WO2024200988A1 - Polypeptides de liaison au tnfr2 et procédés d'utilisation - Google Patents

Polypeptides de liaison au tnfr2 et procédés d'utilisation Download PDF

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WO2024200988A1
WO2024200988A1 PCT/GB2023/052513 GB2023052513W WO2024200988A1 WO 2024200988 A1 WO2024200988 A1 WO 2024200988A1 GB 2023052513 W GB2023052513 W GB 2023052513W WO 2024200988 A1 WO2024200988 A1 WO 2024200988A1
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affimer
polypeptide
amino acid
protein
engineered polypeptide
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Delphine BUFFET
Emma STANLEY
Emma JENKINS
Estelle ADAM
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Avacta Life Sciences Ltd
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Avacta Life Sciences Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • T cell and B cell driven immune reactions have been associated with a wide array of human diseases, as the inappropriate increase of an immune response against various self and foreign antigens plays a causal role in such pathologies as autoimmune disorders, asthma, allergic reactions, graft versus host disease (GvHD), transplantation graft rejection, and a variety of other immunological disorders including cancer.
  • GvHD graft versus host disease
  • Tregs Regulatory T cells
  • MHC self major histocompatability complex
  • Tumor necrosis factor receptor (TNFR) subtypes 1 and 2 have been identified specifically on the Tregs surface.
  • TNFR1 soluble TNF-alpha
  • TNFR2 mainly membrane bound TNF-alpha induces signaling through the mitogen activated protein kinase (MAPK) signaling pathway, which orchestrates the TRAF2/3 and NF- KB mediated transcription of genes that promote cell proliferation and escape from apoptosis.
  • MAPK mitogen activated protein kinase
  • TNFR2 deficient mice had reduced numbers of thymic and peripheral Tregs (2013_Chen X, et al. PMID: 23277487), and TNFR2 -I- Tregs were not able to control inflammatory responses in vivo (2008_Van Mierlo GJ, et al. PMID: 18292492).
  • Tregs were shown to express a higher level of TNFR2 than T effector cells (2013_Okubo Y, et al. PMID: 24193319 & 2002_ Annunziato F, et al.
  • 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 tumour growth by mediating TNF responses in immunosuppressive cells, allowing for immune escape and tumour development (Sheng Y., et al. doi: 10.3389/fimmu.2018.01170). Moreover, in autoimmune diseases affecting the central nervous system (CNS), specifically Multiple sclerosis (MS), pathogenic lymphocytes are triggered in the periphery to infiltrate the CNS and cause local inflammation and demyelination.
  • CNS central nervous system
  • MS Multiple sclerosis
  • Demyelination is the damage to the protective covering (myelin sheath) that surrounds nerve fibers in the brain.
  • Protecting against demyelination or revert it are strategies explored to tackle MS.
  • TNFR2 involvement in neuroprotection For example, in a cuprizone induced mouse model of demyelination, it was shown that TNFR2 is critical for regeneration of oligodendrocyte the cells primarily responsible for maintenance and generation of the myelin sheath that surrounds axons, whereas TNF signaling via TNFR1 promoted nerve demyelination (2001 HA Arnett, et al. PMID: 11600888).
  • TNFR2 Due to its role in directing cell survival and growth, TNFR2 represents an attractive target for treating diseases such as autoimmune disorders, GvHD, allograft rejection, allergic reactions, asthma and cancer.
  • AFFIMER® developed by Avacta Life Sciences Limited, is a small stable protein molecule engineered based on a stefin A protein, which is an in-vivo protein.
  • AFFIMER® includes two short peptide sequences having a random sequence and an N-terminal sequence, and is able to bind 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 to the free peptide library, and has a very small size and high stability compared to antibodies, and is therefore receiving great attention as a next-generation alternative pharmaceutical platform to replace antibodies (U.S. Patent Nos. 9447170, 8853131, etc.).
  • the present invention relates to polypeptides that specifically bind to TNFR2 by engineering the natural stefin A protein, resulting in the development of AFFIMER® polypeptides capable of binding to TNFR2 with excellent affinity and specificity.
  • an engineered polypeptide that binds specifically to TNFR2 with a Kd of 1 x 10-6M or less, wherein the engineered polypeptide is a variant of a Stefin A protein.
  • a fusion protein comprising a dimer, a trimer or a tetramer of the engineered polypeptides of the invention, optionally comprising one or more linkers.
  • a fusion protein comprising one or more of the engineered polypeptides of the invention linked to a therapeutic or diagnostic moiety.
  • a polynucleotide or set of polynucleotides encoding the engineered polypeptide or the fusion protein of the invention.
  • a delivery vehicle comprising the polynucleotide of the invention.
  • a plasmid or minicircle comprising the polynucleotide of the invention.
  • mRNA messenger RNA
  • lipid nanoparticle optionally a cationic lipid nanoparticle, comprising the mRNA of the invention.
  • composition comprising the engineered polypeptide, the fusion protein, or the delivery vehicle of the invention, and optionally a pharmaceutically acceptable excipient.
  • conjugate comprising the engineered polypeptide or the fusion protein of the invention linked to a pharmacologically active moiety.
  • an engineered polypeptide, fusion protein, or delivery vehicle of the invention for use as a medicament.
  • an engineered polypeptide that competes for binding to TNFR2 with an engineered polypeptide according to the invention.
  • an engineered polypeptide which binds the same epitope on TNFR2 as that of 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 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). For the passive selections using Avacta’s proprietary Type 3 AFFIMER® phage library (US Pat. No. 9.932.575), the deselection and competition approaches were simultaneously explored. For the deselection approach, the Fc concentration in round n+1 was the concentration of hTNFR2 in round n.
  • the Fc concentration was lOx molar excess of the hTNFR2 concentration specified in the given round.
  • Avacta proprietary Type 1 AFFIMER® phage library (US Pat. No. 8,841,491)
  • the deselection approach was exclusively performed and a round 4 was introduced since early-stage screening of the round 3 outputs indicated a high diversity and hit rate.
  • both libraries underwent the competition approach for reducing enrichment of Fc-binding phage-displayed AFFIMER® polypeptides. Streptavidin- and neutravi din-coated beads were alternated between rounds to reduce enrichment for streptavidin and/or neutravidin-binding phage-displayed peptides.
  • Stefin polypeptides encompass a subgroup of proteins in the cystatin superfamily, a family which encompasses proteins that contain multiple cystatin-like sequences.
  • the Stefin subgroup of the cystatin family includes relatively small (around 100 amino acids) single domain proteins. They receive no known post-translational modification, 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 solved, facilitating the rational mutation of Stefin A into the AFFIMER® polypeptide.
  • the only known biological activity of cystatins is the inhibition of cathepsin activity, which allowed for exhaustive testing for residual biological activity of the engineered proteins.
  • AFFIMER® polypeptide refers to a small, highly stable protein that is an engineered variant of a Stefin polypeptide.
  • AFFIMER® proteins display 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 similar manner to monoclonal antibodies. Stabilization of the two peptides by the Stefin A protein scaffold constrains the possible conformations that the peptides can take, increasing the binding affinity and specificity compared to libraries of free peptides.
  • These engineered nonantibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications.
  • an AFFIMER® polypeptide includes a sequence derived from Stefin A, sharing substantial identity with a Stefin A wild type sequence, such as human Stefin A. It will be apparent to a person skilled in the art that modifications may be made to the scaffold sequence without departing from the disclosure.
  • an AFFIMER® polypeptide can have an amino acid sequences that is at least 25%, 35%, 45%, 55% or 60% identity to the corresponding sequences to human Stefin A, for example, 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 which are possessed by wild type Stefin A but which are abolished in mutational changes described herein.
  • the desired target such as TFNR2
  • 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 intended for delivery to an individual.
  • An “AFFIMER®-linked conjugate” refers to an AFFIMER® agent having at least one moiety conjugated thereto through a chemical conjugation other than through the formation of a contiguous peptide bond through the C-terminus or N-terminus of the polypeptide portion of the AFFIMER® agent containing 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®-tag conjugate”, which refers to an AFFIMER® agent including at least one detectable moiety (e.g., detectable label) conjugated thereto.
  • An “encoded AFFIMER® polynucleotide” refers to a nucleic acid construct which, when introduced into and expressed by cells, produces an intended AFFIMER® agent.
  • TNFR.2 a nucleic acid construct which, when introduced into and expressed by cells, produces an intended AFFIMER® agent.
  • Tumour necrosis factor receptor 2 (“TNFR2”), along with tumour necrosis factor receptor 1 (TNFR1), is a Type 1 membrane bound receptors that binds tumour necrosis factor alpha (“TNFa”).
  • TNFR2 also known as p75, TNF Receptor Superfamily Member IB 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 long and has a 50 kDa mass.
  • 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 10-6M.
  • Kd dissociation constant
  • the TFNR2 AFFIMER® agent binds TFNR2 with a Kd of 1 x 10-7M or less, Kd of 1> ⁇ 1O-8M or less, Kd of 1X10-9M or less, or a Kd of IxlO-lOM or less.
  • TFNR2 AFFIMER® polypeptide is an engineered polypeptide that binds specifically to TFNR2 with a Kd of 1 x 10-6M or less, wherein the engineered polypeptide is a variant of a Stefin A protein.
  • Polypeptides are polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it 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, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing at least one analog of an amino acid including, for example, unnatural amino acids
  • Amino acids participate in one more peptide bonds of a polypeptide.
  • the abbreviations used herein for designating the amino acids are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11 :1726-1732).
  • Met, He, Leu, Ala and Gly represent "residues" of methionine, isoleucine, leucine, alanine and glycine, respectively.
  • the residue is meant a radical derived from the corresponding a-amino acid by eliminating the OH portion of the carboxyl group and the H portion of the a-amino group.
  • amino acid side chain is that part of an amino acid exclusive of the — CH(NH2)C00H portion, as defined by K. D. Kopple, "Peptides and Amino Acids", W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33.
  • amino acids used in the application of this disclosure are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids which 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 those amino acids and amino acid analogs which have been identified as constituents of peptidylglycan bacterial cell walls.
  • Amino acid residues having “basic sidechains” include Arg, Lys and His.
  • Amino acid residues having “acidic sidechains” include Glu and Asp.
  • Amino acid residues having “neutral polar sidechains” include Ser, Thr, Asn, Gin, Cys and Tyr.
  • Amino acid residues having “neutral non-polar sidechains” include Gly, Ala, Vai, He, Leu, Met, Pro, Trp and Phe.
  • Amino acid residues having “non-polar aliphatic sidechains” include Gly, Ala, Vai, He and Leu.
  • Amino acid residues having “hydrophobic sidechains” include Ala, Vai, He, Leu, Met, Phe, Tyr and Trp.
  • Amino acid residues having “small hydrophobic sidechains” include Ala and Vai.
  • Amino acid residues having “aromatic sidechains” include Tyr, Trp and Phe.
  • Amino acid residues further include analogs, derivatives and congeners of any specific amino acid referred to herein, as for instance, the subject AFFIMER® polypeptide (particularly if generated by chemical synthesis) can include an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3 -phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1 -methylhistidine, 3-methylhistidine, diaminiopimelic acid, ornithine, or diaminobutyric acid.
  • an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3 -phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1 -methylhistidine, 3-methylhistidine, diaminiopimelic acid, ornithine, or diaminobut
  • (D) and (L) stereoisomers of such amino acids when the structure of the amino acid admits of stereoisomeric forms.
  • the configuration of the amino acids and amino acid residues herein are designated by the appropriate symbols (D), (L) or (DL), furthermore when the configuration is not designated the amino acid or residue can have the configuration (D), (L) or (DL).
  • the structure of some of the compounds of this disclosure includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry are included within the scope of this disclosure. Such isomers can be obtained in substantially pure form by classical separation techniques and by sterically controlled synthesis.
  • a named amino acid shall be construed to include both the (D) or (L) stereoisomers.
  • 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 correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity may be measured using sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof.
  • two nucleic acids or polypeptides of the disclosure are substantially identical, meaning 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% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • 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 in length or any integral value there between.
  • identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full 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 in length or any integral value there between.
  • identity exists over a longer region than 60-80 bases, such as at least about 80- 1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.
  • any residues defined as ‘Xaa’ or ‘X’ in a reference sequence herein are included in the percentage identity calculation, i.e. any amino acid in this position in a comparison sequence matches the reference sequence.
  • a conservative amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been generally 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).
  • basic side chains e.g.
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides, soluble proteins, and/or antibodies of the disclosure do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site.
  • Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • a material is considered substantially pure if the material is at least 50% pure (e.g., free from 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 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
  • a linker 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.).
  • a linker is a peptide linker. Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. In some embodiments, linkers are not antigenic and do not elicit an immune response.
  • a transmembrane domain (also called a TM domain) is a sequence that can be added to an AFFIMER® polypeptide, such as through the addition of a polynucleotide sequence encoding the TM domain to the polynucleotide sequence encoding the AFFIMER® polypeptide, to ensure the AFFIMER® polypeptide is retained on the surface of the cell that is expressing it.
  • TM domains are available and readily usable with the AFFIMER® polypeptide.
  • AFFIMER®-antibody fusion is a fusion protein that includes an AFFIMER® polypeptide portion and a variable region of an antibody.
  • AFFIMER®-antibody fusions may include full length antibodies having, for example, at least one AFFIMER® polypeptide sequence appended 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 include at least one AFFIMER® polypeptide sequence as part of a fusion protein with an antigen binding site or variable region of an antibody fragment.
  • An antibody is an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule.
  • antibody encompasses 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 those fragments have been formatted to include an Fc or other FcyRIII 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 FcyRIII binding domain), antibody mimetics, and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological activity.
  • antibody fragments such as Fab, Fab', F(ab')2, and Fv fragments
  • scFv single chain Fv
  • the antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu.
  • the Fc is null binding (does not bind) to FcyR2b.
  • the antibody is not an IgGl Fc.
  • 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.
  • variable region of heavy and light chains include four framework regions (FR) and three complementarity determining regions (CDRs), also known as hypervariable regions.
  • FR framework regions
  • CDRs complementarity determining regions
  • the CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding sites of the antibody.
  • CDRs There are at least two techniques for determining CDRs: (1) an approach based on cross-species 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.
  • cross-species sequence variability e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda Md.
  • crystallographic studies of antigen-antibody complexes Al Lazikani et al., 1997, J. Mol. Biol., 273:927-948.
  • a humanized antibody is a form of a non-human (e.g., murine) antibody that is specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences.
  • humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding capability.
  • the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species.
  • the humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability.
  • the humanized antibody may comprise variable domains containing all or substantially all of the CDRs that correspond to the non-human immunoglobulin whereas all or substantially all of the framework regions are those of a human immunoglobulin sequence.
  • the variable domains comprise the framework regions of a human immunoglobulin sequence.
  • the variable domains comprise the framework regions of a human immunoglobulin consensus sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region or domain
  • An epitope (also referred to herein as an antigenic determinant) is the portion of an antigen capable of being recognized and specifically bound by a particular antibody, a particular AFFIMER® polypeptide or other particular binding domain.
  • the antigen is a polypeptide
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein.
  • Epitopes formed from contiguous amino acids also referred to as linear epitopes
  • epitopes formed by tertiary folding also referred to as conformational epitopes
  • An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.
  • Specifically binds 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, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
  • a target e.g., TFNR2
  • an AFFIMER® polypeptide that specifically binds to TFNR2 is an AFFIMER® polypeptide that binds TFNR2 with greater affinity, avidity (if multimeric formatted), more readily, and/or with greater duration than it binds to other targets.
  • Conjugate refers the joining or linking together of two or more compounds resulting in the formation of another compound, by any joining or linking methods known in the art. It can also refer to a compound that is generated by the joining or linking together two or more compounds.
  • a TFNR2 AFFIMER® polypeptide linked directly or indirectly to at least one chemical moiety or polypeptide is an exemplary conjugate.
  • Such conjugates include fusion proteins, those produced by chemical conjugates and those produced by any other methods.
  • a polynucleotide (also referred to herein as a nucleic acid or a nucleic acid molecule) is a polymer of nucleotides of any length and may comprise DNA, RNA (e.g., messenger RNA (mRNA)) or a combination of DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, 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 deoxyribonucleic acid deoxyribonucleotides. 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 amino acid sequence.
  • a “sequence” may comprise DNA and/or RNA (e.g., messenger RNA) and may be single and/or double stranded.
  • RNA e.g., messenger RNA
  • Nucleic acid sequences may be modified, e.g., mutated, relative to naturally occurring nucleic acid sequences, for example.
  • Nucleic acid sequence may have any length, for example 2 to 000,000 or more nucleotides (or any integral value above or between) a nucleic acid, for example a length of 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 achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE- dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics technology (biolistics).
  • a vector is a construct that is capable of delivering, and usually expressing, at least one gene or sequence of interest in a host cell.
  • 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 to the interior of the cell.
  • a vector may be an autonomously replicating plasmid or virus.
  • the term should also be construed to include facilitate transfer of nucleic acid into cells of the non-plasmid and non- viral compounds, for example, polylysine compounds, liposomes, and the like.
  • Non-limiting examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated virus vectors, and retroviral vectors.
  • An expression vector is a vector comprising a recombinant polynucleotide comprising 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 elements for expression can be supplied by the host cell or in vitro expression system.
  • Expression vectors include, for example, cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentivirus, retroviruses, adenoviruses and adeno-associated viruses).
  • Operably linked refers to functional linkage between the regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the latter.
  • the promoter affects the transcription or expression of the coding sequence, the promoter is operably linked to a coding sequence.
  • DNA sequencing operably linked are contiguous, and may join two protein coding regions in the same reading frame.
  • a promoter is a DNA sequence recognized by the synthetic machinery required for the synthesis machinery of the cell specific transcription of a polynucleotide sequence or introduced.
  • Inducible expression refers to expression under certain conditions, such as activation (or inactivation) of an intracellular signaling pathway or the contacting of the cells harboring the expression construct with a small molecule that regulates the expression (or degree of expression) of a gene operably linked to an inducible promoter sensitive to the concentration of the small molecule.
  • constitutive expression 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 bio-membrane; their presence allows biomolecules such as plasmids or other oligonucleotide to pass from one side of the cellular membrane to the other.
  • Treatment refers to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • Subject refers 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 cessation of a treatment.
  • the sustained response has a duration at least the same as the treatment duration, at least 1.5x, 2. Ox, 2.5x, or 3. Ox length of the treatment duration.
  • a “complete response” or “CR” refers to disappearance of all target lesions; “partial response” or “PR” refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD; 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 as reference the smallest SLD since the treatment started.
  • SLD longest diameters
  • Progression free survival refers to the length of time during and after treatment during which the disease being treated (e.g., inflammatory or autoimmune disease) does not get worse. Progression-free survival may include the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease.
  • ORR Overall response rate
  • “Overall survival” refers to the percentage of individuals in a group who are likely to be alive after a particular duration of time.
  • Antagonistic refer to agents that are capable of, directly or indirectly, substantially inducing, activating, promoting, increasing, or enhancing the biological activity of a target or target pathway.
  • Ant is used herein to include any agent that partially or fully induces, activates, promotes, increases, or enhances the activity of a protein or other target of interest.
  • Antagonist and “antagonistic” refer to or describe an agent that is capable of, directly or indirectly, partially or fully blocking, inhibiting, reducing, or neutralizing a biological activity of a target and/or pathway.
  • antagonist is used herein to include any agent that partially or fully blocks, inhibits, reduces, or neutralizes the activity of a protein or other target of interest.
  • Modulation and modulate refer to a change or an alteration in a biological activity. Modulation includes, but is not limited to, stimulating an activity or inhibiting an activity. Modulation may be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, a pathway, a system, or other biological targets 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.
  • NK natural killer
  • “Pharmaceutically acceptable” refers to a substance approved or approvable by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia 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 which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic effect.
  • a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of 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.
  • an agent such as a TFNR2 AFFIMER.® agent
  • the therapeutically effective amount of an TFNR2 AFFIMER® agent has a therapeutic effect and as such can reduce inflammation; relieve 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 such effects.
  • reference to "about” or “approximately” a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X” includes description of "X”.
  • An AFFIMER® polypeptide is a scaffold based on a Stefin A polypeptide, meaning that it has a sequence which is derived from a Stefin A polypeptide, for example, a mammalian Stefin A polypeptide, for example, a human Stefin A polypeptide.
  • Some aspects of the application provide AFFIMER® polypeptides that bind TFNR2 (also referred to as “TFNR2 AFFIMER® polypeptides”) in which at least one of the solvent accessible loops from the wildtype Stefin A protein having the ability to bind TFNR2, preferably selectively, and preferably with Kd of 10-6M or less.
  • a TFNR2 AFFIMER® polypeptide is derived from the wild-type human Stefin A polypeptide having a backbone sequence and in which 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 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 fewer 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 to 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 to 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.
  • FR1 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% homology with SEQ ID NO: 1. In some embodiments, FR1 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% identity with SEQ ID NO: 1; In some embodiments, FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% homology with SEQ ID NO: 2.
  • FR2 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% identity with SEQ ID NO: 2;
  • FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% homology with SEQ ID NO: 3.
  • FR3 is a polypeptide sequence having at least 80%, 85%, 90%, 95% or even 98% identity with SEQ ID NO: 3.
  • the TNFR2 AFFIMER® polypeptide has an amino acid sequence represented in the general Formula (II): MIP-Xaal-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV- (Xaa)n-Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4-Xaa5-(Xaa)m-Xaa6-D-Xaa7- VLTGYQVDKNKDDELTGF (SEQ ID NO: 4) wherein
  • Xaa individually for each occurrence, is an amino acid residue, and n and m are each, independently, an integer from 3- 20.
  • 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:
  • Xaa individually for each occurrence, is an amino acid residue, and n and m are each, independently, an integer from 3- 20.
  • Xaal is Gly, Ala, Vai, Arg, Lys, Asp, or Glu, more preferably Gly, Ala, Arg or Lys, and more even more preferably Gly or Arg;
  • Xaa2 is Gly, Ala, Vai, Ser or Thr, more preferably Gly or Ser;
  • Xaa3 is Arg, Lys, Asn, Gin, Ser, Thr, more preferably Arg, Lys, Asn or Gin, and even more preferably Lys or Asn;
  • Xaa4 is Gly, Ala, Vai, Ser or Thr, more preferably Gly or Ser;
  • Xaa5 is Ala, Vai, He, Leu, Gly or Pro, more preferably He, Leu or Pro, and even more preferably Leu or Pro;
  • Xaa6 is Gly, Ala, Vai, Asp or Glu, more preferably Ala, Vai, Asp or Glu, and even more preferably Ala or
  • 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.
  • 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.
  • the TNFR2 AFFIMER® polypeptide has an amino acid sequence represented in the general Formula (lib) : MIP-Xaal -GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV (SEQ ID NO: 484) Xaa2-(Xaa)n-Xaa3-TNYYIKVRAGDNKYMHLKVF (SEQ ID NO: 485)-Xaa4- Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF (SEQ ID NO: 486) wherein
  • Xaa individually for each occurrence, is any number of amino acid residues, more suitably three or fewer (preferably, one or two) independently selected amino acids, and n and m are each, independently, an integer from 3- 20.
  • 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 (SEQ ID NO: 487)-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL (SEQ ID NO: 488)-(Xaa)m- EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 489), wherein
  • Xaa individually for each occurrence, is any number of amino acid residues, more suitably three or fewer (preferably, one or two) independently selected amino acids, and n and m are each, independently, an integer from 3- 20.
  • 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.
  • 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.
  • (Xaa)n is an amino acid sequence selected from SEQ ID NOs: 6 to 102, or an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% homology with 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 with a sequence selected from SEQ ID NOs: 6 to 102.
  • (Xaa)m is an amino acid sequence selected from SEQ ID NOs: 103 to 199, or an amino acid sequence having at least 80%, 85%, 90%, 95% or even 98% homology with 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 with a sequence selected from SEQ ID NOs: 103 to 199.
  • the TNFR2 AFFIMER® polypeptide has an amino acid sequence selected from SEQ ID NOs: 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 with a sequence selected from SEQ ID NOs: 200 to 296 relief wherein the sequence optionally excludes one or more of the twenty one carboxy terminal residues.
  • TNFR2 AFFIMER® polypeptide sequences The C-terminal 21 amino acids are extra sequences added for cloning and assay 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.
  • the TNFR2 AFFIMER® polypeptide has an amino acid sequence that is encoded by a nucleic acid having a coding sequence at least 70%, 75% 80%, 85%, 90%, 95% or even 98% identical with a sequence selected from SEQ ID NOs: 297 to 393, optionally excluding the nucleotides encoding the twenty one carboxy terminal amino acids.
  • the TNFR2 AFFIMER® polypeptide has an amino acid sequence that is encoded by a nucleic acid that having a coding sequence that hybridizes to a sequence selected from SEQ ID NOs: 297 to 393 optionally excluding the 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 a wash in 0.2X SSC at 65°C.
  • stringent conditions such as in the presence of 6X sodium chloride/sodium citrate (SSC) at 45°C followed by a wash in 0.2X SSC at 65°C.
  • nucleic acid sequences of the present invention The 5’ 63 nucleotides encoding C- terminal 21 amino acids are extra sequences added for cloning and assay 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.
  • 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 disclosed herein, such as addition or deletion of up to 10 amino acids relative to Stefin A or the Stefin A derived AFFIMER® polypeptide.
  • the AFFIMER® agent is a TNFR2 AFFIMER® agent comprising an AFFIMER® polypeptide portion that binds 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.
  • KD dissociation constant
  • the AFFIMER® agent is a TNFR2 AFFIMER® agent comprising an AFFIMER® polypeptide portion that binds human TNFR2 as a monomer with an off-rate constant (Koff), such as measured by BIACORETM assay, of about 10-3 s-1 (e.g., unit of 1/second) or slower; of about 10-4 s-1 or slower or even of about 10-5 s-1 or slower.
  • Koff off-rate constant
  • the AFFIMER® agent is a TNFR2 AFFIMER® agent comprising an AFFIMER® polypeptide portion that binds human TNFR2 as a monomer with an association constant (Kon), such as measured by BIACORETM assay, of at least about 103 M- ls-1 or faster; at least about 104 M-ls-1 or faster; at least about 105 M-ls-1 or faster; or even at least about 106 M-ls-1 or faster.
  • Kon association constant
  • the AFFIMER® agent is a TNFR2 AFFIMER® agent comprising an AFFIMER® polypeptide portion that binds 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.
  • the AFFIMER® agent has a melting temperature (Tm, e.g., temperature at which both the folded and unfolded states are equally populated) of 65°C or higher, and preferably at least 70°C, 75°C, 80°C or even 85°C or higher. Melting temperature is a particularly useful indicator of protein stability.
  • Tm melting temperature
  • the relative proportions of folded and unfolded proteins can be determined by many techniques known to the skilled person, 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).
  • the AFFIMER® polypeptides may further comprise an additional insertion, substitution and/or deletion that modulates biological activity of the AFFIMER® polypeptide.
  • the additions, substitutions and/or deletions may modulate at least one property or activity of modified AFFIMER® polypeptides.
  • the additions, substitutions or deletions may modulate affinity for the AFFIMER® polypeptide, e.g., for binding to and inhibiting TNFR2, modulate the circulating half-life, modulate the therapeutic half-life, modulate the stability of the AFFIMER® polypeptide, modulate cleavage by proteases, modulate dose, modulate release or bioavailability, facilitate purification, decrease deamidation, improve shelf-life, or improve or alter a particular route of administration.
  • AFFIMER® polypeptides may comprise 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 improve detection, purification or other traits of the polypeptide.
  • 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 so as to confer a desired property, such as secretion from a cell or retention on the cell surface (e.g., for an encoded AFFIMER® polynucleotide), to serve as substrate or other recognition sequences for post-translational modifications, to create multimeric structures aggregating through protein-protein interactions, to alter (often to extend) serum half-life, or to alter tissue localization or tissue exclusion and other ADME properties - merely as examples.
  • a desired property such as secretion from a cell or retention on the cell surface (e.g., for an encoded AFFIMER® polynucleotide), to serve as substrate or other recognition sequences for post-translational modifications, to create multimeric structures aggregating through protein-protein interactions, to alter (often to extend) serum half-life, or to alter tissue localization or tissue exclusion and other ADME properties - merely as examples.
  • fusion domains are particularly useful for isolation and/or purification of the fusion proteins, such as by affinity chromatography.
  • 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.
  • the AFFIMER® agent In order for the AFFIMER® agent to be secreted, it will generally contain a signal sequence that directs the transport of the protein to the lumen of the endoplasmic reticulum and ultimately to be secreted (or retained on the cell surface if a transmembrane domain or other cell surface retention signal).
  • Signal sequences also referred to as signal peptides or leader sequences
  • signal sequences are cleaved from the protein by a signal peptidase after the proteins are transported to the endoplasmic reticulum.
  • the cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is dependent upon amino acid residues within the signal sequence.
  • 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).
  • the signal peptide is a native signal peptide from a human protein.
  • the signal peptide is a non-native signal peptide.
  • the non-native signal peptide is a mutant native signal peptide from the corresponding native secreted human protein, and can include at least one (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitution, insertions and/or deletions.
  • 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 IgG heavy chain or 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 other signal peptide able to efficiently secrete a protein from a cell.
  • exemplary signal peptides include but are not limited to: Table 3. Signal peptide sequences
  • rigid linkers are chosen when the spatial separation of the domains is critical to preserve the stability or bioactivity of the fusion proteins.
  • alpha helix-forming 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 designating any amino acid, preferably Ala, Lys, or Glu.
  • exemplary linkers include GRA, poly(Gly), poly(Ala) and those provided in Table 4.
  • the AFFIMER® polypeptide may further comprise, for example be fused to, a transmembrane domain (TM domain).
  • TM domains ensure that the AFFIMER® polypeptide remains on the surface of the cell expressing the AFFIMER®.
  • TM domains are readily available and include CD3, CD8, CD28, PDGFR TMD, and others.
  • AFFIMER® polypeptide sequence or to a flanking polypeptide moiety provided as part of a fusion protein is at least one sequence that is a site for post-translational modification by an enzyme.
  • These can include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like.
  • an AFFIMER® agent is a multispecific polypeptide including, for example, a first TNFR2 AFFIMER® polypeptide and at least one additional binding domain.
  • the additional binding domain may be a polypeptide sequence selected from amongst, to illustrate, a second AFFIMER® polypeptide (which may be the same or different than the first AFFIMER® polypeptide), an antibody or fragment thereof or other 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), engineered T-cell receptor, an enzyme or catalytic fragment thereof.
  • a second AFFIMER® polypeptide which may be the same or different than the first AFFIMER® polypeptide
  • an antibody or fragment thereof or other antigen binding polypeptide such as a receptor trap polypeptide
  • a receptor-binding ligand such as a cytokine, growth factor or the like
  • an AFFIMER® agent includes at least one additional AFFIMER® polypeptide sequence that is also directed to TNFR2.
  • the additional TNFR2 AFFIMER® polypeptide(s) may be the same or different (or a mixture thereof) as the first TNFR2 AFFIMER® polypeptide in order to create a multispecific AFFIMER® fusion protein.
  • the AFFIMER® agents can bind the same or overlapping sites on TNFR2 or can bind two different sites such that the TNFR2 AFFIMER® agent can simultaneously bind two sites on the same TNFR2 protein (biparatopic) or more than two sites (multiparatopic).
  • an AFFIMER® agent includes at least one antigen binding site from an antibody.
  • the resulting AFFIMER® agent can be a single chain including both the TNFR2 AFFIMER® polypeptide and the antigen binding site (such as in the case of an scFv) or can be a multimeric protein complex such as in antibody assembled with heavy and/or light chains to which the sequence of the anti-TNFR2 antibody has also been fused.
  • the fusion of the AFFIMER® polypeptide sequence to the antibody will preserve the Fc function of the Fc region of the immunoglobulin.
  • the AFFIMER® agent may be capable of binding, via its Fc portion, to the Fc receptor of Fc receptor-positive cells.
  • 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 cytokines and/or co-stimulatory antigens.
  • the AFFIMER® agent may transfer at least a second activation signal required for physiological activation of the T cell to the T cell via the co-stimulatory antigens and/or cytokines.
  • the AFFIMER® agent may possess antibodydependent cellular cytotoxicity (ADCC) function, a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigen has been bound by an antibody, and therefore, trigger tumor cell death via ADCC.
  • ADCC antibodydependent cellular cytotoxicity
  • the AFFIMER® agent is capable of demonstrating ADCC function.
  • the Fc portion may contribute to maintaining the serum levels of the AFFIMER® agent, critical for its stability and persistence in the body. For example, when the Fc portion binds to Fc receptors on endothelial cells and on phagocytes, the AFFIMER® agent may become internalized and recycled back to the blood stream, enhancing its half-life within the body.
  • Exemplary targets of the additional AFFIMER® polypeptides include but are not limited to, another immune checkpoint protein, and immune co-stimulatory receptor (particularly if the additional AFFIMER®(s) can agonize the co-stimulatory receptor), a receptor, a cytokine, a growth factor, or a tumor-associated antigen, merely to illustrate.
  • the immunoglobulin portion may be a monoclonal antibody against at least one autoimmune target (e.g., TNFR2 or IL6-R).
  • the TNFR2 AFFIMER® 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).
  • a protein upregulated in autoimmune conditions e.g., TNFR2 or IL6-R
  • a multispecific a TNFR2 AFFIMER® agent may further comprise a half-life extension moiety, such as any of those described herein.
  • a TNFR2 AFFIMER® agent may comprise at least one TNFR2 AFFIMER® polypeptide linked through a peptide linker to a binding domain specific for at least one immune cell (e.g., T cell and/or NK cell) binding domain (e.g., CD3s chain or CD16) further linked to a half-life extension moiety, such as a fragment crystallizable (Fc) domain (e.g., an FcyR null-binding Fc), human serum albumin (HSA), or an HSA AFFIMER® polypeptide.
  • Fc fragment crystallizable domain
  • HSA human serum albumin
  • the halflife extension moiety is a fragment crystallizable (Fc) domain.
  • the halflife extension moiety is a human serum albumin (HSA).
  • HSA human serum albumin
  • the half-life extension moiety is an HSA AFFIMER® polypeptide.
  • the AFFIMER® agent may not have a half-life and/or PK profile that is optimal for the route of administration, such as parenteral therapeutic dosing.
  • a “halflife” is the amount of time it takes for a substance, such as an AFFIMER® agent of the present disclosure, to lose half of its pharmacologic or physiologic activity or concentration.
  • Biological half-life can be affected by elimination, excretion, degradation (e.g., enzymatic) of the substance, or absorption and concentration in certain organs or tissues of the body.
  • biological half-life can be assessed by determining the time it takes for the blood plasma concentration of the substance to reach half its steady state level (“plasma half-life”).
  • plasma half-life steady state level
  • 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 mitigates in vivo proteolytic degradation or other activity-diminishing modification of the AFFIMER® polypeptide, increases 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 biological activity and/or target selectivity of the modified AFFIMER® polypeptide, increasing manufacturability, and/or reducing immunogenicity of the modified AFFIMER® polypeptide, compared to a comparator such as an unconjugated form of the modified AFFIMER® polypeptide.
  • 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 (HES), 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 proteinaceous half-life extending moieties, such as serum albumin, transferrin, adnectins (e.g., albumin-binding or pharmacokinetics extending (PKE) adnectins), Fc domain, and unstructured polypeptide, such as XTEN and PAS polypeptide (e.g. conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and/or Ser), and a fragment of any combination of the
  • the half-life extending moiety extends the half-life of the resulting AFFIMER® agent circulating in mammalian blood serum compared to the half-life of the protein that is not so conjugated to the moiety (such as relative to the AFFIMER® polypeptide alone). In some embodiments, half-life is extended by greater than or greater than 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, halflife 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 after in vivo administration compared to the protein without the half-life extending moiety.
  • half-life extending moieties that can be used in the generation of AFFIMER® agents of the disclosure include:
  • XTEN also known as recombinant PEG or “rPEG”
  • HAP homoamino acid polymer
  • PAS proline-alanine-serine polymer
  • ELP elastin-like peptide
  • Fc fusions involve the fusion of peptides, proteins or receptor exodomains to the Fc portion of an antibody. Both 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 3-16 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 peptide Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin, the same protein exploited by the fatty acylated peptides is the other popular fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform.
  • Fc and albumin A major difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HSA leading to presentation of a fused peptide as a dimer or a 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 spaced closely enough together or are themselves dimers. This may be desirable or not depending on the target.
  • 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.
  • the Fc region is a FcyR null-binding Fc region.
  • an Fc fusion (“Fc-fusion”), such as a TNFR2 AFFIMER® agent created as an AFFIMER®-Fc fusion protein, is a polypeptide comprising at least one TNFR2 AFFIMER® sequence covalently linked through 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 a TNFR2 AFFIMER® sequence as part of the same polypeptide.
  • An immunoglobulin Fc region may also be linked indirectly to at least one TNFR2 AFFIMER® polypeptide.
  • linkers are known in the art and can optionally be used to link an Fc to a polypeptide including a TNFR2 AFFIMER® sequence to generate an Fc-fusion.
  • Fc-fusions can be dimerized to form Fc-fusion homodimers, or using non-identical Fc domains, to form Fc-fusion heterodimers.
  • an Fc-fusion 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).
  • Fc region of human antibodies for use in generating TNFR2 AFFIMER® agents as TNFR2 AFFIMER® fusion proteins.
  • the principle rationale is to produce a stable protein, large enough to demonstrate a similar pharmacokinetic profile compared with those of antibodies, and to take advantage of the properties imparted by the Fc region; this includes the salvage neonatal FcRn receptor pathway involving FcRn- mediated recycling of the fusion protein to the cell surface post endocytosis, avoiding lysosomal degradation and resulting in release back into the bloodstream, thus contributing to an extended serum half-life.
  • Another obvious advantage is the Fc domain’s binding to Protein A, which can simplify downstream processing during production of the AFFIMER® agent and permit generation of highly pure preparation of the AFFIMER® agent.
  • an Fc domain will include the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • 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 hinge N-terminal to these domains.
  • IgA and IgM Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy2.
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is 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 this region in the context of a whole antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of different Fc positions and are also included as Fc domains as used herein.
  • a “functional Fc region” refers to an Fc domain or fragment thereof which retains the ability to bind FcRn.
  • a functional Fc region binds to FcRn but does not possess 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.
  • Exemplary "effector functions” include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
  • effector functions can be assessed using various assays known in the art for evaluating such antibody effector functions.
  • the Fc domain is derived from an IgGl subclass, however, other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used.
  • IgG2, IgG3, and IgG4 An exemplary sequence of a human IgGl immunoglobulin Fc domain which can be used is:
  • the Fc region used in the fusion protein may comprise the hinge region of an Fc molecule.
  • An exemplary hinge region comprises the core hinge residues spanning positions 1-16 (e.g., DKTHTCPPCPAPELLG ((SEQ ID NO: 536)) of the exemplary human IgGl immunoglobulin Fc domain sequence provided above.
  • the AFFIMER®-containing fusion protein may adopt a multimeric structure (e.g., dimer) owing, in part, to the cysteine residues at positions 6 and 9 within the hinge region of the exemplary human IgGl immunoglobulin Fc domain sequence provided above.
  • the hinge region as used herein may further include residues derived from the CHI and CH2 regions that flank the core hinge sequence of the exemplary human IgGl immunoglobulin Fc domain sequence provided above.
  • the hinge sequence may comprise or consist of GSTHTCPPCPAPELLG (SEQ ID NO: 443) or EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 444).
  • the hinge sequence may include at least one substitution that confer desirable pharmacokinetic, biophysical, and/or biological properties.
  • Some exemplary 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
  • DKTHTCPPCPAPELLGGSS SEQ ID NO: 450
  • the residue P at position 18 of the exemplary human IgGl immunoglobulin Fc domain sequence provided above may be replaced with S to ablate Fc effector function; this replacement is exemplified in hinges having the sequences
  • EPKSSDKTHTCPPCPAPELLGGSS SEQ ID NO: 451
  • EPKSSGSTHTCPPCPAPELLGGSS SEQ ID NO: 452
  • DKTHTCPPCPAPELLGGSS SEQ ID NO: 453
  • residues DK at positions 1-2 of the exemplary human IgGl immunoglobulin Fc domain sequence provided above may be replaced with GS to remove a potential clip site; this replacement is exemplified in the sequence EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 448).
  • the C at the position 103 of the heavy chain constant region of human IgGl may be replaced with S to prevent improper cysteine bond formation in the absence of a light chain; this replacement is exemplified by EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 446), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 451), and EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 452).
  • the Fc is a mammalian Fc such as a human Fc, including Fc domains derived from IgGl, IgG2, IgG3 or IgG4.
  • the Fc region may possess at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a native Fc region and/or with an Fc region of a parent polypeptide.
  • the Fc region may have at least about 90% sequence identity with a native Fc region and/or with an Fc region of a parent polypeptide.
  • the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 454-467 or an Fc sequence from the examples provided by SEQ ID NOs: 454- 467. It should 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 NOs: 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 NOs: 454-467. In some embodiments, the Fc domain comprises the amino acid sequence selected from SEQ ID NOs: 454-467 except the C-terminal lysine thereof is omitted.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., Natural Killer (NK) cells, neutrophils, and macrophages
  • the fusion protein includes an Fc domain sequence for which the resulting AFFIMER® agent has no (or reduced) ADCC and/or complement activation or effector functionality.
  • the Fc domain may comprise a naturally disabled constant region of IgG2 or IgG4 isotype or a mutated IgGl constant region. Examples of suitable modifications are described in EP0307434. One example comprises the substitutions of alanine residues at positions 235 and 237 (EU index numbering).
  • the fusion protein includes an Fc domain sequence for which the resulting AFFIMER® agent will retain some or all Fc functionality for example will be capable of one or both of ADCC and CDC activity, as for example if the fusion protein comprises the Fc domain from human IgGl or IgG3.
  • Levels of effector function can be varied according to known techniques, for example by mutations in the CH2 domain, for example wherein the IgGl CH2 domain has at least one mutation at positions selected from 239 and 332 and 330, for example the mutations are selected from S239D and I332E and A330L such that the antibody has enhanced effector function, and/or for example altering the glycosylation profile of the antigen-binding protein of the disclosure such that there is a reduction in fucosylation of the Fc region.
  • the AFFIMER® agent is a fusion protein comprising, in addition to at least one AFFIMER® sequence, an albumin sequence or an albumin fragment.
  • the AFFIMER® agent is conjugated to the albumin sequence or an albumin fragment through chemical linkage other than incorporation into the polypeptide sequence including the AFFIMER® polypeptide.
  • the albumin, albumin variant, or albumin fragment is human serum albumin (HSA), a human serum albumin variant, or a human serum albumin fragment.
  • HSA human serum albumin
  • Albumin serum proteins comparable to HSA are found in, for example, cynomolgus monkeys, cows, dogs, rabbits and rats.
  • bovine serum albumin is the most structurally 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, albumin sequence derived from cyno serum albumin or bovine serum albumin.
  • Mature HSA a 585 amino acid polypeptide (approx. 67 kDa) having a serum half-life of about 20 days, is primarily responsible for the maintenance of colloidal osmotic blood pressure, blood pH, and transport and distribution of numerous endogenous and exogenous ligands.
  • the protein has three structurally homologous domains (domains I, II and III), is almost entirely in the alpha-helical conformation, and is highly stabilized by 17 disulfide bridges.
  • the AFFIMER® agent can be an albumin fusion protein including at least one AFFIMER® polypeptide sequence and the sequence for mature human serum albumin (SEQ ID NO: 468) or a variant or fragment thereof which maintains the PK and/or biodistribution properties of mature albumin to the extent desired in the fusion protein.
  • the albumin sequence can be set off from the AFFIMER® polypeptide sequence or other flanking sequences in the AFFIMER® agent by use of linker sequences as described above.
  • HSA human serum albumin
  • MKWVTFISLLFLFSSAYS SEQ ID NO: 483
  • RGVFRR pro-domain of 6 amino acids
  • This 24 amino acid residue peptide may be referred to as the pre-pro domain.
  • the AFFIMER®-HSA fusion proteins can be expressed and secreted using the HSA pre-pro-domain in the recombinant proteins coding sequence. Alternatively, the AFFEMER®-HSA fusion can be expressed and secreted through inclusion of other secretion signal sequences, such as described above.
  • the serum albumin polypeptide can be covalently coupled to the AFFIMER®-containing polypeptide through a bond other than a backbone amide bond, such as cross-linked through chemical conjugation between amino acid sidechains on each of the albumin polypeptide and the AFFIMER®-containing polypeptide.
  • lipidation which involves the covalent binding of fatty acids to peptide side chains.
  • PEGylation a basic mechanism for half-life extension as PEGylation, namely increasing the hydrodynamic radius to reduce renal filtration.
  • the lipid moiety is itself relatively small and the effect is mediated indirectly through the non-covalent binding of the lipid moiety to circulating albumin.
  • lipidation 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 the use of glutamate or mini PEGs within the linker.
  • Linker engineering and variation of the lipid moeity can affect self-aggregation which can contribute to increased half-life by slowing down biodistribution, independent of albumin. See, for example, Jonassen et al. (2012) Pharm Res. 29(8):2104-14.
  • PKE2 albumin-binding
  • AdAb albumin binding domain antibody
  • the molecule that binds a serum protein such as HSA comprises an HSA AFFIMER® polypeptide.
  • HSA AFFIMER® polypeptides can be found in W02022/023540.
  • An HSA AFFIMER® polypeptide provided herein, in some embodiments, is linked to another molecule and extend the half-life of that molecule (e.g., a therapeutic polypeptide).
  • HSA AFFIMER® polypeptides have been shown in in vivo pharmacokinetic (PK) studies to extend, in a controlled manner, the serum half-life of any other AFFIMER® polypeptide therapeutic to which it is conjugated in a single genetic fusion, for example, that can be made in E. Coli.
  • AFFIMER® XTTM polypeptides can also be used to extend the half-life of other peptide or protein therapeutics, such as the TNFR2 AFFIMER® of the present invention.
  • an HSA AFFIMER® polypeptide extends the serum half-life of the TNFR2 AFFIMER® polypeptide in vivo.
  • an HSA AFFIMER® polypeptide may extend the half-life of the TNFR2 AFFIMER® polypeptide by at least 2-fold, relative to the half-life of the molecule not linked to an HSA AFFIMER® polypeptide.
  • an HSA AFFIMER® polypeptide extends the half-life of the TNFR2 AFFIMER® 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 TNFR2 AFFIMER® polypeptide not linked to an HSA AFFIMER® polypeptide.
  • an HSA AFFIMER® polypeptide extends the half-life of the TNFR2 AFFIMER® 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 TNFR2 AFFIMER® polypeptide not linked to an HSA AFFIMER® polypeptide.
  • an HSA AFFIMER® polypeptide extends the half-life of the TNFR2 AFFIMER® 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, for example, at least 1 week after in vivo administration, relative to the half-life of the molecule not linked to an HSA AFFIMER® polypeptide.
  • An HSA AFFIMER® polypeptide comprises an AFFIMER® polypeptide in which at least one of the solvent accessible loops is from the wild-type Stefin A protein having amino acid sequences to enable an AFFIMER® polypeptide to bind HSA, selectively, and in some embodiments, with a Kd of 10-6M or less.
  • the HSA AFFIMER® polypeptide is derived from the wild-type human Stefin A protein having a backbone sequence and in which 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 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 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 to 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:
  • each amino acid of (Xaa)n may be the same or selected from any amino acid. The same applies for (Xaa)m.
  • the TNFR2 AFFIMER® polypeptide comprises an HSA AFFIMER® polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 471-477 (Table 6).
  • 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 with a sequence selected from SEQ ID NOs: 471- 477 (Table 6). Additional HSA AFFIMER® polypeptides sequences for use with the present invention can be found in W02022/023540.
  • the subject AFFIMER® agents may also include at least one functional moiety intended to impart detectability or additional pharmacologic activity to the AFFIMER® agent.
  • Functional moieties for detection are those which can be employed to detect association of the AFFIMER® agent with a cell or tissue in vivo.
  • Functional moieties with pharmacologic activity are those agents which are meant 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 pharmacologic consequence to the targeted tissues or cells.
  • AFFIMER® agents including conjugates of substances having a wide variety of functional groups, substituents or moieties, with those Functional Moieties including but not limited to a label; a dye; an immunoadhesion molecule; a radionuclide; a cytotoxic compound; a drug; an affinity label; a photoaffinity label; 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; a 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 moiety; a radioactive moiety; a novel functional group
  • the moiety is a detectable label
  • it can be a fluorescent label, radioactive label, enzymatic label or any other label known to the skilled person.
  • the Functional Moiety is a detectable label that can be included as part of a conjugate to form certain AFFIMER® agents suitable for medical imaging.
  • medical imaging is meant any technique used to visualize an internal region of the human or animal body, for the purposes of diagnosis, research or therapeutic treatment.
  • the AFFIMER® agent can be detected (and quantitated) by radioscintigraphy, magnetic resonance imaging (MRI), computed tomography (CT scan), nuclear imaging, positron emission comprising a metal 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 in enhancing such techniques are those materials that enable visualization of a particular locus, organ or disease site within the body, and/or that lead to some improvement in the quality of the images generated by the imaging techniques, providing improved or easier interpretation of those images.
  • contrast agents Such agents are referred to herein as contrast agents, the use of which facilitates the differentiation of different parts of the image, by increasing the “contrast” between those different regions of the image.
  • contrast agents thus encompasses agents that are used to enhance the quality of an image that may nonetheless be generated in the absence of such an agent (as is the case, for instance, in MRI), as well as agents that are prerequisites for the generation of an image (as is the case, for instance, in nuclear imaging).
  • the detectable label includes a chelate moiety for chelating a metal, e.g., a chelator for a radiometal or paramagnetic ion.
  • the detectable label is a chelator for a radionuclide useful for radiotherapy or imaging procedures.
  • Radionuclides useful within 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.
  • radionuclides useful as toxins in radiation therapy include: 43K, 47Sc, 51Cr, 57Co, 58Co, 59Fe, 64Cu, 67Ga, 67Cu, 68Ga, 71Ge, 75Br, 76Br, 77Br, 77 As, 81Rb, 90Y, 97Ru, 99mTc, lOOPd, lOlRh, 103Pb, 105Rh, 109Pd, l l lAg, U lin, 113In, 119Sb 121Sn, 1231, 1251, 127Cs, 128Ba, 129Cs, 1311, 131Cs, 143Pr, 153Sm, 161Tb, 166Ho, 169Eu, 177Lu, 186Re, 188Re, 189Re, 1910s, 193Pt, 194Ir, 197Hg, 199 Au, 203Pb, 211 At, 212
  • chelators includes, merely to illustrate, l,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).
  • NOTA l,4,7-triazacyclononane-N,N',N"-triacetic acid
  • DOTA 10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid
  • TETA tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid
  • Other detectable isotopes that can be incorporated directly into the amino acid residues of the AFFIMER® polypeptide or which otherwise do
  • paramagnetic ions useful for diagnostic procedures, may also be administered.
  • 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.
  • fluorescent labels include, but are not restricted 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.
  • 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.
  • quantum dots e.g., quantum dots.
  • Non-limiting fluorescent compound 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), the rhodamine derivative JA133, Alexa Fluorescent Dyes (such as Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 555, and Alexa Fluor 647), 4',6-diamidino-2-phenylindole (DAPI),
  • Fluorescent compound 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, mKalamal), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet).
  • fluorescent proteins such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet).
  • GFP green fluorescent protein
  • EGFP enhanced GFP
  • BFP blue fluorescent protein and derivatives
  • BFP blue fluorescent protein and derivatives
  • CFP cyan fluorescent protein and derivatives
  • CFP ECFP
  • Examples of enzymatic labels include, but are not restricted to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase and > -galactosidase.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • glucose oxidase glucose oxidase
  • > -galactosidase examples include, but are not restricted to, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase and > -galactosidase.
  • Biotin labels are typically 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 labelled protein to other moieties which comprise an avidin moiety.
  • the AFFIMER® agent includes at least one therapeutic agent, e.g., to form an AFFIMER® polypeptide-drug conjugate.
  • therapeutic agent refers to a substance that may be used in the cure, mitigation, treatment, or prevention of disease in a human or another animal.
  • therapeutic agents include substances recognized in the official United States Pharmacopeia, official Homeopathic Pharmacopeia of the United States, official National Formulary, or any supplement thereof, and include but are not limited to small molecules, nucleotides, oligopeptides, polypeptides, etc.
  • Therapeutic agents that may be attached to AFFIMER®-containing polypeptides include but are not limited to, cytotoxic agents, anti-metabolites, alkylating agents, antibiotics, growth factor, 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, vincalkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, therapeutic antibodies, tyrosine kinase inhibitors, radiosensitizers, and chemotherapeutic combination therapies, such as illustrations.
  • cytotoxic agents include DNA alkylating agents, topoisomerase inhibitors, microtubule inhibitors (e.g., DM1, DM4, MMAF and MMAE), end
  • any method known in the art for conjugating to antibodies and other proteins may be employed in generating the conjugates of the present disclosure, including those 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 peptide, polypeptide and organic and inorganic moieties to antibodies and other proteins are conventional and very well known in the art and readily adapted for generating those versions of the subject AFFIMER® agents.
  • conjugated moiety is a peptide or polypeptide
  • that moiety can be chemically cross-linked to the AFFIMER®-containing polypeptide or can be included as part of a fusion protein with the AFFIMER®-containing polypeptide.
  • illustrative example would be a diptheria toxin-AFFIMER® fusion protein.
  • the addition to the AFFIMER®-containing polypeptide will generally be by way of chemical conjugation to the AFFIMER®-containing polypeptide - such as through a functional group on an amino acid side chain or the carboxyl group at the C-terminal or amino group at the N-terminal end of the polypeptide.
  • the conjugated moiety will include at least one site that is sensitive to an environmental condition (such as pH) that permits the conjugated moiety to be released from the AFFIMER®- containing polypeptide, such as in a diseased tissue (or tissue to be protected if the conjugated moiety functions to protect healthy tissue).
  • an environmental condition such as pH
  • an AFFIMER® polypeptide-drug conjugate comprises a spacer or bond (LI) between the half-life extension moiety and the substrate recognition sequence (SRS) cleavable by the enzyme, e.g., present in an inflammatory microenvironment.
  • LI spacer or bond
  • SRS substrate recognition sequence
  • the spacer may be any molecule, for example, one or more nucleotides, amino acids, chemical functional groups.
  • the spacer is a peptide linker (e.g., two or more amino acids). Spacers should not adversely affect the expression, secretion, or bioactivity of the polypeptides.
  • spacers are not 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-mediate 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 cell responses are contemplated herein.
  • linkers are non-protein-coding.
  • LI is a hydrocarbon (straight chain or cyclic) such as 6- maleimidocaproyl, maleimidopropanoyl and maleimidom ethyl cyclohexane- 1 -carb oxy late, or LI is N-Succinimidyl 4-(2 -pyridylthio) pentanoate, N- Succinimidyl 4-(N- maleimidomethyl) cyclohexane- 1 carboxylate, N-Succinimidyl (4-iodo-acetyl) aminobenzoate.
  • hydrocarbon straight chain or cyclic
  • LI is N-Succinimidyl 4-(2 -pyridylthio) pentanoate, N- Succinimidyl 4-(N- maleimidomethyl) cyclohexane- 1 carboxylate, N-Succinimidyl (4-iodo-acetyl) aminobenzoate.
  • LI is a polyether such as a poly(ethylene glycol) or other hydrophilic linker.
  • CBM includes a thiol (such as a cysteine residue)
  • LI can be a polyethylene glycol) coupled to the thiol group through a mal eimide moiety.
  • Non-limiting examples of linkers for use in accordance with the present disclosure are described in International Publication No. WO 2019/236567, published December 12, 2019, incorporated by reference herein. Encoded AFFIMER® Polynucleotide for In vivo Delivery
  • AFFIMER® agents such as a TNF2 AFFIMER® agent
  • TNF2 AFFIMER® agent An alternative approach to the delivery of therapeutic AFFIMER® agents, such as a TNF2 AFFIMER® agent, would be to leave the production of the therapeutic polypeptide to the body itself.
  • 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 administer to patients the encoded AFFIMER® polynucleotide, rather than the AFFIMER® agent. This allows the patient’s body to produce the therapeutic AFFIMER® agent of interest for a prolonged period of time, and secrete it either systemically or locally, depending on the production site.
  • Gene-based encoded AFFIMER® polynucleotide can present a labor- and cost-effective alternative to the conventional production, purification and administration of the polypeptide version of the AFFIMER® agent.
  • a number of antibody expression platforms have been pursued in vivo to which delivery of encoded AFFIMER® polynucleotide can be adapted, including viral vectors, naked DNA and RNA.
  • an effective encoded AFFIMER® gene transfer approach results in expression should be at a level that is appropriate to the specific application.
  • Promoters are a major cis-acting element within the vector genome design that can dictate the overall strength of expression as well as cell-specificity.
  • polyadenylation of a transcribed 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 know in the art, such as enhancers, intronic sequences, post transcriptional regulatory agents and the like.
  • Exemplary viral gene therapy system that are readily adapted for use in the present disclosure include plasmid, adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, reovirus, measles virus, Semliki Forest virus, and the like.
  • Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the nucleic acid construct carrying the nucleic acid sequences encoding the epitopes and targeting sequences of interest.
  • encoded AFFIMER® polynucleotides can be delivered in vivo using adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • TNFR2 AFFIMER® polypeptides may be encoded and delivered in vivo using retroviral vectors, such as gammaretroviruses or lentiviruses.
  • the viral vector is pseudotyped with an envelope is chosen from one of amphotropic, polytropic, xenotropic, 10A1, GALV, VSV- G, Baboon endogenous virus, RD114, rhabdovirus, alphavirus, measles or influenza virus envelopes.
  • nucleic acids or polynucleotides for the encoded TNFR2 AFFIMER® agents of the present disclosure include but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P- 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.
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • the encoded TNFR2 AFFIMER® polynucleotide may be delivered by plasmid DNA, minicircle DNA, mRNA, RNA replicons and the like.
  • Means for delivery include transfection using electroporation, the use of lipofection and other transfection reagents, naked DNA delivery, gold particle delivery and other means known in the art.
  • TNFR2 AFFIMER® agent proteins described herein can 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 those sequences in a suitable host. For those recombinant AFFIMER® agent proteins including further modifications, such as a chemical modifications or conjugation, the recombinant AFFIMER® agent protein can be further manipulated chemically or enzymatically after isolation form the host cell or chemical synthesis.
  • the AFFIMER® polypeptide may be secreted or associated with the expressing cell’s membrane, for example through anchoring or 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 condition favorable to expression of the polynucleotide and, (iii) optionally, isolating the AFFIMER® agent from the host cell and/or medium in which the host cell is grown. See e.g., WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.
  • WO 04/041862 WO 2006/122786, WO 2008/020079,
  • a DNA sequence encoding a recombinant AFFIMER® agent protein of interest may 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 those 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. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • the vector for the production of the recombinant AFFIMER® agent protein may be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the recombinant AFFIMER® agent coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • 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.
  • the expression of the recombinant AFFIMER® agent protein is regulated by a constitutive, an inducible or a tissue, specific promoter.
  • the expression vector may include an origin of replication, such as may be selected based upon the type of host cell being used for expression.
  • an origin of replication such as may be selected based upon the type of host cell being used for expression.
  • the origin of replication from the plasmid pBR322 is useful for most Gram- negative bacteria while various origins from SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses (such as HPV or BPV) are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used because it contains the early promoter).
  • the vector may include at least one selectable marker gene, e.g., genetic elements that encode a protein necessary for the survival and growth of a host cell grown in a selective culture medium.
  • 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) supply 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.
  • selection genes may be used to amplify the gene which will be expressed. Amplification is a process where genes which 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. The mammalian cell transformants are placed under selection pressure which only the transformants are uniquely adapted to survive by virtue of the marker present in the vector.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase
  • Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes the recombinant AFFIMER® agent protein.
  • concentration of selection agent in the medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes the recombinant AFFIMER® agent protein.
  • increased quantities of the recombinant AFFIMER® agent 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 including the coding sequence for the recombinant AFFIMER® agent protein.
  • 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 methods set forth above and used in a prokaryotic vector.
  • the 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. Either a native or heterologous promoter may be used depending on the host cell used for expression and the yield desired.
  • Promoters for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems; alkaline phosphatase, a 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 a desired nucleic acid sequence(s), using linkers or adapters as desired to supply restriction sites.
  • Promoters for use with yeast hosts are also known in the art.
  • Yeast enhancers are advantageously used with yeast promoters.
  • Suitable promoters 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 which may be used for expressing the selective binding agents of the disclosure include but are not limited to: the SV40 early promoter region (Bemoist 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.
  • the regulatory sequences of the metallothionine 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).
  • the elastase I gene control region 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 insulin gene control region 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.
  • An enhancer sequence may be inserted into the vector to increase transcription in eukaryotic host cells.
  • enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin).
  • an enhancer from a virus will be used.
  • the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters.
  • Vectors for expressing nucleic acids include those which are compatible with bacterial, insect, and mammalian host cells.
  • Such vectors include, inter alia, 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, cosmids, plasmids or modified viruses, 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, Stratagene Cloning Systems Inc., La Jolla Calif.), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPOTM.
  • TA Cloning® Kit PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, Calif.
  • mammalian, yeast or virus vectors such as a baculovirus expression system (pBacPAK plasmid derivatives, Clontech, Palo Alto, Calif.).
  • the recombinant molecules can be introduced into host cells via 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 disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), 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.
  • Cell lines of particular preference are selected through determining which cell lines have high expression levels.
  • Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells.
  • Fungal cells include yeast and filamentous fungus cells including, for example, 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
  • 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.
  • host-expression vector systems may be utilized to express the recombinant AFFIMER® agent protein of the disclosure.
  • Such host-expression systems represent vehicles by which the coding sequences of the recombinant AFFIMER® agent protein may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the recombinant AFFIMER® agent protein of the disclosure in situ.
  • host-expression systems represent vehicles by which the coding sequences of the recombinant AFFIMER® agent protein may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the recombinant AFFIMER® agent protein of the disclosure in situ.
  • 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 the 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, lymphotic cells (see U.S.
  • 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., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the recombinant AFFIMER® agent protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of the recombinant AFFIMER® agent protein, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include but are not limited, to the E. coli expression vector pUR278 (Ruther et al. (1983) "Easy Identification Of cDNA Clones," EMBO J.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the AFFIMER® agent protein coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • the AFFIMER® agent protein coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts, (see e.g., see Logan et al.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific fashion desired. 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 the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, 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.
  • stable expression is contemplated.
  • cell lines which stably express an antibody of the disclosure may be engineered.
  • 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.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the recombinant AFFIMER® agent proteins of the disclosure. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the recombinant AFFIMER® agent proteins.
  • a number of selection systems may be used, including but not limited to the 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.
  • the expression levels of a recombinant AFFIMER® agent protein can be increased by vector amplification (for a 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, Vol. 3. (Academic Press, New York, 1987)).
  • vector amplification for a 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, Vol. 3. (Academic Press, New York, 1987)
  • a marker in the vector system expressing a recombinant AFFIMER® agent protein is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene.
  • the host cell may be co-transfected with two expression vectors, for instance the first vector encoding a heavy chain and the second vector encoding a light chain derived polypeptide, one or both of which includes an AFFIMER® polypeptide coding sequence.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides.
  • the light chain should 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 coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of the recombinant AFFIMER® agent protein will depend on the particular cell line or transgenic animal used to produce the protein. In some embodiments of AFFIMER®/antibody fusions, a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous, because in the case of antibodies this has been shown to typically exhibit more potent efficacy than fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. NOS: 6,946,292 and 7,214,775).
  • an AFFIMER® agent from production cell lines can be enhanced using a number of known techniques.
  • the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions.
  • the GS system is discussed in whole or part in connection with European Patent NOS: 0216846, 0256055, and 0323997 and European Patent Application No. 89303964.4.
  • the mammalian host cells e.g., CHO
  • the polynucleotide encoding the immunoglobulin chain comprises a glutamine synthetase gene which complements the lack of the gene in the host cell.
  • Such host cells containing the binder or polynucleotide or vector as discussed herein as well as expression methods, as discussed herein, for making the binder using such a host cell are part of the present disclosure.
  • insect cell culture systems e.g., baculovirus
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • the recombinant AFFIMER® agent proteins produced by a transformed host can be purified according to any suitable method.
  • Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S- transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column.
  • Isolated proteins can also be physically characterized using such techniques as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and x-ray crystallography.
  • recombinant AFFIMER® agent proteins produced in bacterial culture can 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 steps. HPLC can be employed for final purification steps.
  • Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • the AFFIMER® agents of the disclosure are useful in a variety of applications including, but not limited to, therapeutic treatment methods, for systemic lupus erythermatosis (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) (relating to stem cell transplants) also called allograft rejection, transplant/Solid Organ Transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic
  • SLE Systemic lupus erythematosus
  • lupus is a chronic autoimmune disease that can cause swelling (inflammation) and pain throughout the body.
  • lupus 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 meaning skin. Individuals with cutaneous lupus erythematosus may experience skin issues like a sensitivity to the sun 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 of systemic lupus erythematosus, but it is usually temporary.
  • Neonatal lupus A rare type of lupus, neonatal lupus is a condition found in infants at birth. Children born with neonatal lupus have antibodies that were passed to them from their mother — who either had lupus at the time of the pregnancy or may have the condition later in life. Not every baby born to a mother with lupus will have the disease.
  • Therapies that may be used in combination with the TNFR2 AFFIMER® polypeptides provided 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 is a frequent complication in people who have systemic lupus erythematosus — more commonly known as lupus.
  • Lupus nephritis occurs when lupus autoantibodies affect structures in your kidneys that filter out waste. This causes kidney inflammation and may lead to blood in the urine, protein in the urine, high blood pressure, impaired kidney function or even kidney failure. As many as half of adults with systemic lupus develop lupus nephritis. Systemic lupus causes immune system proteins to damage the kidneys, harming their ability to filter out waste.
  • Rheumatoid arthritis is a type of chronic (ongoing) arthritis that occurs in joints on both sides of the body, such as hands, wrists and knees.
  • the short-term goals of rheumatoid arthritis medications are to reduce joint pain and swelling and/or to improve joint function.
  • the longterm goal is to slow or stop the disease process, particularly joint damage.
  • Rheumatoid arthritis is a type of chronic (ongoing) arthritis (resulting in pain and swelling) that occurs generally in joints symmetrically (on both sides of the body, such as hands, wrists and knees). This involvement of several joints helps distinguish rheumatoid arthritis from other types of arthritis.
  • rheumatoid arthritis may occasionally affect the skin, eyes, lungs, heart, blood, nerves or kidneys.
  • Therapies that may be used in combination with the TNFR2 AFFIMER® agents provided herein to treat rheumatoid arthritis can include, for example:
  • NSAIDs non-steroidal antiinflammatory drugs
  • MOTRIN® ibuprofen
  • ALEVE® naproxen
  • COX-2 inhibitor Another type of drug - the COX-2 inhibitor - also falls into this drug category, providing relief of the signs and symptoms of rheumatoid arthritis.
  • Celecoxib CELEBREX®
  • the COX- 2 inhibitors were designed to have fewer bleeding side effects on the stomach.
  • DMARDs Disease-modifying antirheumatic drugs
  • Older DMARDs include methotrexate (TREXALL®), gold salts, penicillamine (CUPRIMINE®), hydroxychloroquine (PLAQUENIL®), sulfasalazine (AZULFIDINE®), cyclosporine (SANDIMMUNE®), cyclophosphamide (CYTOXAN®) and leflunomide (ARAVA®).
  • methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine are the most commonly used.
  • cystine, cyclophosphamide, gold salts, and penicillamine are not typically used anymore.
  • the older DMARDs work one step further out than the biologies; they work by modifying the body's own immune response to the inflammation. By attacking the cells at a more specific level of the inflammation itself, biologies are considered to be more effective and more specifically targeted.
  • the biologic agents include etanercept (ENBREL®), infliximab (REMICADE®), adalimumab (HUMIRA®), anakinra (KINARET®), abatacept (ORENCIA®), rituximab (RITUXAN®), certolizumab pegol (CIMZIA®), golimumab (SYMPONI®), tocilizumab (ACTEMRA®) and tofacitinib (XELJANJ®).
  • MS Multiple sclerosis
  • CIS Clinically isolated syndrome
  • RRMS Relapsing-remitting MS
  • PPMS Primary progressive MS
  • SPMS Secondary progressive MS
  • Therapies that may be used in combination with the TNFR2 AFFIMER® polypeptides provided herein include, for example:
  • DMTs Disease-modifying therapies
  • Relapse management medications If there is a severe attack, 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.
  • IBD Inflammatory bowel disease
  • Types include:
  • Crohn’s disease causes pain and swelling in the digestive tract. It can affect any part from the mouth to the anus. It most commonly affects the small intestine and 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’s only detectable with a microscope.
  • Therapies that may be used in combination with the TNFR2 AFFIMER® polypeptides provided herein include, for example: aminosalicylates (an anti-inflammatory medicine like sulfasalazine, mesalamine or balsalazide) minimize irritation to the intestines; antibiotics treat infections and abscesses; biologies interrupt signals from the immune system that cause inflammation; corticosteroids, such as prednisone, keep the immune system in check and manage flares; immunomodulators calm an overactive immune system; antidiarrheal medication; nonsteroidal anti-inflammatory drugs (NSAIDs); vitamins and supplements like probiotics.
  • aminosalicylates an anti-inflammatory medicine like sulfasalazine, mesalamine or balsalazide
  • antibiotics treat infections and abscesses
  • biologies interrupt signals from the immune system that cause inflammation
  • corticosteroids such as prednisone, keep the immune system in check and manage flares
  • GvHD graft versus host disease
  • GvHD Acute graft versus host disease
  • cGvHD Chronic graft versus host disease
  • Psoriasis is a chronic skin disorder, which means a skin condition that doesn’t go away. People with psoriasis have thick, pink or red patches of skin covered with white or silvery scales. The thick, scaly patches are called plaques. Psoriasis usually starts in early adulthood, though it can begin later in life. In addition to red, scaly patches, symptoms of psoriasis include: itchiness, cracked, dry skin, scaly scalp, skin pain, nails that are pitted, cracked or crumbly, and joint pain.
  • Therapies that may be used in combination with the TNFR2 AFFIMER® polypeptides provided herein include, for example: Steroid creams, Moisturizers for dry skin, anthralin (a medication to slow skin cell production), Medicated lotions, shampoos and bath solutions to improve scalp psoriasis, Vitamin D3 ointment, Vitamin A or retinoid creams, light therapy, PUVA (a treatment combines a medication called psoralen with exposure to a special form of UV light), methotrexate, retinoids, cyclosporine, and/or immune therapies.
  • Steroid creams for dry skin
  • anthralin a medication to slow skin cell production
  • Medicated lotions Medicated lotions
  • shampoos and bath solutions to improve scalp psoriasis Vitamin D3 ointment
  • Vitamin A or retinoid creams Vitamin A or retinoid creams
  • light therapy PUVA (a treatment combines a medication called psoralen
  • Sjogren's syndrome is a lifelong autoimmune disorder that reduces the amount of moisture produced by glands in the eyes and mouth. It is named for Henrik Sjogren, a Swedish eye doctor who first described the condition. While dry mouth and dry eyes are the primary symptoms, most people who have these problems don't have Sjogren's syndrome. Dry mouth is also called xerostomia.
  • Sjogren's syndrome There are two forms of Sjogren's syndrome: primary Sjogren's syndrome, which develops on its own, not because of any other health condition, and secondary Sjogren’s syndrome, which develops in addition to other autoimmune diseases like rheumatoid arthritis, lupus and psoriatic arthritis.
  • Therapies that may be used in combination with the TNFR2 AFFIMER® polypeptides provided herein include, for example, 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., pain relievers, anti-rheumatics, immunosuppressants, steroids, antifungals, and treatments for vaginal dryness.
  • 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., pain relievers, anti-rheumatics, immunosuppressants, steroids, antifungals, and treatments for vaginal dryness.
  • MG Myasthenia gravis
  • 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, as some people with mild cases may not know they have the disease. MG mostly affects women aged 20 to 40 and men aged 50 to 80. About one in 10 cases of MG occur in teenagers (juvenile MG). The illness can affect people of all ages but is rare in children.
  • Autoimmune MG is the most common form of this neuromuscular disease. Autoimmune MG may be:
  • Ocular The muscles that move the eyes and eyelids weaken. The eyelids may droop, or you may not be able to keep your eyes open. Some people have double vision. Eye weakness is often the first sign of MG. Nearly half of people with ocular MG evolve into the generalized form within two years of the first symptom. Generalized: Muscle weakness affects the eye and other body parts such as the face, neck, arms, legs and throat. It may be difficult to speak or swallow, lift the arms over the head, stand up from a seated position, walk long distances and climb stairs.
  • Therapies that may be used in combination with the TNFR2 AFFIMER® polypeptides provided herein include, for example, Medications, Monoclonal antibodies, IV immunoglobulin (IVIG), Plasma exchange (plasmapheresis), and/or Surgery.
  • IVIG IV immunoglobulin
  • Plasma exchange plasma exchange
  • TNFR2 AFFIMER® 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 cancer treatments.
  • immunotherapies e.g. CAR-T therapies, checkpoint inhibitors, and conventional cancer treatments.
  • 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).
  • a pharmaceutically acceptable vehicle e.g., a carrier or excipient.
  • pharmaceutically acceptable carriers, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.
  • 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 vehicles 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 octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and 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, arg
  • compositions of the present disclosure can be administered in any number of ways for either local or systemic treatment. Administration can 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 by nebulizer, 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).
  • parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).
  • the therapeutic formulation can be in unit dosage form.
  • Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories.
  • solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier.
  • Conventional tableting ingredients include com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can 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 can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner composition covered by an outer component.
  • the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release.
  • enteric layers or coatings such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • microcapsules can also be entrapped in microcapsules.
  • microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, 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.
  • pharmaceutical formulations include an AFFIMER® agent of the present disclosure complexed with liposomes.
  • Methods to produce liposomes are known to those of skill in the art.
  • some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE).
  • PEG-PE PEG- derivatized phosphatidylethanolamine
  • sustained-release preparations comprising AFFIMER® agents described herein can be produced.
  • Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing an AFFIMER® agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules).
  • 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 lactic acid-glycolic acid copolymers such as the LUPRON DEPOT. TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D- (-)-3 -hydroxybutyric acid.
  • polyesters 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 non-degradable ethylene-vin
  • the method or treatment further comprises administering at least one additional therapeutic agent.
  • An additional therapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the AFFIMER® agent.
  • Pharmaceutical compositions comprising an AFFIMER® agent and the additional therapeutic agent(s) are also provided.
  • 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 work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the AFFIMER® agent.
  • the combination of an AFFIMER® agent described herein and at least one additional therapeutic agent results in additive or synergistic results.
  • the combination therapy results in an increase in the therapeutic index of the AFFIMER® agent.
  • the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s).
  • the combination therapy results in a decrease in the toxicity and/or side effects of the AFFIMER® agent.
  • the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s).
  • Combined administration can include co-administration, 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.
  • an AFFIMER® agent described herein and at least one additional therapeutic agent may be administered in any order or concurrently.
  • the AFFIMER® agent will be administered to patients that have previously undergone treatment with a second therapeutic agent.
  • the AFFIMER® agent and a second therapeutic agent will be administered substantially simultaneously or concurrently.
  • a subject may be given an AFFIMER® agent while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy).
  • a second therapeutic agent e.g., chemotherapy
  • an AFFIMER® agent will be administered within 1 year of the treatment with a second therapeutic agent.
  • an AFFIMER® agent will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain other embodiments, an AFFIMER® agent will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, an AFFIMER® agent will be administered within 5, 4, 3, 2, or 1 days 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).
  • an AFFIMER® agent of the present disclosure depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the AFFIMER® agent is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on, all at the discretion of the treating physician.
  • the AFFIMER® agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is affected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual agent. The administering physician can determine optimum dosages, dosing methodologies, and repetition rates.
  • dosage is from 0.01 ug to 100 mg/kg of body weight, from 0.1 ug to 100 mg/kg of body weight, from lug to 100 mg/kg of body weight, from 1 mg to 100 mg/kg of body weight, 1 mg to 80 mg/kg of body weight from 10 mg to 100 mg/kg of body weight, from 10 mg to 75 mg/kg of body weight, or from 10 mg to 50 mg/kg of body weight.
  • the dosage of the AFFIMER® agent is from about 0.1 mg to about 20 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 0.1 mg/kg of body weight. In some embodiments, the dosage of the AFFIMER® agent is about 0.25 mg/kg of 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
  • the dosage of the AFFIMER® agent is about
  • the dosage of the AFFIMER® agent is about
  • the dosage of the AFFIMER® agent is about
  • 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
  • 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
  • the dosage of the AFFIMER® agent is about 15 mg/kg of body weight.
  • the dosage can be given once or more daily, weekly, monthly, or yearly.
  • the AFFIMER® agent is given once every week, once every two weeks, once every three weeks, or once every four weeks.
  • an AFFIMER® agent may be administered at an initial higher "loading" dose, followed by at least one lower dose.
  • the frequency of administration may also change.
  • a dosing regimen may comprise administering an initial dose, followed by additional doses (or "maintenance" doses) once a week, once every two weeks, once every three weeks, or once every month.
  • a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose.
  • a dosing regimen comprises administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week.
  • a dosing regimen comprises administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.
  • any therapeutic agent may lead to side effects and/or toxicities.
  • the side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose.
  • drug therapy must be discontinued, and other agents may be tried.
  • many agents in the same therapeutic class often display similar side effects and/or toxicities, meaning that the patient either has to stop therapy, or if possible, suffer from the unpleasant side effects associated with the therapeutic agent.
  • the dosing schedule may be limited to a specific number of administrations or "cycles".
  • the AFFIMER® agent is administered for 3, 4, 5, 6, 7, 8, or more cycles.
  • 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 can be decided upon and subsequently modified by those skilled in the art.
  • the present disclosure provides methods of administering to a subject the polypeptides or agents described herein comprising using an intermittent dosing strategy for administering 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.
  • a method for treating inflammatory or autoimmune disease in a human subject comprises administering to the subject a therapeutically effective dose of an AFFIMER® agent in combination with a therapeutically effective dose of another therapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy.
  • the intermittent dosing strategy comprises administering an initial dose of an AFFIMER® agent to the subject and administering subsequent doses of the AFFIMER® agent about 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 about 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 about once every 4 weeks. In some embodiments, the AFFIMER® agent is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly.
  • MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n- GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5), wherein Xaa, individually for each occurrence, is an amino acid residue; and n and m are each, independently, an integer from 3 to 20.
  • MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n- GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5), wherein Xaa, individually for each occurrence, is an amino acid residue; and n and m are each, independently, an integer from 3 to 20.
  • (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.
  • (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.
  • (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.
  • the engineered polypeptide of clause 1 comprising an amino acid sequence at least 75%, at least 85%, or at least 95% identity to the amino acid sequence of SEQ ID Nos: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292 or 293, wherein the sequence optionally excludes one or more of the twenty one carboxy terminal residues.
  • the engineered polypeptide of clause 22 comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID Nos: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292 or 293, wherein the sequence optionally excludes one or more of the twenty one carboxy terminal residues. .
  • a fusion protein comprising a dimer, a trimer or a tetramer of the engineered polypeptide of any one of clauses 1 to 26, optionally comprising one or more linkers.
  • a fusion protein comprising one or more of the engineered polypeptide of any one of clauses 1 to 29 linked to a therapeutic or diagnostic moiety.
  • the polynucleotide or set of polynucleotides of clause 44 comprising a nucleic acid sequence having at least 80%, at least 90%, at least 95, or at least 98% identity to a sequence selected from the group consisting of SEQ ID Nos: 297, 302, 304, 307, 308, 315, 316, 320, 324, 332, 345, 356, 358, 364, 366, 373, 380, 381, 389 and 290, wherein the sequence optionally excludes the nucleotides encoding one or more of the twenty one carboxy terminal residues.
  • a delivery vehicle comprising the polynucleotide of either clause 44 or 45.
  • the delivery vehicle of clause 47 wherein the viral delivery vehicle is an adenoviral vector, a retroviral vector, a lentiviral vector, or an adeno-associated viral (AAV) vector.
  • the viral delivery vehicle is an adenoviral vector, a retroviral vector, a lentiviral vector, or an adeno-associated viral (AAV) vector.
  • a plasmid or minicircle comprising the polynucleotide of any one of clauses 44 to 50.
  • a messenger RNA comprising an open reading frame encoding the engineered polypeptide of any one of clauses 1 to 43.
  • a lipid nanoparticle optionally a cationic lipid nanoparticle, comprising the mRNA of either clause 52 or 53.
  • lipid nanoparticle of clause 54 wherein the lipid nanoparticle is a cationic lipid nanoparticle.
  • a pharmaceutical composition comprising the engineered polypeptide, the fusion protein, or the delivery vehicle of any one of clauses 1 to 50, and optionally a pharmaceutically acceptable excipient.
  • a conjugate comprising the engineered polypeptide or the fusion protein of any one of clauses 1 to 43 linked to a pharmacologically active moiety.
  • a method comprising administering to a subject the pharmaceutical composition of clause 56.
  • the engineered polypeptide, the fusion protein, or the delivery vehicle of clause 60 for use in the treatment of systemic lupus erythermatosis (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) (relating to stem cell transplants) also called allograft rejection, transplant/Solid Organ Transplantation (SOT), primary biliary cholangitis (PBC), psoriasis, psoriatic arthritis, collagen-induced arthritis, experimental allergic encephalomyelitis (EAE), oophoritis, allergic rhinitis, asthma, Sjogren's syndrome, atopic ecze
  • IB An engineered polypeptide that binds specifically to TNFR2 with a Kd of 1 x 10’ 6 M or less, wherein the engineered polypeptide is a variant of a Stefin A protein.
  • the engineered polypeptide of clause IB comprising 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 MIP-Xaal-GLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQV (SEQ ID NO: 484) Xaa2-(Xaa)n-Xaa3-TNYYIKVRAGDNKYMHLKVF (SEQ ID NO: 485)-Xaa4- Xaa5-Xaa6-(Xaa)m-Xaa7-D-Xaa8-VLTGYQVDKNKDDELTGF (SEQ ID NO: 486), wherein Xaa, individually for each occurrence, is any number of amino acid residues; and n and m are each, independently, an integer from 3 to 20.
  • MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n- GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 5), wherein Xaa, individually for each occurrence, is an amino acid residue; and n and m are each, independently, an integer from 3 to 20.
  • (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.
  • the engineered polypeptide of clause IB comprising an amino acid sequence at least 75%, at least 85%, or at least 95% identity to the amino acid sequence of SEQ ID Nos: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292 or 293, wherein the sequence optionally excludes one or more of the twenty one carboxy terminal residues.
  • the engineered polypeptide of clause 22B comprising an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID Nos: 200, 205, 207, 210, 211, 218, 219, 223, 227, 235, 248, 259, 261, 267, 269, 276, 283, 284, 292 or 293, wherein the sequence optionally excludes one or more of the twenty one carboxy terminal residues. .
  • a fusion protein comprising a dimer, a trimer or a tetramer of the engineered polypeptide of any one of clauses IB to 26B, optionally comprising one or more linkers.
  • a fusion protein comprising one or more of the engineered polypeptide of any one of clauses IB to 29B linked to a therapeutic or diagnostic moiety.
  • the polynucleotide or set of polynucleotides of clause 44B comprising a nucleic acid sequence having at least 80%, at least 90%, at least 95, or at least 98% identity to a sequence selected from the group consisting of SEQ ID Nos: 297, 302, 304, 307, 308, 315, 316, 320, 324, 332, 345, 356, 358, 364, 366, 373, 380, 381, 389 and 290, wherein the sequence optionally excludes the nucleotides encoding one or more of the twenty one carboxy terminal residues.
  • a delivery vehicle comprising the polynucleotide of either clause 44B or 45B.
  • the delivery vehicle of clause 47B wherein the viral delivery vehicle is an adenoviral vector, a retroviral vector, a lentiviral vector, or an adeno-associated viral (AAV) vector.
  • the viral delivery vehicle is an adenoviral vector, a retroviral vector, a lentiviral vector, or an adeno-associated viral (AAV) vector.
  • a plasmid or minicircle comprising the polynucleotide of any one of clauses 44B to 50B.
  • 52B A messenger RNA (mRNA) comprising an open reading frame encoding the engineered polypeptide of any one of clauses IB to 43B.
  • mRNA messenger RNA
  • a lipid nanoparticle optionally a cationic lipid nanoparticle, comprising the mRNA of either clause 52B or 53B.
  • a pharmaceutical composition comprising the engineered polypeptide, the fusion protein, or the delivery vehicle of any one of clauses IB to 50B, and optionally a pharmaceutically acceptable excipient.
  • a conjugate comprising the engineered polypeptide or the fusion protein of any one of clauses IB to 43B linked to a pharmacologically active moiety.
  • SLE
  • TNFR2 AFFIMER® polypeptides of the present disclosure were identified by selection from a library of AFFIMER® polypeptides 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.
  • selection procedures have been described (see, e.g., Tiede et al. Protein Eng Des Sei. 2014. 27(5): 145-155 and Hughes et al. Sci Signal. 2018. 10(505): eaaj2005). According to such procedures, suspensions of phage expressing AFFIMER® proteins were incubated with human (or mouse in later experiments) TNFR2 as required, the sequences of which are shown in Table 7.
  • TNFR protein sequences used during selection (from Sino Biologicals).
  • TNFR2 antigens having the sequences shown in Table 7 as well as other proteins shown below were tested in different formats for biotinylation and use in TNFR2 AFFIMER® selection and other assays.
  • the TNFR2 was biotinylated and captured on alternating streptavidin and neutravidin beads, alternatively the TNFR2 was passively absorbed to a surface. Unbound phage particles were then washed away and, following washing, bound phage were eluted. Elution of bound phage was accomplished by i exposure to trypsin. Eluted phage particles were then used to infect Escherichia coli (E. coH . and infected bacteria were incubated under conditions suitable for replication of the bacteriophage.
  • the cycle of allowing phage particles to bind to the target antigen, eluting bound phage particles, propagating eluted phage particles in bacteria, and isolating released phage particles from infected bacteria was repeated to enrich the bacteriophage population for phage particles displaying proteins that bind 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.
  • proteins expressed by phages were expressed and screened by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • AFFIMER® polypeptides were overexpressed from phagemid vectors, the bacterial cells were lysed, and lysates were used as substrates in ELISAs.
  • human, murine and cynomolgus TNFR2 was immobilized on a plate, lysates were added, and the amount of TNFR2 -binding AFFIMER® polypeptide in each plate was measured using a detector antibody specific to the Myc tag expressed on the candidate AFFIMER® polypeptides.
  • AFFIMER® polypeptides with the best human TNFR2- binding activity were sequenced to identify DNA sequences of candidate clones for further development. TNFR1 polypeptides were also used to distinguish specificity. The loop 2 and loop 4 amino acid sequences of each of these candidate clones are shown in Table 1.
  • EXAMPLE 2 AFFIMER® production in mammalian cells as soluble protein and screening
  • telomeres were cloned from the phagemid of Example 1 into the mammalian expression vector pD609kanR (ATUM custom vector) for expression in Expi293FTM cells (ThermoFisher). The average concentration obtained from expression was 2.8 mg/mL, with 19 clones selected based on purity and assessed by for degradation.
  • hTNFR2 mammalian purified AFFIMER® proteins were screened in the HEK BlueTM TNFa SEAP reporter assay (Invivogen) according to manufacturer’s to determine if they could trigger the NFkB pathway and the SEAP production via binding to TNFR2 when they are clustered by an anti-his antibody coated on the plate.
  • 5 of the fast tracked clones (01, 09, 12, 21 and 26) showed the ability to trigger the release of SEAP, with clone 12 showing the most consistent agonism.
  • AFFIMER® polypeptides were cloned from phagemid and similarly characterised. 55 clones were taken forward and binding confirmed by Mirrorball analysis. Further characterisation was done by competition ELSA to test their ability to block the interaction between hTNFR2-hFc-his-avi and recombinant TNF-alpha. A series of experiments were carried out to determine the EC80 of hTNFR2-hFc-his-Avi when binding to coated hTNF alpha (593 pM). AFFIMER® proteins were tested at three different concentrations (10, 1 and 0.1 pM) when possible, in presence of hTNFR2 antigen at its determined EC80.
  • hTNFR2-hFc-his-Avi bound was done by anti-hFc-HRP antibody.
  • clones 06, 21, 26, 27, 44, 108 and 115 showed full inhibition of the interaction between TNFa and hTNFR2 at the tested concentrations.
  • the selected 43 clones were tested in a Biacore kinetic analysis as soluble proteins. Briefly Multicycle kinetics were performed using a CM5 chip on which the dimeric antigen hTNFR2-hFc-his-avi was immobilized at 500RU. AFFIMER® proteins were titrated down in HBS-EP+ buffer from IpM in 1:2 dilution series. Association time and Dissociation used were 200 sec and 400 sec respectively at a 20 pl/min flow rate with a regeneration with 10 mM Glycine pH 1.5 for 30 sec at 30 pl/min. Twelve clones showed very low response and no KD value could be estimated for them.
  • TNFR2 AFFIMER® clones specificity for hTNFR2 was assessed by ELISA when the clones binding to TNFR2 was compared to their binding to TNFR1. None of the clones showed binding to TNFR1 as a recombinant antigen.
  • hTNFRl was also expressed in Expi293FTMcells, and no binding was observed to this cellularly expressed hTNFRl, confirming no crossreactivity between the hTNFR2 AFFIMER® polypeptide binders with hTNFRl .
  • the clones were tested as well for binding to the mouse TNFR2 to verify if any of them was cross reactive. There were very little chances for this to happen as the homology between the two proteins is -68% for the extra cellular domain, which was confirmed as no clone demonstrated binding to mTNFR2 expressing Expi293FTMcells.
  • the 43 lead clones were transiently transfected in HEK 293 cells, along with 4 controls, SQT-Gly, 3tO-Gly, Empty vector, and clone 06 which has shown to be negative. Approximately 29h post transfection the cells viability was determined using a cell counter and the expression of the AFFIMER® was determined by flow cytometry using anti-HA antibody. 48 hours post transfection the AFFIMER® expressing HEK293 cells were co-cultured with HEK Blue reporter cells at 4 different cell densities, 40000, 20000, 10000 and 5000 with a fixed number of HEK Blue cells of 50000 cells/well.
  • the positive control MR2-1 was also incubated with HEK Blue cells for 24h at a range of concentrations. After 24h, the release of SEAP in the supernatant was quantified using Quanti-Blue and measurement of Absorbance 640nm. Staining of the AFFIMER®s with monoclonal anti-HA antibody showed expression levels >90% in both experiments.
  • the effect of the TNFR2 AFFIMER® proteins displayed on HEK293 on the HEK-Blue cells was visible, as well as the MR2-1 clone effect, allowing the selection of 16 clones which were consistently ranked within the best 20 clones in both experiments.
  • the 16 best agonists were characterized in soluble format for its binding to human TNFR2 overexpressing Expi293FTMand cynomolgus TNFR2 over-expressing Expi293F.
  • the experiment was repeated 3 times and 2 different batches of transiently transfected cells were used. All clones showed consistently specific binding to hTNFR2-Expi293FTMcells. Clones 26 and 69 showed the best EC50s on both human and cynomolgus TNFR2 expressing cells. 13 out of 16 tested clones show cross reactivity with cynomolgus TNFR2.
  • the 3 non- crossreactive clones were clones 001, 007 and 009.
  • clones DAW02-013, DAW02-044 and DAW02-112 are the clones with the least difference between the binding to human TNFR2 and cynomolgus TNFR2 over-expressing cells when all 3 experiments are compiled ( Figure 5).
  • Clones 001, 009 and 012 were chosen to be formatted as in-line fusion (ILF) homodimers, to investigate if that format would be an advantage to trigger agonism compared to the monomeric form of the clones, as soluble protein and when displayed on cells.
  • ILF in-line fusion
  • Both constructs were cloned into mammalian secretion vector (pD609-KanR) to purify recombinant protein and assess their expression levels, protein quality, binding kinetics to hTNFR2-hFc-his-avi by Biacore and agonistic activity in reporter assay with HEK reporter cells.
  • both constructs were cloned into pDisplay vector for expression on Expi293FTMcell surface and assessment of their expression levels, binding to hTNFR2-hFc-his-avi and agonistic activity in co culture reporter assay with HEK reporter cells. Codon optimisation did not improve yields or purity of the soluble ILF homodimers, which were confirmed by HPLC.
  • 20 of these clones passed the protein production quality criteria of more than 80% purity by SEC- HPLC and were used to set up various assays as well as being characterised. 5 out of 20 clones demonstrated agonist activity in a preliminary agonist assay where monomeric soluble AFFIMER® protein were clustered with an anti-his antibody via their histidine tag and incubated with the HEK reporter cell line.
  • the phage screening campaign was finalised and a total of 364 human TNFR2 -binding AFFIMER® proteins were purified from the phagemid and tested for binding to a TNFR2 over-expressing cell line.
  • 90 human TNFR2 -binding AFFIMER® proteins were re-formatted into the mammalian expression vector. 55 of these clones passed the protein production quality criteria of more than 80% purity by SEC-HPLC and were carried forward for the rest of the assays.
  • the total number of clones was narrowed down from 75 to 43 following a series of experiment that looked at AFFIMER® soluble proteins binding to hTNFR2 overexpressing cells, agonist assay with HEK reporter cells and preliminary data from agonist assay using HEK reporter cells in co-culture with AFFIMER® proteins expressed at the surface of Expi293FTMcells.
  • These 43 best clones were characterised as soluble proteins for their binding to human TNFR2 by Biacore where 16 out of 43 had their KD successfully calculated using a 1 : 1 binding model. None of the clones showed binding to human TNFR1 by ELISA or mouse TNFR2 by cell binding.
  • AFFIMER® proteins displayed at the surface of HEK293, these 43 clones were tested for their ability to trigger TNFR2 agonism in a HEK reporter cell line when in co-culture. A large majority of the tested clones demonstrated a dose dependent effect on the HEK reporter cells (80%). 16 AFFIMER® proteins were selected as best agonists and taken through additional characterisation to assess cynomolgus TNFR2 binding and human TNFR1 binding on cells. The selected 16 hTNFR2 AFFIMER® polypeptides are clones 001, 007, 009, 012, 013, 026, 027, 037, 044, 059, 069, 079, 100, 103, 112 and 115.
  • in-line fusion homodimers as soluble proteins vs monomers showed advantages for binding to the recombinant TNFR2 by Biacore and possible advantage in the anti-his clustering agonism assay with HEK reporter cells.
  • no advantage was demonstrated compared to the monomers when the in-line fusion AFFIMER® proteins were displayed on cells to trigger the reporter cell line agonism pathway via TNFR2.
  • the reason for the in-line fusion homodimers not performing better than the monomers in this last assay could have been that the in-line 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 limitation of the reporter assay had been reached and it was not possible to discriminate between the two formats.
  • Murine TNFR2 antigens having the sequence as shown in Table 7 but in different formats were tested for suitability for use in assays to select for mTNFR2 AFFIMER® polypeptides.
  • mTNFR2-mFc was chosen for biotinylation and use in the AFFIMER® selections due to its better performance in the assays.
  • the identified mTNFR2 AFFIMER® polypeptides were cloned from phagemid to mammalian vectors for expression in Expi293FTMcells.
  • the expressed proteins were then assessed by SDS- PAGE and HPLC for purity and Biacore for affinity, as well as cell based binding of mTNFR2 in mTNFR2-Expi293FTMcells.
  • Specificity for mTNFR2 was tested using ELISA and cell based assays using mTNFRl and mTNFR2 as the targets, confirming the identified clones’ specificity.
  • the AFFIMER® polypeptides were tested for their ability to block mTNFR2-mFC binding to mTNFa in ELISA, with four clones (157, 160, 171 and 172) showing clear competitive ability at the range tested.
  • 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) the CD25 expression increases.
  • the mTNFR2 AFFIMER® polypeptides were first expressed on the cell surface of HEK293s. Then, these cells were co-cultured for 48 hours with BALB/c splenocytes and finally the CD25 expression increase was evaluated on CD4 and CD8 positive T cells. Expression of the 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 prior to the AFFIMER® proteins and cell anchoring protein.
  • CD25 expression in CD4+ and CD8+ T cells in response to HEK293 cells expressing mTNFR2 AFFIMER® proteins, similar to the response observed for HM102 TNFR2 agonist.
  • the response obtained in CD8+ T cells is stronger than in CD4+ T cells.
  • a similar ranking of the clones was obtained between the CD8+ and CD4+ T cell subsets.
  • the 22 mouse clones were also expressed at the surface of HEK293 cells for a co-culture assay with splenocytes.
  • the main read out for the assay was the CD25 expression increase on CD4+ T cells and CD8+ T cells.
  • a large majority of the tested clones demonstrated a dose dependent effect on the CD4+ T cells and CD8+ T cells (over 70%).
  • Clones were ranked looking at the CD25 expression on both CD4+ and CD8+ T cells. Based on the results of this key assay, six mTNFR2 AFFIMER® proteins were identified: 174, 160, 175, 125, 157 and 179.

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Abstract

La présente invention concerne des variants polypeptidiques de Stefin A de liaison au TNFR2 modifiés, des polynucléotides codant pour les variants polypeptidiques de Stefin A de liaison au TNFR2 modifiés, des cellules exprimant les variants polypeptidiques, des préparations pharmaceutiques des variants polypeptidiques, et des utilisations des variants polypeptidiques dans le traitement de divers états humains, y compris l'inflammation, l'auto-immunité et le cancer.
PCT/GB2023/052513 2023-03-31 2023-09-28 Polypeptides de liaison au tnfr2 et procédés d'utilisation Ceased WO2024200988A1 (fr)

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US12365712B2 (en) 2017-06-06 2025-07-22 Relinia, Inc. Single-chain TNF Receptor 2 agonist fusion proteins
US12410223B2 (en) 2023-09-29 2025-09-09 Trex Bio, Inc. TNF-alpha variant fusion molecules

Citations (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4454106A (en) 1982-06-07 1984-06-12 Gansow Otto A Use of metal chelate conjugated monoclonal antibodies
US4472509A (en) 1982-06-07 1984-09-18 Gansow Otto A Metal chelate conjugated monoclonal antibodies
US4831175A (en) 1986-09-05 1989-05-16 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Backbone polysubstituted chelates for forming a metal chelate-protein conjugate
WO1990014363A1 (fr) 1989-05-19 1990-11-29 Amgen Inc. Inhibiteur de metalloproteinase
US5807715A (en) 1984-08-27 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin
WO1999022026A1 (fr) 1997-10-28 1999-05-06 Applied Imaging Corporation Procede et dispositif de production de caryotypes
WO2004041862A2 (fr) 2002-11-08 2004-05-21 Ablynx N.V. Anticorps a domaine unique diriges contre le facteur de necrose tumorale alpha et leurs utilisations
US6946292B2 (en) 2000-10-06 2005-09-20 Kyowa Hakko Kogyo Co., Ltd. Cells producing antibody compositions with increased antibody dependent cytotoxic activity
WO2006122786A2 (fr) 2005-05-18 2006-11-23 Ablynx Nv Nanocorpstm; utilises contre le facteur-alpha de necrose tumorale
US7214775B2 (en) 1999-04-09 2007-05-08 Kyowa Hakko Kogyo Co., Ltd. Method of modulating the activity of functional immune molecules
WO2008020079A1 (fr) 2006-08-18 2008-02-21 Ablynx N.V. Séquences d'acides aminés dirigées contre l'il-6r et polypeptides les contenant utilisés pour le traitement de maladies et de troubles associés au signal médié par il-6
WO2008142164A2 (fr) 2007-05-24 2008-11-27 Ablynx N.V. Séquences d'acides aminés dirigées contre rank-l et polypeptides comprenant ces dernières, destinés au traitement de maladies et affections osseuses
WO2008142571A2 (fr) 2007-05-21 2008-11-27 Humboldt-Universität Zu Berlin Sonde pour détecter une séquence d'acide nucléique particulière
WO2009056282A1 (fr) 2007-10-29 2009-05-07 Autoimmun Diagnostika Gmbh Procédé elispot faisant appel à deux systèmes de filtration
WO2009068627A2 (fr) 2007-11-27 2009-06-04 Ablynx N.V. Séquences d'acides aminés dirigés contre des cytokines hétérodimères et/ou leurs récepteurs et polypeptides les comprenant
WO2010097385A1 (fr) * 2009-02-24 2010-09-02 Glaxo Group Limited Constructions de liaison d'antigène
WO2011140086A2 (fr) 2010-05-03 2011-11-10 Bristol-Myers Squibb Company Molécules de liaison à l'albumine sérique
US8841491B2 (en) 2009-04-13 2014-09-23 Taoka Chemical Co., Ltd. Method for producing fluorene derivative
US8853131B2 (en) 2008-04-18 2014-10-07 Aptuscan Limited Modified Stefin A scaffold proteins
WO2015143199A1 (fr) 2014-03-20 2015-09-24 Bristol-Myers Squibb Company Domaines de fibronectine de type iii se liant à l'albumine sérique
WO2017053617A1 (fr) 2015-09-23 2017-03-30 Bristol-Myers Squibb Company Domaines de la fibronectine de type iii se liant à l'albumine sérique à vitesse de dissociation élevée
US9932575B2 (en) 2013-02-14 2018-04-03 University Of Leeds Scaffold proteins derived from plant cystatins
WO2018227018A1 (fr) * 2017-06-07 2018-12-13 Silverback Therapeutics, Inc. Conjugués d'anticorps constitués de composés immunomodulateurs et leurs utilisations
WO2019008335A1 (fr) * 2017-07-07 2019-01-10 Avacta Life Sciences Limited Protéines d'échafaudage
WO2019236567A2 (fr) 2018-06-04 2019-12-12 Trustees Of Tufts College Conjugué médicament-liant activé par un micro-environnement tumoral et utilisations associées
WO2021249786A1 (fr) * 2020-06-09 2021-12-16 Avacta Life Sciences Limited Polypeptides et procédés de diagnostic du sars-cov2
WO2022023540A1 (fr) 2020-07-30 2022-02-03 Avacta Life Sciences Limited Polypeptides de liaison de sérumalbumine

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0216846B2 (fr) 1985-04-01 1995-04-26 Celltech Limited Lignee cellulaire de myelomes transformee et procede d'expression d'un gene codant un polypeptide eucaryotique employant cette lignee
GB8601597D0 (en) 1986-01-23 1986-02-26 Wilson R H Nucleotide sequences
JP3101690B2 (ja) 1987-03-18 2000-10-23 エス・ビィ・２・インコーポレイテッド 変性抗体の、または変性抗体に関する改良
GB8717430D0 (en) 1987-07-23 1987-08-26 Celltech Ltd Recombinant dna product

Patent Citations (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4454106A (en) 1982-06-07 1984-06-12 Gansow Otto A Use of metal chelate conjugated monoclonal antibodies
US4472509A (en) 1982-06-07 1984-09-18 Gansow Otto A Metal chelate conjugated monoclonal antibodies
US5807715A (en) 1984-08-27 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin
US4831175A (en) 1986-09-05 1989-05-16 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Backbone polysubstituted chelates for forming a metal chelate-protein conjugate
WO1990014363A1 (fr) 1989-05-19 1990-11-29 Amgen Inc. Inhibiteur de metalloproteinase
WO1999022026A1 (fr) 1997-10-28 1999-05-06 Applied Imaging Corporation Procede et dispositif de production de caryotypes
US7214775B2 (en) 1999-04-09 2007-05-08 Kyowa Hakko Kogyo Co., Ltd. Method of modulating the activity of functional immune molecules
US6946292B2 (en) 2000-10-06 2005-09-20 Kyowa Hakko Kogyo Co., Ltd. Cells producing antibody compositions with increased antibody dependent cytotoxic activity
WO2004041862A2 (fr) 2002-11-08 2004-05-21 Ablynx N.V. Anticorps a domaine unique diriges contre le facteur de necrose tumorale alpha et leurs utilisations
WO2006122786A2 (fr) 2005-05-18 2006-11-23 Ablynx Nv Nanocorpstm; utilises contre le facteur-alpha de necrose tumorale
WO2008020079A1 (fr) 2006-08-18 2008-02-21 Ablynx N.V. Séquences d'acides aminés dirigées contre l'il-6r et polypeptides les contenant utilisés pour le traitement de maladies et de troubles associés au signal médié par il-6
WO2008142571A2 (fr) 2007-05-21 2008-11-27 Humboldt-Universität Zu Berlin Sonde pour détecter une séquence d'acide nucléique particulière
WO2008142164A2 (fr) 2007-05-24 2008-11-27 Ablynx N.V. Séquences d'acides aminés dirigées contre rank-l et polypeptides comprenant ces dernières, destinés au traitement de maladies et affections osseuses
WO2009056282A1 (fr) 2007-10-29 2009-05-07 Autoimmun Diagnostika Gmbh Procédé elispot faisant appel à deux systèmes de filtration
WO2009068627A2 (fr) 2007-11-27 2009-06-04 Ablynx N.V. Séquences d'acides aminés dirigés contre des cytokines hétérodimères et/ou leurs récepteurs et polypeptides les comprenant
US8853131B2 (en) 2008-04-18 2014-10-07 Aptuscan Limited Modified Stefin A scaffold proteins
US9447170B2 (en) 2008-04-18 2016-09-20 Avacta Life Sciences Limited Modified Stefin A scaffold proteins
WO2010097385A1 (fr) * 2009-02-24 2010-09-02 Glaxo Group Limited Constructions de liaison d'antigène
US8841491B2 (en) 2009-04-13 2014-09-23 Taoka Chemical Co., Ltd. Method for producing fluorene derivative
WO2011140086A2 (fr) 2010-05-03 2011-11-10 Bristol-Myers Squibb Company Molécules de liaison à l'albumine sérique
US9932575B2 (en) 2013-02-14 2018-04-03 University Of Leeds Scaffold proteins derived from plant cystatins
WO2015143199A1 (fr) 2014-03-20 2015-09-24 Bristol-Myers Squibb Company Domaines de fibronectine de type iii se liant à l'albumine sérique
WO2017053617A1 (fr) 2015-09-23 2017-03-30 Bristol-Myers Squibb Company Domaines de la fibronectine de type iii se liant à l'albumine sérique à vitesse de dissociation élevée
WO2018227018A1 (fr) * 2017-06-07 2018-12-13 Silverback Therapeutics, Inc. Conjugués d'anticorps constitués de composés immunomodulateurs et leurs utilisations
WO2019008335A1 (fr) * 2017-07-07 2019-01-10 Avacta Life Sciences Limited Protéines d'échafaudage
WO2019236567A2 (fr) 2018-06-04 2019-12-12 Trustees Of Tufts College Conjugué médicament-liant activé par un micro-environnement tumoral et utilisations associées
WO2021249786A1 (fr) * 2020-06-09 2021-12-16 Avacta Life Sciences Limited Polypeptides et procédés de diagnostic du sars-cov2
WO2022023540A1 (fr) 2020-07-30 2022-02-03 Avacta Life Sciences Limited Polypeptides de liaison de sérumalbumine

Non-Patent Citations (74)

* Cited by examiner, † Cited by third party
Title
"CURRENT PROTOCOLS IN HUMAN GENETICS", 1994, JOHN WILEY & SONS
"CURRENT PROTOCOLS IN MOLECULAR BIOLOGY", 1993, JOHN WILEY & SONS
"Hoboken", 2013, WILEY, article "ELP-fusion technology for biopharmaceuticals. In: Schmidt S, editor. Fusion protein technologies for biopharmaceuticals: application and challenges."
"Remington: The Science and Practice of Pharmacy", 2012, PHARMACEUTICAL PRESS
AL LAZIKANI ET AL., J. MOL. BIOL., vol. 273, 1997, pages 927 - 948
ALEXANDER ET AL., MOL. CELL. BIOL., vol. 7, 1987, pages 1436 - 44
ANDERSEN ET AL.: "Extending half-life by indirect targeting of the neonatal Fc receptor (FcRn) using a minimal albumin binding domain", J BIOL CHEM., vol. 286, 2011, pages 5234 - 41, XP055019819, DOI: 10.1074/jbc.M110.164848
BEBBINGTONHENTSCHEL: "DNA CLONING", vol. 3, 1987, ACADEMIC PRESS, article "The Use of Vectors Based On Gene Amplification For The Expression Of Cloned Genes In Mammaian Cells"
BECK ET AL.: "Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies", MABS., vol. 3, 2011, pages 1 - 2
BERNOISTCHAMBON, NATURE, vol. 290, 1981, pages 304 - 310
BIOCHEMISTRY, vol. 11, 1972, pages 1726 - 1732
BITTER ET AL.: "Expression and Secretion Vectors For Yeast", METHODS IN ENZYMOL., vol. 153, 1987, pages 516 - 544
BRINSTER ET AL., NATURE, vol. 296, 1982, pages 39 - 42
CALICETI ET AL.: "Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates", ADV DRUG DELIVERY REV., vol. 55, 2003, pages 1261 - 77, XP003001867, DOI: 10.1016/S0169-409X(03)00108-X
COLBERE-GARAPIN ET AL.: "A New Dominant Hybrid Selective Marker For Higher Eukaryotic Cells", J. MOL. BIOL., vol. 150, 1981, pages 1 - 14, XP024018978, DOI: 10.1016/0022-2836(81)90321-1
CROUSE ET AL.: "Expression and Amplification of Engineered Mouse Dihydrofolate Reductase Minigenes", MOL. CELL. BIOL., vol. 3, 1983, pages 257 - 266
CZAJKOWSKY ET AL.: "Fc-fusion proteins: new developments and future perspectives.", EMBO MOL MED., vol. 4, 2012, pages 1015 - 28, XP055542855, DOI: 10.1002/emmm.201201379
DAVID ET AL., BIOCHEMISTRY, vol. 13, 1974, pages 1014
DEBOER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 80, 1983, pages 21 - 5
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", HUM REPROD., vol. 17, pages 1987 - 93, XP055409330, DOI: 10.1093/humrep/17.8.1987
FARES ET AL.: "Design of a longacting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit", PROC NATL ACAD SCI USA., vol. 89, 1992, pages 4304 - 8
FLOSS ET AL.: "Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application.", TRENDS BIOTECHNOL., vol. 28, 2012, pages 37 - 45
GEORGE ET AL.: "An analysis of protein domain linkers: their classification and role in protein folding", PROTEIN ENG., vol. 15, no. 11, 2002, pages 871 - 9, XP002374925, DOI: 10.1093/protein/15.11.871
GREGORIADIS ET AL.: "Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids", INT J PHARM., vol. 300, 2005, pages 125 - 30, XP005013268, DOI: 10.1016/j.ijpharm.2005.06.007
HAMMER ET AL., SCIENCE, vol. 235, 1987, pages 53 - 8
HUANG ET AL.: "Receptor-Fc fusion therapeutics, traps, and Mimetibody technology", CURR OPIN BIOTECHNOL, vol. 20, 2009, pages 692 - 9, XP026778880, DOI: 10.1016/j.copbio.2009.10.010
HUGHES ET AL., SCI SIGNAL., vol. 10, no. 505, 2018, pages eaaj2005
HUNTER ET AL., NATURE, vol. 144, 1962, pages 945
INOUYE ET AL.: "Up-Promoter Mutations In The Lpp Gene Of Escherichia coli", NUCLEIC ACIDS RES., vol. 13, 1985, pages 3101 - 3110, XP001316824
JEVSEVAR ET AL.: "PEGylation of therapeutic proteins.", BIOTECHNOL J, vol. 5, 2010, pages 113 - 28, XP072399620, DOI: 10.1002/biot.200900218
JONASSEN ET AL., PHARM RES., vol. 29, no. 8, 2012, pages 2104 - 14
K. D. KOPPLE: "Peptides and Amino Acids", 1966, W. A. BENJAMIN INC.
KANG ET AL.: "Emerging PEGylated drugs", EXPERT OPIN EMERG DRUGS., vol. 14, 2009, pages 363 - 80, XP008164958, DOI: 10.1517/14728210902907847
KOHLER: "Immunoglobulin Chain Loss In Hybridoma Lines", PROC. NATL. ACAD. SCI., 1980
KOLLIAS ET AL., CELL, vol. 46, 1986, pages 485 - 94
KONTERMANN: "Strategies to extend plasma half-lives of recombinant antibodies", BIODRUGS., vol. 23, 2009, pages 93 - 109, XP008124089, DOI: 10.2165/00063030-200923020-00003
KOSA ET AL., J PHARM SCI., vol. 96, no. 11, 2007, pages 3117 - 24
KRUMLAUF ET AL., MOL. CELL. BIOL., vol. 5, 1985, pages 1639 - 48
LOGAN ET AL.: "Adenovirus Tripartite Leader Sequence Enhances Translation Of mRNAs Late After Infection", PROC. NATL. ACAD. SCI., 1984
MACDONALD, HEPATOLOGY, vol. 7, 1987, pages 425 - 515
MASON ET AL., SCIENCE, vol. 234, 1986, pages 1372 - 8
MERO ET AL.: "Conjugation of hyaluronan to proteins", CARB POLYMERS., vol. 92, 2013, pages 2163 - 70, XP028972870, DOI: 10.1016/j.carbpol.2012.11.090
MOGRAM ET AL., NATURE, vol. 314, 1985, pages 283 - 340
MORGAN ET AL.: "Human gene therapy", ANN. REV. BIOCHEM., vol. 62, 1993, pages 191 - 217, XP002031972, DOI: 10.1146/annurev.bi.62.070193.001203
MULLIGAN ET AL.: "Selection For Animal Cells That Express The Escherichia coli Gene Coding For Xanthine-Guanine Phosphoribosyltransferase", PROC. NATL. ACAD. SCI., 1981
MULLIGAN: "The Basic Science of Gene Therapy", SCIENCE, vol. 260, 1993, pages 926 - 932, XP002925334, DOI: 10.1126/science.8493530
NAT BIOTECHNOL., vol. 27, 2009, pages 1186 - 90
NYGREN, J., HISTOCHEM. AND CYTOCHEM., vol. 30, 1982, pages 407
O'CONNOR-SEMMES ET AL.: "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., vol. 96, 2014, pages 704 - 12
O'HARE ET AL.: "Transformation Of Mouse Fibroblasts To Methotrexate Resistance By A Recombinant Plasmid Expressing A Prokaryotic Dihydrofolate Reductase", PROC. NATL. ACAD. SCI., 1981
ORNITZ ET AL., COLD SPRING HARBOR SYMP. QUANT. BIOL., vol. 50, 1986, pages 399 - 409
PACE ET AL.: "Measuring the conformational stability of a protein", PROTEIN STRUCTURE: A PRACTICAL APPROACH, vol. 2, 1997, pages 299 - 321
PAIN ET AL., J. IMMUNOL. METH., vol. 40, 1981, pages 219
PINKERT ET AL., GENES AND DEVEL., vol. 1, 1987, pages 161 - 71
PROUDFOOT: "Expression and Amplification Of Engineered Mouse Dihydrofolate Reductase Minigenes", NATURE, vol. 322, 1986, pages 562 - 565
READHEAD ET AL., CELL, vol. 48, 1987, pages 703 - 12
RUTHER ET AL.: "Easy Identification Of cDNA Clones", EMBO J., vol. 2, 1983, pages 1791 - 1794, XP055413083, DOI: 10.1002/j.1460-2075.1983.tb01659.x
SAMBROOK ET AL.: "GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL", 1990, COLD SPRING HARBOR LABORATORY
SANTERRE ET AL.: "Expression Of Prokaryotic Genes For Hygromycin B And G418 Resistance As Dominant-Selection Markers In Mouse L Cells", GENE, vol. 30, 1984, pages 147 - 156, XP025696524, DOI: 10.1016/0378-1119(84)90115-X
SCHLAPSCHY ET AL.: "Fusion of a recombinant antibody fragment with a homoamino-acid polymer: effects on biophysical properties and prolonged plasma half-life.", PROTEIN ENG DES SEL., vol. 20, 2007, pages 273 - 84, XP002498446, DOI: 10.1093/protein/gzm020
SCHLAPSCHY: "PASylation: a biological alternative to PEGylation for extending the plasma halflife of pharmaceutically active proteins.", PROTEIN ENG DES SEL., vol. 26, 2013, pages 489 - 501, XP055195431, DOI: 10.1093/protein/gzt023
SHINKAWA ET AL., J. BIOL. CHEM., vol. 278, 2003, pages 3466 - 3473
SOCKOLOSKY ET AL.: "Fusion of a short peptide that binds immunoglobulin G to a recombinant protein substantially increases its plasma half-life in mice", PLOS ONE., vol. 9, 2014, pages e102566
SWIFT ET AL., CELL, vol. 38, 1984, pages 647 - 58
SZYBALSKA ET AL.: "Genetics Of Human Cess Line. IV. DNA-Mediated Heritable Transformation of a Biochemical Trait", PROC. NATL. ACAD. SCI., 1962
TACHIBANA ET AL.: "Altered Reactivity Of Immunoglobutin Produced By Human-Human Hybridoma Cells Transfected By pSV.2-Neo Gene", CYTOTECHNOLOGY, vol. 6, no. 3, 1991, pages 219 - 226
TIEDE ET AL., PROTEIN ENG DES SEL., vol. 27, no. 5, 2014, pages 145 - 155
TOLSTOSHEV: "Gene Therapy, Concepts, Current Trials And Future Directions", ANN. REV. PHARMACOL. TOXICOL., vol. 32, 1993, pages 573 - 596
VAN HEEKE ET AL.: "Expression Of Human Asparagine Synthetase In Escherichia coli", J. BIOL. CHEM., vol. 24, 1989, pages 5503 - 5509, XP002438811
VILLA-KAMAROFF ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 75, 1978, pages 3727 - 3731
WIGLE ET AL.: "Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells", CELL, vol. 11, 1977, pages 223 - 232
WIGLER ET AL.: "Transformation Of Mammalian Cells With An Amplfiable Dominant-Acting Gene", PROC. NATL. ACAD. SCI., 1980
WILLIAM BA ET AL., J. CLIN. MED., vol. 8, no. 8, 2019, pages 1261
YAMAMOTO ET AL.: "Isolation Of Transforming DNA: Cloning The Hamster Aprt Gene", CELL, vol. 22, 1980, pages 817 - 823

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US12410223B2 (en) 2023-09-29 2025-09-09 Trex Bio, Inc. TNF-alpha variant fusion molecules

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