the selective linkers of the disclosure include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, Nature, 290:304-310, 1981); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980), Cell 22:787-97); the herpes thymidine kinase promoter (Wagner et al. (1981), Proc. Natl. Acad. Sci. U.S.A. 78:1444-5); the regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296;39-42, 1982); prokaryotic expression vectors such as the beta-lactamase promoter (Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. U.S.A., 75; 3727-3731, 1978); or the tac promoter (DeBoer, et al. (1983), Proc. Natl. Acad. Sci. U.S.A., 80: 21-5). Also of interest are the following animal transcriptional control regions, which demonstrate tissue specificity and have been used in transgenic animals: the control region of the elastase I gene which is active in pancreatic acinar cells (Swift 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); the control region of the insulin gene which is active in pancreatic beta cells (Hanahan (1985), Nature 315: 115-22); the immunoglobulin gene control region which is active 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); the mouse mammary tumor virus control region which is active in testicular, mammary, lymphoid, and mast cells (Leder et al. (1986), Cell 45: 485-95); the albumin gene control region which is active in the liver (Pinkert et al. (1987), Genes and Devel. 1: 268-76); the control region of the alpha-fetoprotein gene which is active in the liver (Krumlauf et al. (1985), MoI. Cell. Biol. 5: 1639-48; Hammer et al. (1987), Science, 235: 53-8); the control region of the alpha 1-antitrypsin gene which is active in the liver (Kelsey et al. (1987), Genes and Devel. 1: 161-71); the control region of the beta-globin gene which is active in myeloid cells (Mogram et al., Nature, 315 338-340, 1985; Kollias et al. (1986), Cell 46: 89-94); the myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al. (1987), Cell, 48: 703-12); the myosin light chain-2 gene control region which is active in skeletal muscle (Sani (1985), Nature, 314: 283-6); and the gonadotropin-releasing hormone gene control region which is active in the hypothalamus (Mason et al. (1986), Science 234: 1372-8).

An enhancer sequence can be inserted into the vector to increase transcription in eukaryotic host cells. Several enhancer sequences are known to be available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, an enhancer from a virus will be used. Examples of enhancer elements for activation of eukaryotic promoters are the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and the adenovirus enhancers.

While an enhancer may be spliced into the vector at a position 5' or 3' relative to the polypeptide coding region, it is typically located at a site 5' of the promoter.

Vectors for expressing nucleic acids include those that are compatible with bacterial, insect, and mammalian host cells. These vectors include, but are not limited to, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, San Diego, Calif.), pBSII (Stratagene Company, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BlueBacII; Invitrogen), pDSR-alpha (PCT Publication No. WO90/14363), and pFastBacDual (Gibco/BRL, Grand Island, N.Y.).

Additional possible vectors include, but are not limited to, engineered viruses, cosmids, or plasmids, but the vector system must be compatible with the selected host cell. Such vectors include, but are not limited to, plasmids such as Bluescript® plasmid derivatives (a high copy number ColEl-based phagemid, from Stratagene Cloning Systems Inc., La Jolla, Calif.), PCR cloning plasmids designed to clone Taq-amplified PCR products (e.g., TOPO™, TA Cloning® Kit, PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, Calif.), and mammalian, yeast, or viral vectors such as a baculovirus expression system (pBacPAK plasmid derivatives, Clontech, Palo Alto, Calif.). Recombinant molecules may be introduced into host cells by transformation, transfection, infection, electroporation, or other known techniques.

Eukaryotic and prokaryotic host cells, including mammalian cells as hosts for expression of the recombinant AFFIMER® agent protein described herein are well known in the art and include numerous immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma (e.g., Hep G2) cells, A549 cells, 3T3 cells, HEK-293 cells, and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that can be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. Fungal cells include cells of yeasts and filamentous fungi including, for example, cells of Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (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., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurospora crassa.

A variety of host expression vector systems may be used to express the recombinant AFFIMER® agent protein of the disclosure. Such host expression systems represent vehicles by which the recombinant AFFIMER® agent protein coding sequences can be produced and then purified, but also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express the recombinant AFFIMER® agent protein of the disclosure in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing AFFIMER® agent protein coding sequences; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing AFFIMER® agent protein coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g. baculovirus) containing AFFIMER® agent protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g. cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmid) containing AFFIMER® agent protein coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells, lymphatic cells (see U.S. Patent No. 5,807,715), Per C.6 cells (rat retinal cells developed by Crucell)) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending on the intended use for the expressed recombinant AFFIMER® agent protein. For example, where a large quantity of such protein is to be produced, for the generation of pharmaceutical compositions of the recombinant AFFIMER® agent protein, vectors that direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al. (1983) "Easy Identification Of cDNA Clones," EMBO J. 2:1791-1794), wherein the coding sequence of the AFFIMER® agent protein may be individually ligated into the vector in frame with the lac Z coding region such that a fusion protein is produced; pIN vectors (Inouye et al. (1985) "Up-Promoter 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); etc. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, these fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to a glutathione-agarose bead matrix followed by elution in the presence of free glutathione. pGEX vectors are designed to include factor Xa protease or thrombin cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence of the AFFIMER® agent protein can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems can be used. In cases where an adenovirus is used as the expression vector, the sequence of interest encoding the AFFIMER® agent protein can be ligated to an adenovirus transcription/translation control complex, e.g. the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g. the E1 or E3 region) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts. (See e.g. Logan et al. (1984) "Adenovirus Tripartite Leader Sequence Enhances Translation Of mRNAs Late After Infection," Proc. Natl. Acad. Sci. (U.S.A.) 81:3655-3659). Specific initiation signals may also be required for efficient translation of the inserted sequences encoding the AFFIMER® agent protein. These signals include the ATG initiation codon and flanking sequences. In addition, the initiation codon must be in frame with the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons may have a variety of origins, both natural and synthetic. Efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bitter et al. (1987) "Expression and Secretion Vectors For Yeast," Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be selected which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific desired manner. Such modifications (e.g. glycosylation) and processing (e.g. cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected to ensure the correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells which possess the cellular machinery for the correct processing of primary transcription, glycosylation and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is contemplated. For example, cell lines that stably express an antibody of the disclosure can be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. Following introduction of the foreign DNA, the engineered cells can be allowed to grow for 1 to 2 days in enriched medium and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can in turn be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines that express the recombinant AFFIMER® agent proteins of the disclosure. Such engineered cell lines can be particularly useful in screening and evaluating compounds that interact directly or indirectly with the recombinant AFFIMER® agent proteins.

A number of selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al. (1977) "Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells," Cell 11:223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al. (1962) "Genetics Of Human Cess Line. IV. DNA-Mediated Heritable Transformation of a Biochemical Trait," Proc. Natl. Acad. Sci. (U.S.A.) 48:2026-2034), and adenine phosphoribosyltransferase (Lowy et al. (1980) "Isolation Of Transforming DNA: Cloning The Hamster Aprt Gene," Cell 22:817-823) whose genes can be employed in cells tk-, hgprt- or aprt-, respectively. Also, resistance to antimetabolites can be used as a basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. (1980) "Transformation Of Mammalian Cells With An Amplfiable Dominant-Acting Gene," Proc. Natl. Acad. Sci. (U.S.A.) 77:3567-3570; O'Hare et al. (1981) "Transformation Of Mouse Fibroblasts To Methotrexate Resistance By A Recombinant Plasmid Expressing A Prokaryotic Dihydrofolate Reductase," Proc. Natl. Acad. Sci. (U.S.A.) 78:1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan et al. (1981) "Selection For Animal Cells That Express The Escherichia coli Gene Coding For Xanthine-Guanine Phosphoribosyltransferase," Proc. Natl. Acad. Sci. (U.S.A.) 78:2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Tachibana et al. (1991) "Altered Reactivity Of Immunoglobutin Produced By Human-Human Hybridoma Cells Transfected By pSV.2-Neo Gene," Cytotechnology 6(3):219-226; Tolstoshev (1993) "Gene Therapy, Concepts, Current Trials And Future Directions," Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993) "The Basic Science of Gene Therapy," Science 260:926-932; and Morgan et al. (1993) "Human gene therapy," Ann. Rev. Biochem. 62:191-217). Methods commonly known in the art of recombinant DNA technology that can be used are 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) “A New Dominant Hybrid Selective Marker For Higher Eukaryotic Cells,” J. Mol. Biol. 150:1-14; and hygro, which confers resistance to hygromycin (Santerre et al. (1984) "Expression Of Prokaryotic Genes For Hygromycin B And G418 Resistance As Dominant-Selection Markers In Mouse L Cells," Gene 30:147-156).

Expression levels of a recombinant AFFIMER®-containing protein can be increased by vector amplification (for review, see Bebbington and Hentschel, "The Use of Vectors Based On Gene Amplification For The Expression Of Cloned Genes In Mammaian Cells," in DNA CLONING, Volume 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing a recombinant AFFIMER®-containing protein is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the recombinant AFFIMER®-containing protein, production of the recombinant AFFIMER®-containing protein will also increase (Crouse et al. (1983) "Expression and Amplification of Engineered Mouse Dihydrofolate Reductase Minigenes," Mol. Cell. Biol. 3:257-266).

Where the AFFIMER® agent is an AFFIMER® antibody fusion or other multiprotein complex, the host cell may be cotransfected with two expression vectors, for example, the first vector encoding a heavy chain and the second vector encoding a light chain-derived polypeptide, one or both of which comprise a sequence encoding the AFFIMER® polypeptide. Both vectors may contain identical selectable markers that allow for equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used that encodes both the heavy and light chain polypeptides. In such situations, the light chain must be placed before the heavy chain to avoid an excess of 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. (U.S.A.) 77:2197-2199). The sequences encoding the heavy and light chains may include cDNA or genomic DNA.

In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic of the glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation profile of the recombinant AFFIMER® agent protein will depend on the particular cell line or transgenic animal used to produce the protein. In certain embodiments of AFFIMER®/antibody fusions, a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous, because in the case of antibodies, it has been shown to generally exhibit more potent efficacy than fucosylated counterparts both in vitro and in vivo (See, e.g., Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Patent NOS: 6,946,292 and 7,214,775).

In addition, expression of an AFFIMER® agent from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos.: 0216846, 0256055 and 0323997 and European Patent Application No. 89303964.4. Thus, in certain embodiments of the invention, mammalian host cells (e.g., CHO) lack the glutamine synthetase gene and are cultured in the absence of glutamine in the medium wherein, however, the polynucleotide encoding the immunoglobulin chain comprises a glutamine synthetase gene that complements the absence of the gene in the host cell. Such host cells containing the binder or polynucleotide or vector as described herein as well as expression methods, as described herein, for making the binder using such a host cell are part of the present disclosure.

Expression of recombinant proteins in insect cell culture systems (e.g. baculovirus) also provides a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for the production of heterologous proteins in insect cells are well known to those skilled in the art.

AFFIMER®-enhanced recombinant proteins produced by a transformed host may be purified by any suitable method. Standard methods include chromatography (e.g., size exclusion chromatography, affinity, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza envelope sequence, and glutathione-S-transferase may be attached to the protein to allow easy purification by passage through an appropriate affinity column. Isolated proteins may 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.

In some embodiments, recombinant AFFIMER®-agent proteins produced in bacterial culture may be isolated, for example, by initial extraction from cell pellets, followed by at least one concentration, salting out, aqueous ion exchange, or size exclusion chromatography step. HPLC may be used for final purification steps. Microbial cells used in expression of a recombinant protein may be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

Methods of use and pharmaceutical compositions

The AFFIMER® agents of the disclosure are useful in a variety of applications including, but not limited to, therapeutic treatment methods for systemic lupus erythematosis (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/colitis), graft-versus-host disease (GvHD) (related to stem cell transplants) also known as allograft rejection, solid organ transplantation/transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, inflammatory bowel disease (IBD) and osteoarthritis. collagen, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, glomerulosclerosis and/or cancer. The methods of use may be in vitro, ex vivo or in vivo methods.

Systemic Lupus Erythermatosis

Systemic lupus erythematosus (SLE), commonly referred to simply as lupus, is a chronic autoimmune disease that can cause swelling (inflammation) and pain throughout the body. There are several different types of lupus. Systemic lupus erythematosus is the most common. Other types of lupus include:

Cutaneous lupus erythematosus: This type of lupus affects the skin - cutaneous is a term that means skin. People with cutaneous lupus erythematosus may experience skin problems such as sun sensitivity and rashes. Hair loss can also be a symptom of this condition.

Drug-induced lupus: These cases of lupus are caused by certain medications. People with drug-induced lupus may have many of the same symptoms as systemic lupus erythematosus, but it is usually temporary.

Neonatal lupus: A rare type of lupus, neonatal lupus is a condition that is found in infants at birth. Children born with neonatal lupus have antibodies that were passed down to them from their mothers—who either had lupus at the time of pregnancy or may develop the condition later in life. Not all babies born to a mother with lupus will develop the disease.

Therapies that may be used in combination with the AFFIMER® TNFR2 polypeptides offered herein include, for example: steroids (corticosteroids, including prednisone); Hydroxychloroquine (Plaquenil®); Azathioprine (Imuran®); Methotrexate (Rheumatrex®); Cyclophosphamide (Cytoxan®) and Mycophenolate Mofetil (CellCept®); Belimumab (Benlysta®); and/or Rituximab (Rituxan®).

Lupus nephritis

Lupus nephritis is a common complication in people with systemic lupus erythematosus, more commonly known as lupus. Lupus nephritis occurs when lupus autoantibodies affect the structures in the kidneys that filter waste. This causes inflammation of the kidneys and can lead to blood in the urine, protein in the urine, high blood pressure, impaired kidney function, or even kidney failure. Up to half of adults with systemic lupus develop lupus nephritis. Systemic lupus causes immune system proteins to damage the kidneys, impairing their ability to filter waste.

Rheumatoid arthritis

Rheumatoid arthritis is a chronic (lifelong) type of arthritis that affects joints on both sides of the body, such as the hands, wrists, and knees. The short-term goals of rheumatoid arthritis medications are to reduce joint pain and swelling and/or improve joint function. The long-term goal is to slow or stop the disease process, especially joint damage.

Arthritis is a general term that describes inflammation of the joints. Rheumatoid arthritis is a chronic (permanent) type of arthritis (causing pain and swelling) that usually occurs in the joints symmetrically (on both sides of the body, such as the hands, wrists, and knees). This involvement of multiple joints helps distinguish rheumatoid arthritis from other types of arthritis.

In addition to affecting the joints, rheumatoid arthritis can occasionally affect the skin, eyes, lungs, heart, blood, nerves, or kidneys.

Therapies that may be used in combination with the TNFR2 AFFIMER® agents proposed herein to treat rheumatoid arthritis may include, for example:

Medications that reduce pain and inflammation. These products include nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen (MOTRIN®), naproxen (ALEVE®), and similar products. Another type of medication - a COX-2 inhibitor - also falls into this category of medications, providing relief from the signs and symptoms of rheumatoid arthritis. Celecoxib (CELEBREX®), a COX-2 inhibitor, is available and used in the United States. COX-2 inhibitors were designed to have fewer side effects of stomach bleeding.

Disease-modifying antirheumatic drugs (DMARDs). Unlike other NSAIDs, DMARDs can actually slow the disease process by altering the immune system. Older DMARDs include methotrexate (TREXALL®), gold, penicillamine (CUPRIMINE®), hydroxychloroquine (PLAQUENIL®), sulfasalazine (AZULFIDINE®), cyclosporine (SANDIMMUNE®), cyclophosphamide (CYTOXAN®), and leflunomide (ARAVA®). Currently, methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine are the most commonly used. (Cyclosporine, cyclophosphamide, gold, and penicillamine are generally no longer used.)

Biologics. Beyond these more “traditional” DMARDs, newer drugs have been approved. Currently, there are seven different classes of drugs, and in some cases, different kinds exist within each class. (Some of these, like the TNF blockers, have been in use since 2000.) Collectively, these DMARDs are known by another name – biologics (or biologic response agents). Compared to traditional DMARDs, these drugs target the molecules that cause inflammation in rheumatoid arthritis. Inflammatory cells in the joints are implicated in the development of rheumatoid arthritis itself. Biologics reduce the inflammatory process that ultimately causes the joint damage seen in rheumatoid arthritis. Older DMARDs work a bit further than biologics; they work by altering the body’s own immune response to inflammation. By attacking cells at a more specific level than the inflammation itself, biologics are considered more effective and more specifically targeted. Biologics include etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), anakinra (KINARET®), abatacept (ORENCIA®), rituximab (RITUXAN®), certolizumab pegol (CIMZIA®), golimumab (SYMPONI ®), tocilizumab (ACTEMRA®), and tofacitinib (XELJANJ®). Some of the biologics are used in combination with traditional DMARDs, particularly methotrexate.

Multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease. With these conditions, the immune system mistakenly attacks healthy cells. In people with MS, the immune system attacks cells in myelin, the protective sheath that surrounds nerves in the brain and spinal cord. Damage to the myelin sheath interrupts nerve signals from the brain to other parts of the body. The damage can cause symptoms affecting the brain, spinal cord, and eyes.

There are four types of multiple sclerosis:

Clinically isolated syndrome (CIS): When a person has a first episode of MS symptoms, health care providers often classify it as CIS. Not everyone with CIS develops multiple sclerosis.

Relapsing-remitting MS (RRMS): This is the most common form of multiple sclerosis. People with RRMS have attacks—also called relapses or exacerbations—of new or worsening symptoms. Periods of remission follow (when symptoms stabilize or disappear).

Primary progressive multiple sclerosis (PPMS): People diagnosed with PPMS have symptoms that slowly and gradually worsen without any periods of relapse or remission.

Secondary progressive multiple sclerosis (SPMS): In many cases, people initially diagnosed with RRMS eventually progress to SPMS. With secondary progressive multiple sclerosis, nerve damage continues to accumulate. Symptoms gradually worsen. Although you may still experience relapses or flare-ups (when symptoms increase), you do not have periods of remission (when symptoms stabilize or disappear) afterward.

Therapies that may be used in combination with the TNFR2 AFFIMER® polypeptides offered herein include, for example:

Disease-modifying therapies (DMTs): Several medications have FDA approval for the long-term treatment of MS. These medications help reduce relapses (also called attacks or relapses). They slow the progression of the disease. And they can prevent new lesions from forming in the brain and spinal cord.

Relapse management medications: In severe attacks, a neurologist may recommend a high dose of corticosteroids. The medication can quickly reduce inflammation. They slow damage to the myelin sheath surrounding nerve cells.

Physical rehabilitation: Multiple sclerosis can affect physical functions. Staying physically fit and strong helps maintain mobility.

Mental health counseling: Coping with a chronic condition can be emotionally challenging. MS can sometimes affect mood and memory. Working with a neuropsychologist or having other emotional support is an essential part of managing the disease.

Chronic inflammatory bowel disease

Inflammatory bowel diseases (IBD) are a group of disorders that cause chronic inflammation (pain and swelling) in the intestines.

Crohn's disease and ulcerative colitis are the main types of IBD. Types include:

Crohn's disease causes pain and swelling in the digestive tract. It can affect anywhere from the mouth to the anus. It most commonly affects the small intestine and the upper part of the large intestine.

Ulcerative colitis causes swelling and sores (ulcers) in the large intestine (colon and rectum).

Microscopic colitis causes intestinal inflammation that is only detectable under a microscope.

Therapies that may be used in combination with the AFFIMER® TNFR2 polypeptides offered here include, for example: aminosalicylates (an anti-inflammatory medication such as sulfasalazine, mesalamine, or balsalazide) minimize intestinal irritation; antibiotics treat infections and abscesses; biologics interrupt signals from the immune system that cause inflammation; corticosteroids, such as prednisone, keep the immune system in check and manage flare-ups; immunomodulators calm an overactive immune system; antidiarrheal medications; nonsteroidal anti-inflammatory drugs (NSAIDs); and vitamins and supplements such as probiotics.

Graft versus host disease

Graft-versus-host disease (GvHD) is a condition that can occur after an allogeneic transplant. In GvHD, donated bone marrow or peripheral blood stem cells perceive the recipient's body as foreign, and the donated cells/bone marrow attack the body.

There are two forms of GvHD: acute graft versus host disease (aGvHD); and chronic graft versus host disease (cGvHD).

Psoriasis

Psoriasis is a chronic skin condition, meaning a skin condition that does not go away. People with psoriasis have thick, pink or red patches of skin covered in white or silvery scales. The thick, scaly patches are called plaques. Psoriasis usually begins in early adulthood, although it can start later in life. In addition to red, scaly patches, symptoms of psoriasis include: itchy, dry, cracked skin, scaly scalp, skin pain, pitted, cracked, or brittle nails, and joint pain.

Therapies that may be used in combination with the AFFIMER® TNFR2 polypeptides offered herein include, for example: Steroid creams, moisturizers for dry skin, anthralin (a drug that slows the production of skin cells), medicated lotions, shampoos and baths to improve scalp psoriasis, vitamin D3 ointments, vitamin A or retinoid creams, light therapy, PUVA (a treatment that combines a drug called psoralen with exposure to a special form of UV light), methotrexate, retinoids, cyclosporine and/or immune therapies.

Sjogren's syndrome

Sjögren's syndrome is a lifelong autoimmune disease that reduces the amount of moisture produced by the glands in the eyes and mouth. It is named after Henrik Sjögren, a Swedish oculist who first described the condition. Although dry mouth and dry eyes are the main symptoms, most people who have these problems do not have Sjögren's syndrome. Dry mouth is also called xerostomia.

There are two forms of Sjögren's syndrome: primary Sjögren's syndrome, which develops on its own, not because of another health problem, and secondary Sjögren's syndrome, which develops in addition to other autoimmune diseases such as rheumatoid arthritis, lupus, and psoriatic arthritis.

Therapies that may be used in combination with the TNFR2 AFFIMER® polypeptides provided herein include, e.g., treatments for dry eyes (e.g., artificial tears, prescription eye drops, punctal plugs, surgery, autologous serum drops), treatments for dry mouth (e.g., saliva producers), treatments for joint or organ problems (e.g., analgesics, antirheumatic drugs, immunosuppressants, steroids, antifungals, and treatments for vaginal dryness.

Autoimmune myasthenia gravis

Autoimmune myasthenia gravis (MG) is an autoimmune disease, which means that the body's immune system mistakenly attacks its own parts. MG affects the communication between nerves and muscles (the neuromuscular junction).

People with MG lose the ability to voluntarily control their muscles. They experience muscle weakness and fatigue of varying severity. They may not be able to move the muscles of the eyes, face, neck, and limbs. MG is a lifelong neuromuscular disease.

MG affects about 20 out of every 100,000 people. Experts estimate that 36,000 to 60,000 Americans have this neuromuscular disease. The actual number of people affected may be higher because some people with mild cases may not know they have the disease. MG most commonly affects women ages 20 to 40 and men ages 50 to 80. About one in 10 cases of MG occurs in adolescents (juvenile MG). The disease can affect people of all ages but is rare in children.

Autoimmune MG is the most common form of this neuromuscular disease. Autoimmune MG can be:

Eye: The muscles that move the eyes and eyelids become weak. The eyelids may droop or it may not be possible to keep the eyes open. Some people experience double vision. Eye weakness is often the first sign of MG. About half of people with ocular MG progress to the generalized form within two years of the first symptom.

Generalized: Muscle weakness affects the eyes and other parts of the body such as the face, neck, arms, legs, and throat. It may be difficult to speak or swallow, raise the arms above the head, stand up from a sitting position, walk long distances, and climb stairs.

Therapies that may be used in combination with the TNFR2 AFFIMER® polypeptides provided herein include, for example, drugs, monoclonal antibodies, IV immunoglobulin (IVIG), plasma exchange (plasmapheresis), and/or surgery.

Cancer

TNF binding to TNFR2 has been shown to promote activation of tumor cells and tumor-associated cell types, while affecting the immune response, such as tumor-infiltrating lymphocytes, to create an environment in which the tumor can progress. Indeed, TNFR2 expression in tumors is associated with disease progression and poor clinical outcomes (Sheng Y, et al.). Therefore, the AFFIMER® TNFR2 polypeptides of the present invention may be useful in the treatment of cancer, either as monotherapy or in conjunction with other cancer treatments such as immunotherapies, e.g., CAR-T therapies, checkpoint inhibitors, and conventional anticancer therapies.

Pharmaceutical Preparations Formulations are prepared for storage and use by combining a purified AFFIMER® agent of the present disclosure with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Pharmaceutically acceptable carriers, excipients and/or stabilizers are generally considered by those skilled in the art to be inactive ingredients of a pharmaceutical formulation or composition.

In some embodiments, an AFFIMER® agent described herein is lyophilized and/or stored in a lyophilized form. In some embodiments, a formulation comprising an AFFIMER® agent described herein is lyophilized.

Suitable pharmaceutically acceptable carriers include, but are not limited to, nontoxic buffers such as phosphate, citrate 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, phenol, butyl or benzyl alcohol, alkylparabens such as methyl- or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, 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 dextrins; 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 TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London.).

The pharmaceutical compositions of the present disclosure may be administered in any number of ways for either local or systemic treatment. Administration may be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including nebulization, intratracheal and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g. injection or infusion) or intracranial (e.g. intrathecal or intraventricular).

The therapeutic formulation may be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier. Typical tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These may be used to form a solid preformulation composition containing a homogeneous mixture of a compound of the present disclosure, or a non-toxic pharmaceutically acceptable salt thereof. The solid preformulation composition is then subdivided into unit dosage forms of a type described above. The tablets, pills, etc. of the formulation or composition may be coated or otherwise mixed to provide a dosage form having the advantage of prolonged action. For example, the tablet or pill may include an inner composition coated with an outer component. In addition, the two components may be separated by an enteric layer which serves to resist disintegration and allows the inner component to pass through the stomach intact or to be delayed in its release. A variety of materials may be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

The AFFIMER® agents described herein may also be entrapped in microcapsules. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London.

In some embodiments, the pharmaceutical formulations comprise an AFFIMER® agent of the present disclosure complexed with liposomes. Methods of producing the liposomes are known to those skilled in the art. For example, some liposomes may be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes may be extruded through filters of defined pore size to produce liposomes of the desired diameter.

In some embodiments, sustained release preparations comprising AFFIMER® agents described herein may be produced. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing an AFFIMER® agent, wherein the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained release matrices include 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 copolymers of lactic acid and glycolic acid such as LUPRON DEPOT™. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate and poly-D-(-)-3-hydroxybutyric acid.

In some embodiments, in addition to administering an AFFIMER® agent described herein, the method or treatment further comprises administering at least one additional therapeutic agent. An additional therapeutic agent may be administered prior to, concurrently with, and/or subsequent to administration of the AFFIMER® agent. Pharmaceutical compositions comprising an AFFIMER® agent and the additional therapeutic agent(s) are also provided. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

Combination therapy with two or more therapeutic agents often uses agents that act by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergistic effects. Combination therapy may allow a lower dose of each agent than that used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the AFFIMER® agent.

In certain embodiments of the methods described herein, the combination of an AFFIMER® agent described herein and at least one additional therapeutic agent results in additive or synergistic results. In certain embodiments, the combination therapy results in an increase in the therapeutic index of the AFFIMER® agent. In certain embodiments, the combination therapy results in an increase in the therapeutic index of the therapeutic agent(s). In certain embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the AFFIMER® agent. In certain embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the therapeutic agent(s).

Combined administration may include coadministration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.

It will be understood that the combination of an AFFIMER® agent described herein and at least one additional therapeutic agent may be administered in any order or concurrently. In some embodiments, the AFFIMER® agent will be administered to patients who have previously undergone treatment with a second therapeutic agent. In some other embodiments, the AFFIMER® agent and a second therapeutic agent will be administered substantially simultaneously or concurrently. For example, a subject may receive an AFFIMER® agent while undergoing a treatment trajectory with a second therapeutic agent (e.g., chemotherapy). In some embodiments, an AFFIMER® agent will be administered within 1 year of treatment with a second therapeutic agent. In some alternative embodiments, an AFFIMER® agent will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In some other embodiments, an AFFIMER® agent will be administered within 4, 3, 2, or 1 week(s) of any treatment with a second therapeutic agent. In some embodiments, an AFFIMER® agent will be administered within 5, 4, 3, 2, or 1 day(s) of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments may be administered to the subject within a matter of hours or minutes (e.g., substantially simultaneously).

For the treatment of disease, the appropriate dosage of an AFFIMER® agent of this disclosure depends on the type of disease being treated, the severity and course of the disease, the responsiveness of the disease, whether the AFFIMER® agent is being administered for therapeutic or preventive purposes, prior therapies, the patient's clinical history, etc., all at the discretion of the treating physician. The AFFIMER® agent may be administered as a single dose or over a series of treatments lasting from several days to several months, or until cure is achieved or abatement of the disease state is achieved. Optimal dosage regimens can be calculated from measurements of drug accumulation in the patient's body and will vary depending on the relative potency of each individual agent. The treating physician can determine optimal dosages, dosing methodologies, and repetition frequencies. In some embodiments, the dosage is from 0.01 ug to 100 mg/kg body weight, from 0.1 ug to 100 mg/kg body weight, from 1 ug to 100 mg/kg body weight, from 1 mg to 100 mg/kg body weight, from 1 mg to 80 mg/kg body weight, from 10 mg to 100 mg/kg body weight, from 10 mg to 75 mg/kg body weight, or from 10 mg to 50 mg/kg body weight. In some embodiments, the dosage of the AFFIMER® agent is from about 0.1 mg to about 20 mg/kg body weight. In some embodiments, the dosage of the AFFIMER® agent is about 0.1 mg/kg body weight. In some embodiments, the dosage of the AFFIMER® agent is about 0.25 mg/kg body weight. In some embodiments, the dosage of the AFFIMER® agent is about 0.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 1 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 1.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 2 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 2.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 7.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 10 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 12.5 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 15 mg/kg of body weight. In some embodiments, the dosage may be administered once or more per day, per week, per month, or per year. In some embodiments, the AFFIMER® agent is given once per week, once every two weeks, once every three weeks, or once every four weeks.

In some embodiments, an AFFIMER® agent may be administered at a higher initial “loading” dose, followed by at least one lower dose. In some embodiments, the frequency of administration may also vary. In some embodiments, a dosage regimen may include administering an initial dose, followed by additional doses (or “maintenance” doses) once a week, every two weeks, every three weeks, or every month. For example, a dosage regimen may include administering an initial loading dose, followed by a weekly maintenance dose of, for example, half the initial dose. In some embodiments, a dosage regimen includes administering an initial loading dose, followed by maintenance doses of, for example, half the initial dose every two weeks. In some embodiments, a dosage regimen includes administering three initial doses over 3 weeks, followed by maintenance doses, for example, of the same amount every two weeks.

As is known to those skilled in the art, the administration of any therapeutic agent may result in side effects and/or toxicities. In some cases, the side effects and/or toxicities are so severe that they preclude the administration of the particular agent at a therapeutically effective dose. In some cases, drug therapy must be discontinued, and other agents may be tried. However, many agents within the same therapeutic class often exhibit similar side effects and/or toxicities, meaning that the patient must either discontinue therapy or, if possible, suffer the unpleasant side effects associated with the therapeutic agent.

In some embodiments, the dosing regimen may be limited to a specific number of administrations or "cycles." In some embodiments, the AFFIMER® agent is administered for 3, 4, 5, 6, 7, 8, or more cycles. For example, the AFFIMER® agent is administered every 2 weeks for 6 cycles, the AFFIMER® agent is administered every 3 weeks for 6 cycles, the AFFIMER® agent is administered every 2 weeks for 4 cycles, the AFFIMER® agent is administered every 3 weeks for 4 cycles, etc. Dosing schedules may be decided and subsequently modified by those skilled in the art.

Thus, the present invention provides methods of administering to a subject the polypeptides or agents described herein comprising using an intermittent dosing strategy to administer at least one agent (e.g., two or three agents), which may reduce side effects and/or toxicities associated with administration of an AFFIMER® agent. In some embodiments, a method of treating an inflammatory or autoimmune disease in a human subject comprises administering to the subject a therapeutically effective amount of an AFFIMER® agent in combination with a therapeutically effective amount of another therapeutic agent, wherein one or both agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an AFFIMER® agent to the subject and administering subsequent doses of the AFFIMER® agent approximately once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an AFFIMER® agent to the subject and administering subsequent doses of the AFFIMER® agent approximately once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of an AFFIMER® agent to the subject and administering subsequent doses of the AFFIMER® agent approximately once every 4 weeks. In some embodiments, the AFFIMER® agent is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly.

Other embodiments of the invention are set forth in the clauses below.

1. An engineered polypeptide that specifically binds to TNFR2 with a Kd of 1×10 ^-6 M or less, wherein the engineered polypeptide is a variant of a Stefin A protein.

2. An engineered polypeptide according to clause 1 comprising an amino acid sequence having at least 80%, at least 90%, at least 95% or at least 98% identity with an amino acid sequence of MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa)m-Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4),

where Xaa, individually for each occurrence, is an amino acid residue; and n and m are each, independently, an integer from 3 to 20.

3. An engineered polypeptide according to clause 2 wherein the amino acid sequence has 100% identity with an amino acid sequence of MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa)m-Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF (SEQ ID NO: 4),

where Xaa, individually for each occurrence, is an amino acid residue; and n and m are each, independently, an integer from 3 to 20.

4. An engineered polypeptide according to clause 1 comprising an amino acid sequence having at least 80%, at least 90%, at least 95% or at least 98% identity with an amino acid sequence of

(SEQ ID NO: 5),

where Xaa, individually for each occurrence, is an amino acid residue; and n and m are each, independently, an integer from 3 to 20.

5. An engineered polypeptide according to clause 4 where the amino acid sequence has 100% identity with an amino acid sequence of

(SEQ ID NO: 5),

where Xaa, individually for each occurrence, is an amino acid residue; and n and m are each, independently, an integer from 3 to 20.

6. An engineered polypeptide according to any one of clauses 1 to 5, wherein n is an integer from 5 to 18.

7. An engineered polypeptide according to clause 6, wherein n is an integer from 7 to 15.

8. An engineered polypeptide according to clause 7, wherein n is an integer from 8 to 12.

9. An engineered polypeptide according to any one of clauses 1 to 8, wherein n is an integer from 5 to 18.

10. An engineered polypeptide according to clause 9, wherein m is an integer from 7 to 15.

11. An engineered polypeptide according to clause 10, wherein m is an integer from 8 to 12.

12. An engineered polypeptide according to any one of clauses 1 to 11 that binds to TNFR2 with a Kd of 1×10 ^-7 M.

13. Engineered polypeptide according to clause 12 that binds to TNFR2 with a Kd of 1×10 ^-8 M.

14. An engineered polypeptide according to any one of clauses 1 to 13, wherein (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6-102.

15. An engineered polypeptide according to clause 14, wherein (Xaa)n consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 6-102.

16. An engineered polypeptide according to any one of clauses 1 to 15, wherein (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 103-199.

17. An engineered polypeptide according to clause 16, wherein (Xaa)m consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 103-199.

18. An engineered polypeptide according to clause 14, wherein (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 11, 13, 16, 17, 24, 25, 29, 33, 41, 54, 65, 67, 73, 75, 82, 89, 90, 98 and 99.

19. An engineered polypeptide according to clause 18, wherein (Xaa)n consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 11, 13, 16, 17, 24, 25, 29, 33, 41, 54, 65, 67, 73, 75, 82, 89, 90, 98 and 99.

20. An engineered polypeptide according to clause 16, wherein (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 103, 108, 110, 113, 114, 121, 122, 126, 130, 138, 151, 162, 164, 170, 172, 179, 186, 187, 195 and 196.

21. An engineered polypeptide according to clause 20, wherein (Xaa)m consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 103, 108, 110, 113, 114, 121, 122, 126, 130, 138, 151, 162, 164, 170, 172, 179, 186, 187, 195 and 196.

22. An engineered polypeptide according to clause 1 comprising an amino acid sequence having at least 75%, at least 85%, or at least 95% identity with an amino acid sequence of SEQ ID No: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292 to 293, wherein the sequence optionally excludes one or more of the twenty-one carboxy terminal residues.

23. An engineered polypeptide according to clause 22 comprising an amino acid sequence having 100% identity with an amino acid sequence of SEQ ID No: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292 to 293, wherein the sequence optionally excludes one or more of the twenty-one carboxy terminal residues.

24. An engineered polypeptide according to any one of clauses 1 to 23, wherein the Stefin A polypeptide is a mammalian Stefin A polypeptide.

25. An engineered polypeptide according to any one of clauses 1 to 24, wherein the Stefin A polypeptide is a human Stefin A polypeptide.

26. An engineered polypeptide according to any one of clauses 1 to 25, further comprising a signal peptide.

27. A fusion protein comprising a dimer, trimer or tetramer of the engineered polypeptide according to any one of clauses 1 to 26, optionally comprising one or more linkers.

28. A fusion protein according to clause 10, wherein the linker is a rigid linker.

29. A fusion protein according to clause 28, wherein the rigid linker is a peptide linker comprising the sequence (EAAAK)n, where n is 1-6.

30. A fusion protein comprising one or more of the engineered polypeptides according to any one of clauses 1 to 29 linked to a therapeutic or diagnostic moiety.

31. Fusion protein according to clause 30, wherein the therapeutic moiety is a therapeutic protein or peptide.

32. Fusion protein according to clause 31, wherein the therapeutic group is an antibody.

33. Fusion protein according to clause 30, wherein the diagnostic moiety is a fluorescent protein.

34. An engineered polypeptide according to any one of clauses 1 to 26 or the fusion protein according to any one of clauses 27 to 33 further comprising a half-life extension moiety.

35. An engineered polypeptide or fusion protein according to clause 34, wherein the half-life extension moiety is selected from antibody Fc domains, human serum albumin, serum binding proteins.

36. An engineered polypeptide or fusion protein according to clause 34, wherein the half-life extension moiety is a variant of a Stefin A protein that specifically binds to human serum albumin with a Kd of 1×10 ^-6 M or less.

37. An engineered polypeptide or fusion protein according to any of clauses 1 to 36, wherein the Kd is measured by Biacore kinetic analysis.

38. An engineered polypeptide or fusion protein according to clause 37, wherein the Kd is measured as set forth in EXAMPLE 3, “Biacore assay”.

39. An engineered polypeptide or fusion protein according to any of clauses 1 to 38, wherein the engineered polypeptide binds to TNFR2 as a monomer with an IC50 in a competitive binding assay with TNFR2 of 1 uM 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.

40. An engineered polypeptide or fusion protein according to any of clauses 1 to 39, wherein the TNFR2 is a mammalian TNFR2.

41. An engineered polypeptide or fusion protein according to clause 40, wherein the TNFR2 is a human TNFR2.

42. An engineered polypeptide or fusion protein according to clause 40, wherein the TNFR2 is a mouse TNFR2.

43. An engineered polypeptide or fusion protein according to clause 34, wherein the half-life extension moiety is selected from the group consisting of SEQ ID NO: 471-477.

44. Polynucleotide or set of polynucleotides encoding the engineered polypeptide or fusion protein according to any of clauses 1 to 43.

45. A polynucleotide or set of polynucleotides according to clause 44, comprising a nucleic acid sequence having at least 80%, at least 90%, at least 95% or at least 98% identity with a sequence selected from the group consisting of SEQ ID NO: 297, 302, 304, 307, 308, 315, 316, 320, 324, 332, 345, 356, 358, 364, 366, 373, 380, 381, 389 and 290, wherein the sequence optionally excludes nucleotides encoding one or more of the twenty-one carboxy terminal residues.

46. Delivery vehicle comprising the polynucleotide of one of clauses 44 or 45.

47. Delivery vehicle as per clause 46, which is a viral distribution vehicle.

48. Delivery vehicle according to clause 47, wherein the viral delivery vehicle is an adenoviral vector, a retroviral vector, a lentiviral vector or an adeno-associated viral (AAV) vector.

49. Delivery vehicle as per clause 46, which is a non-viral delivery vehicle.

50. Delivery vehicle according to clause 49, wherein the non-viral delivery vehicle is a liposome or a lipid nanoparticle.

51. Plasmid or minicircle comprising the polynucleotide according to any one of clauses 44 to 50.

52. Messenger RNA (mRNA) comprising an open reading frame encoding the engineered polypeptide according to any of clauses 1 to 43.

53. The mRNA according to clause 52 further comprising an open reading frame encoding binding to immune cells.

54. Lipid nanoparticle, optionally cationic lipid nanoparticle, comprising mRNA according to clause 52 or 53.

55. A lipid nanoparticle according to clause 54, wherein the lipid nanoparticle is a cationic lipid nanoparticle.

56. A pharmaceutical composition comprising the engineered polypeptide, fusion protein or delivery vehicle according to any one of clauses 1 to 50, and optionally a pharmaceutically acceptable excipient.

57. A conjugate comprising the engineered polypeptide or fusion protein according to any one of clauses 1 to 43 linked to a pharmacologically active moiety.

58. A method comprising administering to a subject the pharmaceutical composition according to clause 56.

59. The procedure of clause 58 where the subject suffers from systemic lupus erythematosis (SLE), lupus nephritis (e.g. drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), chronic inflammatory bowel disease (IBD) (e.g. Crohn’s disease and colitis/ulcerative colitis), graft versus host disease (GvHD) (related to stem cell transplants) also known as allograft rejection, solid organ transplantation/transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, glomerulosclerosis or cancer.

60. Engineered polypeptide, fusion protein or delivery vehicle according to any of clauses 1 to 50 for use as a medicament.

61. The engineered polypeptide, fusion protein or delivery vehicle of clause 60 for use in the treatment of systemic lupus erythematosis (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) (related to stem cell transplantation) also known as allograft rejection, solid organ transplantation/transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, glomerulosclerosis or cancer.

62. Use of the engineered polypeptide, fusion protein or delivery vehicle according to any of clauses 1 to 50 in the manufacture of a medicament for the treatment of systemic lupus erythematosis (SLE), lupus nephritis (e.g. drug-induced lupus nephritis), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), multiple sclerosis (MS), chronic inflammatory bowel diseases (IBD) (e.g. Crohn's disease and colitis/ulcerative colitis), graft-versus-host disease (GvHD) (related to stem cell transplantation) also known as allograft rejection, solid organ transplantation/transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, erythema multiforme (EPM)-induced arthritis, collagen, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, glomerulosclerosis or cancer.

63. An engineered polypeptide that competes for binding to TNFR2 with an engineered polypeptide according to any of clauses 1 to 43.

64. An engineered polypeptide that binds to the same epitope on TNFR2 as an engineered polypeptide according to any of clauses 1 to 43.

65. An engineered polypeptide or fusion protein according to any one of clauses 1 to 64 which comprises a signal sequence and/or a transmembrane domain.

EXAMPLES

The aim of these experiments was to identify AFFIMER® polypeptides that are specific agonists of human and mouse TFNR2.

Example 1. Selection of TNFR2-binding AFFIMER® polypeptides from a phage display library

Identification of candidate clones

The AFFIMER® TNFR2 polypeptides of the present invention were identified by screening from an AFFIMER® polypeptide library with two random loop sequences, each loop having a length of about 9 amino acids displayed in a constant AFFIMER® polypeptide framework backbone based on the amino acid sequence of Stefin A. Such screening procedures have been described (see, e.g., Tiede et al. Protein Eng Des Sel. 2014. 27(5): 145–155 and Hughes et al. Sci Signal. 2018. 10(505): eaaj2005). According to such procedures, phage suspensions expressing AFFIMER® proteins were incubated with human (or mouse in subsequent experiments) TNFR2 as needed, the sequences of which are shown in Table 7.

Table 7. TNFR protein sequences used in screening (from Sino Biologicals). Protein Target sequence Plasmid catalog number SEQ ID NO: Human TNFR2 MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQN RICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPT PEPSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTR NQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS Cat: HG10417-CY 478 Human TNFR1 MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVE ISSCVTRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLSL LFIGLMYRYQRWKSKLYSIVCGKSTPEKEGELEGTTTKPLAPNPSFSPTPGFTPTLGFSPVPSSTFTSSSTYTPGDCPNFAAPRREVAPPYQGADPILATALASDPIPNPLQKW EDSAHKPQSLDTDDPATLYAVVENVPPLRWKEFVRRLGLSDHEIDRLELQNGRCLREAQYSMLATWRRRTPRREATLELLGRVLRDMDLLGCLEDIEEALCGPAALPPAPSLLR HG10872-CY 479 Cynomolgus TNFR2 MAPAAVWAALAVGLELWAAGHALPAQVAFTPYAPEPGGTCRLREYYDQTAQMCCSKCPPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQN RICTCRPGWYCALSKQEGCRLCAQLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICHVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTP APSTAPGTSFLLPVGPSPPAEGSTGDIVLPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQRETKVPHLPADKARGAQGPEQQHLLTTVPSSSSSSLESSASALDRRAPTRN QPQAPGAEKASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTTGDTDASPSGSPKDEQVPFSKEECAFRSQLETPETLLGSTGEKPLPLGVPDAGMKPS CG90102-ACR 480 Mouse TNFR2 MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQISQEYYDRKAQMCCAKCPPGQYVKHFCNKTSDTVCADCEASMYTQVWNQFRTCLSCSSSCTTDQVEIRACTKQQNRV CACEAGRYCALKTHSGSCRQCMRLSKCGPGFGVASSRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDAVCAPESPTLSAIPRTLYVSQPEPTRSQPLDQEPGPSQ TPSILTSLGSTPIIEQSTKGGISLPIGLIVGVTSLGLLMLGLVNCIILVQRKKKPSCLQRDAKVPHVPDEKSQDAVGLEQQHLLTTAPSSSSSSLESSASAGDRRAPPGGHPQARVMA EAQGFQEARASSRISDSSHGSHGTHVNVTCIVNVCSSSDHSSQCSSQASATVGDPDAKPSASPKDEQVPFSQEECPSQSPCETTETLQSHEKPLPLGVPDMGMKPSQAGWFDQIAVKVA
MG50128-CY 481 Mouse TNFR1 MGLPTVPGLLLSLVLLALLMGIHPSGVTGLVPSLGDREKRDSLCPQGKYVHSKNNSICCTKCHKGTYLVSDCPSPGRDTVCRECEKGTFTASQNYLRQCLSCKTCRKEMSQVE ISPCQADKDTVCGCKENQFQRYLSETHFQCVDCSPCFNGTVTIPCKETQNTVCNCHAGFFLRESECVPCSHCKKNEECMKLCLPPPLANVTNPQDSGTAVLLPLVILLGLCLLS FIFISLMCRYPRWRPEVYSIICRDPVPVKEEKAGKPLTPAPSPAFSPTSGFNPTLGFSTPGFSSPVSSTPISPIFGPSNWHFMPPVSEVVPTQGADPLLYESLCSVPAPTSVQ KWEDSAHPQRPDNADLAILYAVVDGVPPARWKEFMRFMGLSEHEIERLEMQNGRCLREAQYSMLEAWRRRTPRHEDTLEVVGLVLSKMNLAGCLENILEALRNPAPSSTTRLPR
MG50496-CY 482

Table 8. Commercial antigens used in screening. TNFR2 antigens with the sequences shown in Table 7 and other proteins shown below were tested in various formats for biotinylation and use in TNFR2 AFFIMER® screening and other assays. Antigen Supplier Catalog number Label Source Expected PM (kDa) hTNFR2-hFc-his-Avi (biotin-conjugated) BPS 100205 Human IgG1, 6x His, Avi, Biotin HEK293 75 hTNFR2-hFc-his-Avi BPS 79363 Human IgG1, 6x His, Avi HEK293 75 hTNFR2-his Acro Biosystems AMS.TN2-H5227 6X His label HEK293 26 hTNFR2-his-avi (biotin-conjugated) Acro Biosystems AMS.TN2-H82E3 6X His and Avi Label, Biotin HEK293 26 hTNFR2-his Sino Biologicals 10417-H08H 6x His HEK293 45 hTNFR2 Peprotech 310-12 N / A E. coli 20 mTNFR2-mFc R&D Biosystems 9707-R2-050 Mouse IgG2a NS0 52 mTNFR2-his Sino Biologicals 50128-M08H 6x His HEK293 27 mTNFR2-hFc Sino Biologicals 50128-M02H Human IgG1 Fc HEK293 52 hTNFa Peprotech 300-01-A N / A E. coli 17.4 mTNFa Peprotech 315-01A N / A E. coli 17.3 hFc (IgG1) AcroBiosystems FCC-H5214 N / A HEK293 26 hFc-Avi (IgG1) (conjugated to biotin) AcroBiosystems IG1-H8213 Avi label, biotin HEK293 28.4 mFc (IgG2a) AcroBiosystems IGA-M5207 N / A HEK293 26.4 Biotinylated mFc (IgG2a) AcroBiosystems IGA-M8210 N / A HEK293 28.2 hTNFR1-hFc AcroBiosystems TN1-H5251 Human IgG1 Fc HEK293 47.9 mTNFR1-hFc-His R&D Systems 430-RI/CF Human IgG1 Fc NS0 48.6

In some cases, TNFR2 was biotinylated and captured on streptavidin and neutravidin beads alternately, alternatively TNFR2 was passively absorbed to a surface. Unbound phage particles were then removed by washing, and following washing, bound phage were eluted. Elution of bound phage was accomplished by exposure to trypsin. The eluted phage particles were then used to infect Escherichia coli (E. coli) and the infected bacteria were incubated under conditions suitable for bacteriophage replication. After release of bacteriophage particles from these infected bacteria, the cycle of allowing the phage particles to bind to the target antigen, eluting the bound phage particles, propagating the eluted phage particles in the bacteria, and isolating the released phage particles from the infected bacteria was repeated to enrich the bacteriophage population for phage particles displaying proteins that bind to the target antigen. Specific conditions were modified in these cycles, such as increasing the number of wash steps, reducing the amount of available antigen, or adding a blocking reagent, to select for phage particles displaying proteins that bind more tightly or specifically to the target antigen.

After multiple rounds of phage display library selection and amplification, proteins expressed by the phages were expressed and screened by enzyme-linked immunosorbent assay (ELISA). Briefly, AFFIMER® polypeptides were overexpressed from phagemid vectors, bacterial cells were lysed, and the lysates were used as substrates in the ELISAs. In these ELISAs, human, murine, and cynomolgus TNFR2 were immobilized on a plate, lysates were added, and the amount of AFFIMER® polypeptide binding to TNFR2 in each plate was measured using a detector antibody specific for the Myc tag expressed on the candidate AFFIMER® polypeptides. The phagemid vectors encoding the AFFIMER® polypeptides were AFFIMER® with the best binding activity to human TNFR2 were sequenced to identify the DNA sequences of candidate clones for further development. TNFR1 polypeptides were also used to distinguish specificity. The amino acid sequences of loop 2 and loop 4 of each of these candidate clones are shown in Table 1.

EXAMPLE 2: Production of AFFIMER® in mammalian cells as a soluble protein and screening

Cloning into a vector of interest

To assess the ability of the selected AFFIMER® proteins to bind to TNFR2 when expressed on the surface of a mammalian cell, transiently transfected cell pools were generated for 30 “fast-tracked” clones based on the most repeated sequences during passive selection. These clones were used for initial characterization and optimization of the assay. The AFFIMER® TNFR2 sequences and TNFR2 targets were cloned from the phagemid of Example 1 into the mammalian expression vector pD609kanR (ATUM custom vector) for expression in Expi293F™ cells (ThermoFisher). The average concentration obtained from expression was 2.8 mg/mL, with 19 clones selected for purity and assayed for degradation.

These 19 clones were screened on cells expressing Expi293F™hTNFR2 by flow cytometry at 1 µM and 0.1 µM while being compared to the negative controls SQT-GLY and 3T0-GLY which contain glycines only in Loop 2 and Loop 4 and are therefore not specific binders. The signal detected on the transfected cells was subtracted from the signal obtained on the untransfected Expi293F™ cells. Of these, 16 clones demonstrated binding to hTNFR2-Expi293F™ cells at 1 µM > 10% of the binding to the parental cell line. Clones 15, 22 and 23 did not demonstrate such binding ( ).

Purified AFFIMER® mammalian hTNFR2 proteins were screened in the HEK Blue™ TNFα SEAP reporter assay (Invivogen) according to the manufacturer’s instructions to determine whether they could trigger the NFkB pathway and SEAP (Secreted embryonic alkaline phosphatase) production via binding to TNFR2 when clustered by an anti-His antibody coated on the plate. In this experiment, 5 of the accelerated clones (01, 09, 12, 21, and 26) demonstrated the ability to trigger SEAP release, with clone 12 showing the most consistent agonism.

90 other AFFIMER® polypeptides were cloned from the phagemid and characterized similarly. 55 clones were taken up and binding confirmed by Mirrorball analysis. Further characterization was performed by competitive ELSA to test their ability to block the interaction between hTNFR2-hFc-his-avi and recombinant TNF-alpha. A series of experiments were performed to determine the EC80 of hTNFR2-hFc-his-Avi upon binding to coated hTNF-alpha (593 pM). AFFIMER® proteins were tested at three different concentrations (10, 1 and 0.1 µM) where possible, in the presence of hTNFR2 antigen at its determined EC80. Detection of bound hTNFR2-hFc-his-Avi was performed by anti-hFc-HRP antibody. Among the 75 clones tested, 32 clones, which showed an inhibition % greater than 65% at 10 µM, were selected to be further characterized in order to determine their IC50 ( ). Clones 06, 21, 26, 27, 44, 108 and 115 showed complete inhibition of the interaction between TNFα and hTNFR2 at the concentrations tested.

At this stage, these 55 clones as well as the 14 clones from the accelerated group (13 agonists, one negative, clone 06) were cloned into the pDisplay vector (Thermo Fisher). This vector contains a PDGFR protein transmembrane domain in C-term to allow Affimer expression on the surface of the expression cells and as soluble proteins. Additional tags such as HA in N-term are also encoded by this vector allowing easy verification of Affimer expression. Once cloned, these Affimers were transiently transfected into Expi293F™ cells for SEAP analysis as previously described. Taking all the data together, 43 clones were selected for further characterization.

EXAMPLE 3: Characterization of 43 selected clones

Biacore Test

The 43 selected clones were tested in a Biacore kinetic assay as soluble proteins. Briefly, multicycle kinetics were performed using a CM5 chip on which the dimeric hTNFR2-hFc-his-avi antigen was immobilized at 500RU. AFFIMER® proteins were downtitrated in HBS-EP+ buffer from 1 µM in a 1:2 dilution series. The association and dissociation times used were 200 s and 400 s, respectively, at a flow rate of 20 µl/min with regeneration with 10 mM glycine at pH 1.5 for 30 s at 30 µl/min. Twelve clones showed a very weak response and no KD values could be estimated for these. Clone 09 was fitted using the 1:1 binding model, however, the reported KD value was too high (>E-05) to be reliable with the concentration range used. For 16 clones, the signal achieved was adequate and they could be successfully fitted using the 1:1 binding model, the curve and their fit are compiled. Two clones (21 and 69) that are dimers were fitted using both the 1:1 binding model and a steady state to estimate their KD, while in 12 clones, a low Rmax made it difficult to fit them using the 1:1 binding model and the steady state model was used instead. In this case, the obtained KD value was not reported; instead, an order of magnitude was. The results are presented in .

ELISA cross-reactivity of binding

The specificity of the TNFR2 AFFIMER® clones for hTNFR2 was assessed by ELISA when the clones binding to TNFR2 were compared to their binding to TNFR1. None of the clones showed binding to TNFR1 as a recombinant antigen. hTNFR1 was also expressed in Expi293F™ cells, and no binding was observed to this cellularly expressed hTNFR1, confirming the absence of cross-reactivity between the hTNFR2 AFFIMER® polypeptide binders with hTNFR1.

In addition to their binding to human TNFR2, the binding of the clones to mouse TNFR2 was also tested to see if any of them were cross-reactive. This was very unlikely to occur as the homology between the two proteins is approximately 68% for the extracellular domain, which was confirmed as no clones demonstrated binding to Expi293F™ cells expressing mTNFR2.

HEK293 – Characterization by HEK Blue test

The 43 core clones were transiently transfected into HEK 293 cells, along with 4 controls, SQT-Gly, 3t0-Gly, Empty Vector and clone 06 which was found to be negative. Approximately 29h after transfection, cell viability was determined using a cell counter, and AFFIMER® expression was determined by flow cytometry using an anti-HA antibody. 48h after transfection, HEK293 cells expressing AFFIMER® were cocultured with HEK Blue reporter cells at 4 different cell densities, 40000, 20000, 10000 and 5000 with a fixed HEK Blue cell number of 50,000 cells/well. In parallel, the positive control MR2-1 was also incubated with HEK Blue cells for 24h at a range of concentrations. After 24 h, SEAP release into the supernatant was quantified using Quanti-Blue and 640 nm absorbance measurement. Staining of AFFIMER® with an anti-HA monoclonal antibody showed expression levels >90% in each of the two experiments. In the coculture reporter assay, the effect of the TNFR2 AFFIMER® proteins exhibited on HEK293 on HEK-Blue cells was visible, as well as the effect of the MR2-1 clone, allowing the selection of 16 clones that were consistently ranked among the top 20 clones in each of the two experiments. These results are compiled in , with the following clones identified: 001, 007, 009, 012, 013, 026, 027, 037, 044, 059, 069, 079, 100, 103, 112 and 115.

Cellular binding to human and cynomolgus TNFR2

The top 16 agonists were characterized in soluble format for their binding to human TNFR2 overexpressing Expi293F™ and cynomolgus TNFR2 overexpressing Expi293F. The experiment was repeated 3 times and 2 different batches of transiently transfected cells were used. All clones showed consistent specific binding to hTNFR2-Expi293F™ cells. Clones 26 and 69 showed the best EC50 on both human and cynomolgus TNFR2 expressing cells. 13 of the 16 clones tested showed cross-reactivity with cynomolgus TNFR2. The 3 clones without cross-reactivity were clones 001, 007 and 009. Without necessarily being the best binders, clones DAW02-013, DAW02-044 and DAW02-112 are the clones with the least difference between binding to cells overexpressing human TNFR2 and cynomolgus TNFR2 when the 3 experiments are compiled ( ).

EXAMPLE 4. In-line fusion homodimer constructions

Clones 001, 009, and 012 were selected to be formatted as in-line fusion (ILF) homodimers to determine whether this format would be an advantage for eliciting agonism over the monomeric form of the clones, both as a soluble protein and when displayed on cells. Two constructs were used for the generation of ILF homodimers with a flexible linker, a codon optimized for mammalian expression, and a non-optimized codon. Both constructs were cloned into a mammalian secretion vector (pD609-KanR) to purify the recombinant protein and assess their expression levels, protein quality, hTNFR2-hFc-his-avi binding kinetics by Biacore, and agonist activity in a reporter assay with HEK reporter cells. In addition, both constructs were cloned into the pDisplay vector for expression on the Expi293F™ cell surface and assessment of their expression levels, binding to hTNFR2-hFc-his-avi and agonist activity in a coculture reporter assay with HEK reporter cells. Codon optimization did not improve the yields or purity of soluble ILF homodimers, which was confirmed by HPLC.

When analyzed by Biacore, clones 001 and 009 showed increased affinity in their soluble ILF format compared to the soluble monomeric format. Clone 12 showed no response. No differences were observed between the codon optimization constructs. In SEAP assays, only clone 12 showed an improved profile as a soluble ILF format, but when displayed on cells, in both SEAP assays and flow cytometric display assays, no differences could be determined between the ILF and monomeric formats.

Summary of hTNFR2 AFFIMER® polypeptide selection and characterization.

More than 2500 monoclonal phages were screened by phage ELISA with a success rate of over 60% with good diversity (20-40%). When screened in the ELISA as crude cell extract, more than 400 clones showed binding to the hTNFR2 antigen. At this stage, it was decided to introduce another step in the selection screening campaign to reduce the number of clones to be cloned into the mammalian expression vector. In parallel, it was also decided to accelerate 30 clones from passive selection directly to expression as soluble AFFIMER® proteins to facilitate assay development and construction of the screening cascade for the remaining clones. 20 of these clones passed the quality criteria of protein production of more than 80% purity by SEC-HPLC (size exclusion chromatography - high performance liquid chromatography) and were used to set up different assays as well as to be characterized. 5 clones out of 20 demonstrated agonist activity in a preliminary agonist assay where the monomeric soluble AFFIMER® protein was pooled with an anti-his antibody via their histidine tag and incubated with the HEK reporter cell line.

In parallel with the development of accelerated clone assays, the phage screening campaign was finalized and a total of 364 human AFFIMER® TNFR2-binding proteins were purified from the phagemid and tested for binding to a TNFR2-overexpressing cell line. Following this assay, 90 human AFFIMER® TNFR2-binding proteins were reformatted into the mammalian expression vector. 55 of these clones passed the protein production quality criteria of >80% purity by SEC-HPLC and were taken forward for the remainder of the assays.

The total number of clones (both accelerated and non-accelerated) was narrowed from 75 to 43 following a series of experiments investigating the binding of soluble AFFIMER® proteins to hTNFR2-overexpressing cells, the agonist assay with HEK reporter cells and preliminary data from the agonist assay using HEK reporter cells co-cultured with AFFIMER® proteins expressed on the surface of Expi293F™ cells. These top 43 clones were characterized as soluble proteins for their binding to human TNFR2 by Biacore where 16 out of 43 had their KD calculated successfully using a 1:1 binding model. None of the clones demonstrated binding to human TNFR1 by ELISA or mouse TNFR2 by cell binding. As AFFIMER® proteins displayed on the surface of HEK293, the ability of these 43 clones to trigger TNFR2 agonism in a HEK reporter cell line when cocultured was tested. A large majority of the clones tested demonstrated a dose-dependent effect on HEK reporter cells (80%). 16 AFFIMER® proteins were selected as the best agonists and subjected to further characterization to assess cynomolgus TNFR2 binding and human TNFR1 binding on the cells. The 16 selected AFFIMER® hTNFR2 polypeptides are clones 001, 007, 009, 012, 013, 026, 027, 037, 044, 059, 069, 079, 100, 103, 112 and 115.

The use of inline fusion homodimers as soluble proteins vs. monomers showed advantages for binding to recombinant TNFR2 by Biacore and a possible advantage in the anti-His clustering agonism assay with HEK reporter cells. However, no advantage was demonstrated over monomers when the inline fusion AFFIMER® proteins were displayed on cells to trigger the agonism pathway of the reporter cell line via TNFR2. The reason why inline fusion homodimers did not perform better than monomers in the latter assay could be that the inline fusion format was not well displayed on the cell surface and only one of the two AFFIMER® proteins was available for binding or that the limit of the reporter assay had been reached and it was not possible to discriminate between the two formats.

EXAMPLE 5. Mouse TNFR2 AFFIMER® polypeptides

Phage selection and screening campaign

The suitability of murine TNFR2 antigens, having the sequence as shown in Table 7 but in different formats, for use in assays to select AFFIMER® mTNFR2 polypeptides was tested. Among the three formats (mFc, hFc and His-tagged), mTNFR2-mFc was chosen for biotinylation and use in AFFIMER® selections because of its better performance in the assays.

Selections

Passive and solution selections were performed on both AFFIMER® phage libraries as described for AFFIMER® hTNFR2 selection. Enrichment was observed when the mTNFR2 concentration was maintained at 10 nM in round 3. Over 2000 AFFIMER® phage-displayed proteins were screened by monoclonal phage ELISA from the results of round 2 and round 3. Of these clones, over 900 demonstrated binding to mTNFR2, with no detectable nonspecific binding to mouse Fc, plastic, streptavidin, or neutravidin. These were defined as “hits” based on the criteria of 450nm-630nm absorbance above 0.5 on mTNFR2-coated plates and below 0.05 on control antigen-coated plates (i.e. mouse Fc, plastic, neutravidin). Sequencing of phage ELISA hits identified 281 unique clone sequences, 109 of which demonstrated binding to mTNFR2 following ELISA on crude cell extracts. Further analysis of binding using mTNFR2-Expi293F™ cells narrowed this field to 58 clones, identified as clones 122-179 in Table 2.

Characterization of soluble proteins

As described for the characterization of the hTNFR2 AFFIMER® polypeptide above, the identified mTNFR2 AFFIMER® polypeptides were cloned from phagemids into mammalian vectors for expression in Expi293F™ cells. The purity of the expressed proteins was then assessed by SDS-PAGE and HPLC, and the affinity by Biacore, as well as the cell-based binding of mTNFR2 in mTNFR2-Expi293F™ cells. Specificity for mTNFR2 was tested using ELISA and cell-based assays using mTNFR1 and mTNFR2 as targets, confirming the specificity of the identified clones. To complete the characterization of soluble proteins, the ability of AFFIMER® polypeptides to block mTNFR2-mFC binding to mTNFα in an ELISA was tested, with four clones (157, 160, 171 and 172) showing clear competitive ability in the range tested.

T-cell stimulation assays

T cell stimulation assay was used as a screening tool since activated T cells express high levels of TNFR2, when stimulated with HM102 (TNFR2 agonist) CD25 expression increases.

The mTNFR2 AFFIMER® polypeptides were first expressed on the cell surface of HEK293 cells. Then, these cells were cocultured for 48 hours with BALB/c splenocytes and finally the increase in CD25 expression was assessed on CD4 and CD8 positive T lymphocytes. The expression of AFFIMER® proteins on the HEK293 cell surface was assessed by flow cytometry staining of the HA tag present at the beginning of the open reading frame before the AFFIMER® proteins and the cell anchor protein. In order to assess the effect of AFFIMER®-HEK proteins on CD4+ and CD8+ cells, after 48h of incubation, the cells were stained with the following panel of markers, Live/Dead staining, CD90.2, CD4, CD8 and CD25.

A dose-response was observed in CD25 expression in CD4+ and CD8+ T cells in response to HEK293 cells expressing AFFIMER® mTNFR2 proteins, similar to the response observed for the TNFR2 agonist HM102. The response obtained in CD8+ T cells is stronger than in CD4+ T cells. However, focusing on the two highest cell numbers used, a similar ranking of clones was obtained between CD8+ and CD4+ T cell subsets.

In two cycles of this test experiment, the six top-ranked AFFIMER® proteins were consistent 174, 160, 175, 125, 157, and 179.

Summary of mTNFR2 AFFIMER® polypeptide selection and characterization.

More than 2000 monoclonal phages were screened by phage ELISA with a moderate success rate of 35-60%, with good diversity (25-35%). When screened in the ELISA as crude cell extract, 109 clones showed binding to the mTNFR2 antigen. Using the same screening cascade as for the human program, it was decided to purify the identified binders from the phagemid and test their binding to a TNFR2 overexpressing cell line. Following this assay, 60 AFFIMER® mouse TNFR2-binding proteins were reformatted into the mammalian expression vector, produced and tested for their biophysical properties as soluble proteins. 22 Affimer proteins passed the protein production quality criteria of >80% purity by SEC-HPLC. The 22 clones were characterized in different assays as soluble proteins and then were expressed on the surface of HEK293 for a coculture assay with splenocytes.

Although only 10 of the 22 Affimer proteins showed triple-digit nanomolar binding affinities (KD) when tested on Biacore, it was considered that even an Affimer protein with a higher KD value could be a good agonist when displayed on the cell surface. Additionally, the performance of the clones as soluble proteins also depended on whether they were a natural dimer or trimer. For these reasons, the Biacore data were considered in the context of mTNFR2 agonist activity and were not used to discriminate between clones. When tested on cells expressing mouse TNFR2, 20 of the 22 clones demonstrated specific binding with a wide range of EC50 values. For the Biacore data, the cell binding results were considered only in the context of mouse TNFR2 agonist activity and were not used to discriminate between clones. None of the clones bound to TNFR1 as recombinant proteins or expressed on the cell surface. 5 of the 22 clones demonstrated complete inhibition of the interaction between TNF-alpha and TNFR2 in a competitive ELISA.

The 22 mouse clones were also expressed on the surface of HEK293 cells for a coculture assay with splenocytes. The primary readout of the assay was the increase in CD25 expression on CD4+ T cells and CD8+ T cells. A large majority of the clones tested demonstrated a dosage-dependent effect on CD4+ T cells and CD8+ T cells (over 70%). Clones were ranked with respect to CD25 expression on CD4+ and CD8+ T cells. Based on the results of this key assay, six AFFIMER® mTNFR2 proteins were identified: 174, 160, 175, 125, 157 and 179.

Claims (31)

An engineered polypeptide that specifically binds to TNFR2 with a Kd of 1×10 ^-6 M or less, wherein the engineered polypeptide is a variant of a Stefin A protein and wherein the engineered polypeptide comprises an amino acid sequence having at least 80%, at least 90%, at least 95% or at least 98% identity to an amino acid sequence of
(SEQ ID NO: 4),
where Xaa, individually for each occurrence, is one or two amino acid residues; and n and m are each, independently, an integer from 3 to 20. An engineered polypeptide according to claim 1, comprising an amino acid sequence having at least 80%, at least 90%, at least 95% or at least 98% identity with an amino acid sequence of
MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5),
where Xaa, individually for each occurrence, is an amino acid residue; and n and m are each, independently, an integer from 3 to 20. An engineered polypeptide according to any preceding claim which binds to TNFR2 with a Kd of 1×10 ^-7 M, preferably a Kd of 1×10 ^-8 M. An engineered polypeptide according to claim 1 or claim 2, wherein (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-102, and/or (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103-199. An engineered polypeptide according to claim 4, wherein (Xaa)n comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 11, 13, 16, 17, 24, 25, 29, 33, 41, 54, 65, 67, 73, 75, 82, 89, 90, 98 and 99 and/or (Xaa)m comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 103, 108, 110, 113, 114, 121, 122, 126, 130, 138, 151, 162, 164, 170, 172, 179, 186, 187, 195 and 196. An engineered polypeptide according to claim 1 comprising an amino acid sequence having at least 75%, at least 85%, or at least 95% identity with an amino acid sequence of SEQ ID NO: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292 and 293, wherein the sequence optionally excludes one or more of the twenty-one carboxy terminal residues. An engineered polypeptide according to any preceding claim, further comprising a signal peptide. An engineered polypeptide according to any preceding claim, further comprising a transmembrane domain. A fusion protein comprising a dimer, trimer or tetramer of the engineered polypeptide according to any preceding claim, optionally comprising a linker. The fusion protein of claim 9, wherein the linker is a rigid linker. The fusion protein of claim 10, wherein the rigid linker is a peptide linker comprising the sequence (EAAAK)n, where n is 1-6. A fusion protein comprising one or more of the engineered polypeptide(s) of any one of claims 1 to 8 linked to a therapeutic or diagnostic moiety. The fusion protein of claim 12, wherein the therapeutic moiety is a therapeutic protein or peptide, optionally an antibody. The fusion protein of claim 12, wherein the diagnostic moiety is a fluorescent protein. An engineered polypeptide according to any one of claims 1-8 or the fusion protein according to any one of claims 9-14 further comprising a half-life extending moiety selected from antibody Fc domains, human serum albumin, serum binding proteins. An engineered polypeptide or fusion protein according to claim 15, wherein the half-life extending moiety of the engineered polypeptide is a variant of a Stefin A protein that specifically binds to human serum albumin with a Kd of 1×10 ^-6 M or less. An engineered polypeptide or fusion protein according to claim 16, wherein the half-life extension moiety is selected from the group consisting of SEQ ID NO: 471-477. Polynucleotide or set of polynucleotides encoding the engineered polypeptide according to any one of claims 1 to 8 or 15 to 17 or the fusion protein according to any one of claims 9 to 14. A polynucleotide or set of polynucleotides according to claim 18 comprising a nucleic acid sequence having at least 80%, at least 90%, at least 95% or at least 98% identity with a sequence selected from the group consisting of SEQ ID NO: 297, 302, 304, 307, 308, 315, 316, 320, 324, 332, 345, 356, 358, 364, 366, 373, 380, 381, 389 and 290, wherein the sequence optionally excludes nucleotides encoding twenty carboxy-terminal residues. A delivery vehicle comprising the polynucleotide of any one of claims 18 or 19. The delivery vehicle of claim 20, which is a viral delivery vehicle. The delivery vehicle of claim 21, wherein the viral delivery vehicle is an adenoviral vector, a retroviral vector, a lentiviral vector or an adeno-associated viral (AAV) vector. The delivery vehicle of claim 20, which is a non-viral delivery vehicle. The delivery vehicle of claim 23, wherein the non-viral delivery vehicle is a liposome or a lipid nanoparticle. Plasmid or minicircle comprising the polynucleotide according to any one of claims 18 or 19. Messenger RNA (mRNA) comprising an open reading frame encoding the engineered polypeptide of any one of claims 1 to 8 or 15 to 17. Lipid nanoparticle, optionally cationic lipid nanoparticle, comprising the mRNA of claim 26. A pharmaceutical composition comprising the engineered polypeptide of any one of claims 1 to 8 or 15 to 17, the fusion protein of any one of claims 9 to 14, or the delivery vehicle of any one of claims 20 to 24, and optionally a pharmaceutically acceptable excipient. A conjugate comprising the engineered polypeptide of any one of claims 1 to 8 or 15 to 17 or the fusion protein of any one of claims 9 to 14 linked to a pharmacologically active moiety. Pharmaceutical composition according to claim 28 for use as a medicament. A pharmaceutical composition according to claim 30 for use in the treatment of systemic lupus erythematosis (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) (related to stem cell transplantation) also known as allograft rejection, solid organ transplantation/transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic eczema, myasthenia gravis, Graves' disease, glomerulosclerosis and/or cancer.