Neonatal Fc receptor binding affimers

Abstract

In some embodiments, the present application provides AFFIMER® polypeptides that bind to the neonatal Fc receptor (FcRn) and extend the half-life of a polypeptide, as well as compositions containing the polypeptides, methods of using the polypeptides, and methods of producing the polypeptides.

Description

[Technical field]

[0001] The present invention relates to polypeptides comprising an FcRn-binding AFFIMER® sequence that bind to human FcRn. [Background technology]

[0002] The neonatal Fc receptor (FcRn) binds with high affinity to IgG and albumin through non-overlapping sites at slightly acidic pH (e.g., 5.0-6.5) but does not bind IgG or albumin at neutral pH. FcRn expression has been detected in nearly all tissues, including epithelial, endothelial, and cells of hematopoietic origin. FcRn facilitates monitoring of IgG and serum albumin turnover because its expression is upregulated in response to the proinflammatory cytokine, TNF-α, and downregulated in response to IFN-γFcRn has been used therapeutically to shuttle biological traffic across mucosal surfaces to improve drug absorption or distribution. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent Publication No. 200510250854 [Patent Document 2] U.S. Patent No. 6,080,728 [Non-patent literature]

[0004] [Non-Patent Document 1] Kuo TT et al. mAbs 2011;3(5):422-430 [Non-Patent Document 2] Roopenian DC et al. Nature Reviews 2007;7(9):715-725 [Non-Patent Document 3] Kuo TT et al. Journal of Clinical Immunology 2010;30(6):777-89

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[0005] The neonatal Fc receptor (FcRn) binds with high affinity to IgG and albumin via non-overlapping sites at slightly acidic pH (e.g., 5.0-6.5) but does not bind to IgG or albumin at neutral pH. [Means for solving the problem]

[0006] An object of the present invention is to provide a polypeptide comprising an FcRn-binding AFFIMER® sequence that binds to human FcRn.

[0007] It is another object of the present invention to provide a pharmaceutical formulation.

[0008] It is yet another object of the present invention to provide a method comprising administering to a subject having an autoimmune and / or inflammatory disease.

[0009] It is yet another object of the present invention to provide a method for increasing the serum half-life of a therapeutic molecule.

[0010] It is yet another object of the present invention to provide uses of polynucleotides that target FcRn.

[0011] It is yet another object of the present invention to provide the use of polynucleotides to increase the serum half-life of therapeutic molecules. Effect of the Invention

[0012] The present disclosure is based on the generation of AFFIMER® polypeptides that bind to the human neonatal Fc receptor (FcRn), thereby extending the serum half-life of any other therapeutic molecule (e.g., a therapeutic AFFIMER® polypeptide, protein, nucleic acid, or drug) to which said polypeptide is conjugated in a controlled manner. [Brief description of the drawings]

[0013] [Figure 1] Example of LGC01 clone binding at pH 6 in direct huFcRn ELISA. [Diagram 2] FIG. 13 is an example of differential binding of LGC01 clones at pH 6 and 7.4 in a direct huFcRn ELISA. [Diagram 3] 3A-3C are analytical SEC-HPLC traces of purified FcRn AFFIMER® monomer and AVA04-FcRn binding AFFIMER® fusion. [Figure 4] 4A-4B are SDS-PAGE analyses of purified FcRn AFFIMER® monomer and AVA04-FcRn binding AFFIMER® fusion. [Diagram 5] 5A-5B are FcRn binding ELISAs showing the binding activity of purified FcRn AFFIMER® monomer and AVA04-FcRn binding AFFIMER® fusions at pH 6 and 7. [Figure 6] 13 is an FcRn competition ELISA showing activity of FcRn AFFIMER® monomer and AVA04-FcRn binding AFFIMER® fusion. [Figure 7] 13 is a flow cytometry histogram of AFFIMER® clones with high cell binding affinity at pH 6.0 and varying binding affinity at pH 7.4. [Figure 8] This is confirmation of cell binding of Affimer using hFcRn-overexpressing CHO monoclonal cell lines (pH 6.0 and pH 7.4). [Figure 9] Demonstration of FcRn-mediated recirculation of FcRn-binding AFFIMER® polypeptides as measured using a human endothelial cell-based recirculation assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] In some aspects, the present application provides a half-life extension platform based on AFFIMER® polypeptides that bind (e.g., competitively or non-competitively) to neonatal Fc receptors (FcRn, e.g., human FcRn). A series of human FcRn-binding AFFIMER® polypeptides (referred to as anti-human FcRn AFFIMER® polypeptides) with a range of binding affinities have been developed. These polypeptides have been shown in in vivo pharmacokinetic (PK) studies to extend in a controlled manner the serum half-life of other polypeptides to which any other AFFIMER® polypeptide may be conjugated (e.g., as a single gene fusion), e.g., produced in bacterial cells (e.g., Escherichia coli). The FcRn-binding AFFIMER® polypeptides provided herein may also be used to extend the half-life of other polypeptides, such as therapeutic proteins.

[0015] In some aspects, the present invention provides a method for the preparation of human FcRn at 1×10 -6Kd less than or equal to M and (optionally) K for binding at pH 7.4 d K for human FcRn binding is at least half log greater than d The present invention relates to a polypeptide comprising an FcRn-binding AFFIMER® sequence that binds FcRn.

[0016] In some embodiments, the FcRn-binding AFFIMER® sequence is at a concentration of 1×10 -7 K below M d , 1 x 10 at pH 6.0 -8 K below M d , or 1 x 10 at pH 6.0 -9 K below M d and binds to FcRn.

[0017] In some embodiments, the polypeptide has a K for binding to human FcRn at pH 6 and a K for binding to human FcRn at pH 7.4. d at least 1 log lower than that of the K for binding to human FcRn at pH 7.4 d at least 1.5 log lower than that of the K for binding to human FcRn at pH 7.4 d or a K for binding to human FcRn at pH 7.4 d At least 2.5 log less than K d It binds to human FcRn.

[0018] In some embodiments, the FcRn-binding AFFIMER® sequence has a K d K for binding to FcRn at pH 6 that is at least 1 log greater than d K for binding to FcRn at pH 6 is at least 1.5 logs greater than d or a K for binding to FcRn at pH 6 d At least 2.5 logs greater than K d and binds to FcRn.

[0019] In some embodiments, the FcRn-binding AFFIMER® polypeptide sequence binds to human FcRn, and the protein / polypeptide has a circulating half-life in a human patient of at least 7 days, preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or even 21 days.

[0020] In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 10 hours, greater than 24 hours, greater than 48 hours, greater than 72 hours, greater than 96 hours, greater than 120 hours, greater than 144 hours, greater than 168 hours, greater than 192 hours, greater than 216 hours, greater than 240 hours, greater than 264 hours, greater than 288 hours, greater than 312 hours, greater than 336 hours, or greater than 360 hours.

[0021] In some embodiments, the polypeptide has a serum half-life in a human patient that is greater than 50%, greater than 60%, greater than 70%, or greater than 80% of the serum half-life of IgG.

[0022] In some embodiments, the polypeptide has a serum half-life in a human patient that is greater than 50%, greater than 60%, greater than 70%, or greater than 80% of the serum half-life of serum albumin.

[0023] In some embodiments, the polypeptide does not inhibit the binding of human serum albumin to human FcRn.

[0024] In some embodiments, the polypeptide does not inhibit binding of IgG to human FcRn.

[0025] In some embodiments, binding of a polypeptide to human FcRn promotes transport of the polypeptide from apical to basal across an epithelial cell layer.

[0026] Yet another embodiment relates to a protein comprising an FcRn-binding AFFIMER® polypeptide sequence that binds to human FcRn and promotes transport of the protein across an epithelial tissue barrier.

[0027] In some embodiments, the AFFIMER® polypeptide sequence has an amino acid sequence represented by formula (I): FR1-(Xaa)n-FR2-(Xaa)m-FR3 (I) wherein FR1 is an amino acid sequence having at least 70% identity to MIPGGLSEAK PATPEIQEIV DKVKPQLEEK TNETYGKLEA VQYKTQVLA (SEQ ID NO: 1); FR2 is an amino acid sequence having at least 70% identity to GTNYYIKVRA GDNKYMHLKV FKSL (SEQ ID NO: 2); FR3 is an amino acid sequence having at least 70% identity to EDLVLTGYQV DKNKDDELTG F (SEQ ID NO: 3); Xaa is, in each instance, an amino acid; n is an integer from 3 to 20, and m is an integer from 3 to 20.

[0028] For example, FR1 may be at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% identical to SEQ ID NO: 1; FR2 has at least 80%, at least 84%, at least 88%, at least 92%, or at least 96% identical to SEQ ID NO: 2; and / or FR3 has at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 3. In some embodiments, FR1 comprises the amino acid sequence of SEQ ID NO: 1, FR2 comprises the amino acid sequence of SEQ ID NO: 2, and FR3 comprises the amino acid sequence of SEQ ID NO: 3.

[0029] In some embodiments, the AFFIMER® polypeptide sequence has an amino acid sequence, where (Xaa)n is an amino acid sequence represented by the formula: -Xaa-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa-Xaa- (Sequence number 4) wherein Xaa, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, and Xaa7 are, individually in each occurrence, amino acid residues, provided that (i) at least two of Xaa2, Xaa3, Xaa4, or Xaa5 are selected from His, Lys, or Arg, or (ii) at least two of Xaa4, Xaa5, Xaa6, or Xaa7 are selected from His, Lys, or Arg. In some preferred embodiments, at least three, and preferably four, of Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, or Xaa7 are selected from His, Lys, or Arg.

[0030] In some embodiments, the AFFIMER® polypeptide sequence has an amino acid sequence, where (Xaa)n is set forth in SEQ ID NO:6. ~ 299 and 1182. In some embodiments, the loop 2 sequence is an amino acid sequence that is at least 75% identical, and more preferably at least 80%, 85%, 90%, or 95% identical to a loop 2 sequence selected from SEQ ID NO:6. ~ 299 and 1182.

[0031] In some embodiments, the AFFIMER® polypeptide sequence has an amino acid sequence, where (Xaa)m is an amino acid sequence represented by the formula -Xaa-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa- (SEQ ID NO:5), where Xaa, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, and Xaa14 are each independently In each case, the amino acid residues are, but not limited to, at least three of Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, and Xaa14 are selected from His, Lys, or Arg, and at least two of Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13, and Xaa14 are further selected from His, Lys, Arg, Phe, Tyr, or Trp.

[0032] In some embodiments, the AFFIMER® polypeptide sequence has an amino acid sequence where (Xaa)m is at least 75% identical, and more preferably at least 80%, 85%, 90%, or 95% identical to a loop 4 sequence selected from SEQ ID NOs: 300-593 and 1183. In some embodiments, the loop 4 sequence is selected from SEQ ID NOs: 300-593 and 1183.

[0033] Yet another aspect relates to a protein comprising an FcRn-binding AFFIMER® polypeptide sequence that binds to human FcRn and has an amino acid sequence at least 75% identical, more preferably 90%, 85%, 90% or even 95% identical, to an AFFIMER® polypeptide sequence selected from SEQ ID NOs: 594-887 and 1184. In some embodiments, the FcRn-binding AFFIMER® polypeptide sequence binds to human FcRn and has an amino acid sequence that is identical to an AFFIMER® polypeptide sequence selected from SEQ ID NOs: 594-887 and 1184.

[0034] Yet another embodiment relates to a protein that comprises an FcRn-binding AFFIMER® polypeptide sequence having an amino acid sequence that binds to human FcRn and can be encoded by a nucleic acid having a coding sequence that hybridizes to any one of SEQ ID NOs: 888-1181 under stringent conditions of 6× sodium chloride / sodium citrate (SSC) at 45° C., followed by washing with 0.2× SSC at 65° C.

[0035] Yet another embodiment relates to a protein comprising (i) an FcRn-binding AFFIMER® polypeptide sequence that binds to human FcRn, and (ii) a heterologous polypeptide covalently associated to the FcRn-binding AFFIMER® polypeptide sequence (optionally as a fusion protein or chemically conjugated), which confers therapeutic activity in a human patient.

[0036] In some embodiments, the polypeptide further comprises a heterologous polypeptide covalently linked via an amide bond to form a contiguous fusion protein.

[0037] In some embodiments, the heterologous polypeptide comprises a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is selected from the group consisting of a polypeptide hormone, a cytokine polypeptide, a chemokine polypeptide, a growth factor, a hemostatically active polypeptide, an enzyme, and a toxin. In some embodiments, the therapeutic polypeptide is selected from the group consisting of a receptor trap and a receptor ligand. In some embodiments, the therapeutic polypeptide sequence is selected from the group consisting of an angiogenic agent and an anti-angiogenic agent. In some embodiments, the therapeutic polypeptide is a neurotransmitter, optionally the neurotransmitter is neuropeptide Y. In some embodiments, the therapeutic polypeptide is an erythropoiesis stimulator, optionally the erythropoiesis stimulator is erythropoietin or an erythropoietin mimetic. In some embodiments, the therapeutic polypeptide is an incretin, optionally selected from the group consisting of glucagon, gastric inhibitory peptide (GIP), glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), peptide YY (PYY), and oxyntomodulin (OXM). In some embodiments, the therapeutic polypeptide is an anti-cancer immune enhancer, such as a checkpoint inhibitor, a costimulatory receptor agonist, or an innate immune inducer. In some embodiments, the therapeutic polypeptide is an anti-inflammatory immune suppressant, such as a checkpoint agonist, a costimulatory receptor antagonist, or an innate immune suppressant.

[0038] In some embodiments, the polypeptide extends the serum half-life of the heterologous polypeptide in vivo, e.g., the heterologous polypeptide may have an extended half-life that is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 30-fold greater than the half-life of the heterologous polypeptide not linked to an AFFIMER® polypeptide. In some embodiments, the polypeptide comprises a loop 2 amino acid sequence of any one of SEQ ID NOs: 6-299 and 1182. In some embodiments, the polypeptide comprises a loop 4 amino acid sequence of any one of SEQ ID NOs: 300-593 and 1183.

[0039] In some embodiments, the polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to any one of SEQ ID NOs: 594-887 or 1184.

[0040] In some embodiments, the polypeptide is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to any one of SEQ ID NOs: 888-1181.

[0041] Another aspect of the present disclosure provides a pharmaceutical formulation comprising any one of the AFFIMER® polypeptides described herein and a pharma- ceutically acceptable excipient (e.g., a carrier, a buffer, and / or a salt, etc.), for example, for therapeutic use in a human patient. In some embodiments, the pharmaceutical composition is formulated for pulmonary delivery. For example, the pharmaceutical composition may be formulated as an intranasal formulation. In other embodiments, the pharmaceutical composition is formulated for topical (e.g., transdermal) delivery.

[0042] Another aspect of the present disclosure provides a polynucleotide comprising a sequence that encodes an AFFIMER® polypeptide described herein.

[0043] In some embodiments, the sequence encoding the polypeptide is operably linked to a transcriptional regulatory sequence, which may be, for example, a promoter or enhancer. Other transcriptional regulatory sequences are contemplated herein.

[0044] In some embodiments, the polynucleotide further comprises an origin of replication, a minichromosome maintenance element (MME), and / or a nuclear localization element. In some embodiments, the polynucleotide further comprises a polyadenylation signal sequence operably linked to and transcribed together with the polypeptide coding sequence. In some embodiments, the polynucleotide further comprises at least one intron sequence. In some embodiments, the polynucleotide further comprises at least one ribosome binding site transcribed together with the polypeptide coding sequence.

[0045] In some embodiments, the polynucleotide is a deoxyribonucleic acid (DNA). In some embodiments, the polynucleotide is a ribonucleic acid (RNA).

[0046] Further aspects of the present disclosure provide viral vectors, plasmids and / or minicircles comprising the AFFIMER® polypeptides described herein.

[0047] Another aspect of the present disclosure provides a cell comprising the polypeptide, polynucleotide, viral vector, plasmid and / or minicircle described herein.

[0048] A further aspect of the present disclosure provides a method comprising administering to a subject having an autoimmune and / or inflammatory disease a therapeutically effective amount of an AFFIMER® polypeptide described herein.

[0049] Yet another aspect of the present disclosure provides a method comprising administering to a subject having cancer a therapeutically effective amount of an AFFIMER® polypeptide described herein.

[0050] Yet another aspect of the present disclosure provides a method for increasing the serum half-life of a therapeutic molecule, the method comprising conjugating the therapeutic molecule to an AFFIMER® polypeptide described herein.

[0051] A further aspect of the present disclosure provides a method for producing a polypeptide as described herein, said method comprising expressing a nucleic acid encoding said polypeptide in a host cell and optionally isolating said polypeptide from said host cell.

[0052] It goes without saying that any one of the AFFIMER® polypeptides described herein may include or exclude a signal sequence (e.g., from 15 to 30 amino acids present at the N-terminus of the polypeptide) or a tag sequence (e.g., a C-terminal polyhistidine (e.g., HHHHHH (SEQ ID NO: 1185))).

[0053] Another aspect of the present disclosure provides the use of a polynucleotide to target FcRn.

[0054] Another aspect of the present disclosure provides the use of a polynucleotide to increase the serum half-life of a therapeutic molecule.

[0055] <Modes of the Invention> The present disclosure is based on the generation of AFFIMER® polypeptides that bind to the human neonatal Fc receptor (FcRn) and extend the serum half-life of any other therapeutic molecule (e.g., a therapeutic AFFIMER® polypeptide, protein, nucleic acid, or drug) to which said polypeptide is conjugated in a controlled manner.

[0056] The human FcRn-binding AFFIMER® polypeptides of the present disclosure are based on a natural protein (cystatin) engineered to stably display two loops that create a binding surface, and offer a number of advantages compared to antibodies, antibody fragments, and other non-antibody molecule binding proteins. One is the small size of the AFFIMER® polypeptide itself, which is approximately 14 kDa in its monomeric form, or 1 / 10 the size of an antibody. This small size offers the potential for increased tissue penetration, especially in target tissues that are poorly vascularized and / or fibrotic (such as tumors). AFFIMER® polypeptides may be produced in prokaryotic and eukaryotic systems, since they have a simple protein structure (compared to multi-domain antibodies) and do not require disulfide bonds or other post-translational modifications for function. In addition to libraries of AFFIMER® polypeptides (e.g., phage display techniques as described in the accompanying Examples), site-directed mutagenesis can be used to generate AFFIMER® polypeptides with tunable binding kinetics that are ideal for therapeutic applications. For example, AFFIMER® polypeptides have high affinity for human FcRn, e.g., single-digit nanomolar or lower K for monomeric AFFIMER® polypeptides. d , and picomolar K in multivalent format d and binding activity. AFFIMER® polypeptides can be linked to human FcRn with tight binding kinetics, e.g., 10 -4 ~10 -5 (s-1) Slow range K off It can be produced rapidly, which is advantageous for target tissue localization.

[0057] The human FcRn-binding AFFIMER® polypeptides of the present disclosure include AFFIMER® polypeptides with exquisite selectivity.

[0058] Furthermore, the human FcRn-binding AFFIMER® polypeptides can be easily formatted, allowing Fc fusions, whole antibody fusions, and in-line multimers to be readily generated and manufactured.

[0059] The lack of requirement for disulfide bonds and post-translational modifications also allows numerous embodiments of proteins, including human FcRn-binding AFFIMER® polypeptides, to be delivered therapeutically by expression of gene transfer constructs introduced into a patient's tissue, including formats in which the protein is delivered systemically (e.g., expression from muscle tissue) or locally (e.g., via gene transfer within a tumor).

[0060] AFFIMER® polypeptides (also referred to simply as AFFIMER®) are small, highly stable polypeptides (e.g., proteins) that are recombinantly engineered variants of stefin polypeptides. Thus, the term "AFFIMER® polypeptides" may be used interchangeably herein with the term "recombinantly engineered variants of stefin polypeptides." The term "affimer" may be used interchangeably with AFFIMER®, etc., and any term may be used without limitation. Stefin polypeptides are a protein subgroup of the cystatin superfamily (a family that includes proteins that contain multiple cystatin-like sequences). The stefin subgroup of the cystatin family are relatively small (~100 amino acids), single domain proteins. They undergo no known post-translational modifications and lack disulfide bonds, implying that they can fold identically in a wide range of extracellular and intracellular environments. Stefin A is a monomeric, single chain, single domain protein of 98 amino acids. The structure of Stefin A was analyzed, facilitating rational mutation of Stefin A into the AFFIMER® polypeptide. The only known biological activity of cystatins is the inhibition of cadepsin activity, which allowed for thorough testing of the residual biological activity of the engineered proteins.

[0061] AFFIMER® polypeptides exhibit two peptide loops and an N-terminal sequence that can all be randomized to bind to a target protein of interest with high affinity and specificity in a manner similar to monoclonal antibodies. Stabilization of the two peptides by the Stefin A protein scaffold can constrain the possible conformations that the peptides can adopt, increasing binding affinity and specificity compared to libraries of free peptides. Such engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in various applications. Mutations can be made to other parts of the Stefin A polypeptide sequence, which can then improve the properties of these affinity reagents, e.g., increase stability, making them rigid over a range of temperatures and pH. In some embodiments, AFFIMER® polypeptides include sequences derived from Stefin A that share significant identity with the Stefin A wild-type sequence, such as human Stefin A. In some embodiments, AFFIMER® polypeptides have an amino acid sequence that shares at least 25%, 35%, 45%, 55%, or 60% identity with a sequence corresponding to human Stefin A. For example, AFFIMER® polypeptides may have an amino acid sequence that shares at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95% identity, e.g., where the sequence variation does not adversely affect the ability of the scaffold to bind to a target of interest, and where, e.g., a biological function possessed by wild-type Stefin A is eliminated by the mutational changes described herein without restoring or producing the same.

[0062] As used herein, the term AFFIMER® may be used interchangeably with "recombinantly engineered variants of stefin polypeptides."

[0063] Human Neonatal Fc Receptor (FcRn) Binding AFFIMER® Polypeptides One aspect of the present disclosure provides AFFIMER® polypeptides (referred to as anti-human FcRn AFFIMER® polypeptides) that bind to human neonatal Fc receptor (FcRn). Human neonatal Fc receptor (also known as Brambell receptor) is a protein encoded by the FCGRT gene. Such Fc receptors are similar in structure to MHC class I molecules and associate with β-2-microglobulin. FcRn contains a 40 kDa α heavy chain that non-covalently associates with the 120 kDa light chain β-2-microglobulin. The FcRn heavy chain contains three extracellular domains (α1, α2, and α3), a transmembrane domain, and a 44 amino acid cytoplasmic tail. In humans, FcRn plays a role in monitoring IgG and serum albumin turnover (Non-Patent Documents 1 and 2). Neonatal Fc receptor expression is upregulated by the proinflammatory cytokine TNF-α and downregulated by IFN-γ. An exemplary human FcRn sequence is provided by UniProtKB primary accession number X and may include other human isoforms.

[0064] FcRn-mediated transcytosis of IgG across epithelial cells is possible because FcRn binds IgG at acidic pH (<6.5) but not at neutral or higher pH. Thus, FcRn can bind IgG in the slightly acidic intestinal lumen and ensure efficient unidirectional transport to the basolateral side, where the pH is neutral to slightly basic (Non-Patent Document 3).

[0065] FcRn extends the half-life of IgG and serum albumin by reducing lysosomal degradation in endothelial cells (Non-Patent Document 4) and bone marrow-derived cells (Non-Patent Document 5). IgG, serum albumin, and other serum proteins are continuously internalized by pinocytosis. In general, serum proteins are transported from endosomes to lysosomes, where they are degraded. IgG and serum albumin, the two most abundant serum proteins, are bound by FcRn at a slightly acidic pH (<6.5) and recycled to the cell surface, where they are released at the neutral pH of blood (>7.0). In this way, IgG and serum albumin avoid lysosomal degradation. This mechanism provides an explanation for the longer serum circulating half-life of IgG and serum albumin (Non-Patent Document 6 and Non-Patent Document 4).

[0066] Anti-human FcRn AFFIMER® polypeptides include at least one solvent accessible loop that selectively binds to human FcRn, and in some embodiments, at least one solvent accessible loop that selectively binds to human FcRn. -6 K below M d The present invention includes AFFIMER® polypeptides derived from wild-type Stefin A protein having an amino acid sequence that enables it to bind to Stefin A at a 5' end.

[0067] In some embodiments, the polypeptide binds to human FcRn at a concentration of 1×10 -9 M~1×10 -6 K of M d In some embodiments, the polypeptide binds to human FcRn at a concentration of 1×10 -6 K below M d In some embodiments, the polypeptide binds to human FcRn at a concentration of 1×10 -7 K below M d In some embodiments, the polypeptide binds to human FcRn at a concentration of 1×10 -8 K below M d In some embodiments, the polypeptide binds to human FcRn at a concentration of 1×10-9 K below M d In some embodiments, the polypeptide binds to human FcRn at a concentration of 1×10 at pH 7.4. -9 M~1×10 -6 K of M d In some embodiments, the polypeptide binds to human FcRn at a concentration of 1×10 at pH 7.4. -6 K below M d In some embodiments, the polypeptide binds to human FcRn at a concentration of 1×10 at pH 7.4. -7 K below M d In some embodiments, the polypeptide binds to human FcRn at a concentration of 1×10 at pH 7.4. -8 K below M d In some embodiments, the polypeptide binds to human FcRn at a concentration of 1×10 at pH 7.4. -9 K below M d Combine with.

[0068] In some embodiments, the polypeptide has a K for binding to human FcRn at pH 5.8-6.2 and a K for binding to human FcRn at pH 7.4-7.6. d K less than hafl log~2.5log d In some embodiments, the polypeptide binds to human FcRn at a K of 5.8-6.2 and a K of 7.4-7.6. d At least half log less than K d In some embodiments, the polypeptide binds to human FcRn at a K of 5.8-6.2 and a K of 7.4-7.6. d At least 1 log less than K d In some embodiments, the polypeptide binds to human FcRn at a K of 5.8-6.2 and a K of 7.4-7.6. d At least 1.5 log less than K d In some embodiments, the polypeptide binds to human FcRn at a K of 5.8-6.2 and a K of 7.4-7.6. dAt least 2 log less than K d In some embodiments, the polypeptide binds to human FcRn at a K of 5.8-6.2 and a K of 7.4-7.6. d At least 2.5 log less than K d In some embodiments, the polypeptide has a K for binding to human FcRn at pH 6 and a K for binding to human FcRn at pH 7.4. d K less than half log to 2.5 log d In some embodiments, the polypeptide has a K for binding to human FcRn at pH 6 and a K for binding to human FcRn at pH 7.4. d At least half log K d In some embodiments, the polypeptide has a K for binding to human FcRn at pH 6 and a K for binding to human FcRn at pH 7.4. d At least 1 log less than K d In some embodiments, the polypeptide has a K for binding to human FcRn at pH 6 and a K for binding to human FcRn at pH 7.4. d At least 1.5 log less than K d In some embodiments, the polypeptide has a K for binding to human FcRn at pH 6 and a K for binding to human FcRn at pH 7.4. d At least 2 log less than K d In some embodiments, the polypeptide has a K for binding to human FcRn at pH 6 and a K for binding to human FcRn at pH 7.4. d At least 2.5 log less than K d And people Binds to FcRn.

[0069] In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 10 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 24 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 48 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 72 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 96 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 120 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 144 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 168 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 192 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 216 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 240 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 264 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 288 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 312 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 336 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of greater than 360 hours. In some embodiments, the polypeptide has a serum half-life in a human patient of 24-360 hours, 48-360 hours, 72-360 hours, 96-360 hours, or 120-360 hours.

[0070] In some embodiments, the anti-human FcRn AFFIMER® polypeptide comprises a loop 2 amino acid sequence selected from any one of SEQ ID NOs: 6-299 and 1182 (Table 1). In some embodiments, the anti-human FcRn AFFIMER® polypeptide comprises a loop 4 amino acid sequence selected from any one of SEQ ID NOs: 300-593 and 1183 (Table 1).

[0071] In some embodiments, (Xaa)n comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 6-299 and 1182. In some embodiments, (Xaa)n comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOs: 6-299 and 1182. In some embodiments, (Xaa)n comprises an amino acid sequence of any one of SEQ ID NOs: 6-299 and 1182.

[0072] In some embodiments, (Xaa)m comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 300-593 and 1183. In some embodiments, (Xaa)m comprises an amino acid sequence having 80% to 90% identity to the amino acid sequence of any one of SEQ ID NOs: 300-593 and 1183. In some embodiments, (Xaa)m comprises an amino acid sequence of any one of SEQ ID NOs: 300-593 and 1183. [Table 1A] [Table 1B] [Table 1C] [Table 1D] [Table 1E] [Table 1F] [Table 1G]

[0073] In some embodiments, the anti-human FcRn AFFIMER® polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 594-887 and 1184 (Table 2).

[0074] In some embodiments, the anti-human FcRn AFFIMER® polypeptide comprises an amino acid sequence having at least 80% or at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 594-887 and 1184. In some embodiments, the anti-human FcRn AFFIMER® polypeptide comprises an amino acid sequence having 80%-90% identity to the amino acid sequence of any one of SEQ ID NOs: 594-887 and 1184. In some embodiments, the anti-human FcRn AFFIMER® polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 594-887 and 1184. [Table 2A] [Table 2B] [Table 2C] [Table 2D] [Table 2E] [Table 2F] [Table 2G] [Table 2H]

Table 2I

Table 2J

Table 2K

Table 2L

Table 2M

Table 3A

Table 3B

Table 3C

Table 3D

Table 3E

Table 3F

Table 3G

Table 3H

Table 3I

Table 3J

Table 3K

Table 3L

Table 3M

Table 3N

Table 3O

Table 3P

Table 3Q

Table 3R

Table 3S

Table 3T

Table 3U

Table 3V

Table 3W

Table 3X

Table 3Y

Table 3Z

Table 3AA

Table 3AB

Table 3AC

Table 3AD

[0075] The anti-human FcRn AFFIMER® polypeptides provided herein, in some embodiments, are also linked to another molecule to extend the half-life of the molecule (e.g., a therapeutic polypeptide). The term half-life refers to the amount of time it takes for a substance, such as a therapeutic AFFIMER® polypeptide, to lose half of its pharmacological or physiological activity or concentration. Biological half-life may be affected by the elimination, excretion, degradation (e.g., enzymatic degradation), or absorption and concentration of the substance in some organs or tissues of the body. Biological half-life can be assessed, for example, by measuring the time it takes for the plasma concentration of the substance to reach half of its normal state level ("plasma half-life").

[0076] In some embodiments, the anti-human FcRn AFFIMER® polypeptides increase the serum half-life of a molecule (e.g., a therapeutic polypeptide) in vivo. For example, the anti-human FcRn AFFIMER® polypeptides can increase the half-life of a molecule by at least 1.2-fold compared to the half-life of the molecule not bound to the anti-human FcRn AFFIMER® polypeptide. In some embodiments, the anti-human FcRn AFFIMER® polypeptides increase the half-life of a molecule by at least 1.5-fold, at least 2-fold, 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 compared to the half-life of the molecule not bound to the anti-human FcRn AFFIMER® polypeptide. In some embodiments, the anti-human FcRn AFFIMER® polypeptide extends the half-life of the molecule by 1.2-5 fold, 1.2-10 fold, 1.5-5 fold, 1.5-10 fold, 2-5 fold, 2-10 fold, 3-5 fold, 3-10 fold, 15-5 fold, 4-10 fold, or 5-10 fold, compared to the half-life of the molecule not bound to the anti-human FcRn AFFIMER® polypeptide. In some embodiments, the anti-human FcRn AFFIMER® polypeptide extends the half-life of the molecule by at least 6 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, e.g., at least one week, following in vivo administration, compared to the half-life of the molecule not bound to the anti-human FcRn AFFIMER® polypeptide.

[0077] Polypeptides A polypeptide is a polymer of amino acids (natural or non-natural, e.g., amino acid analogs) of any length. The terms "polypeptide" and "peptide" are used interchangeably herein unless otherwise indicated. A protein is an example of a polypeptide. A polypeptide may be linear or branched, and may contain natural and / or non-natural (e.g., modified) amino acids, or may contain non-amino acids (e.g., interspersed throughout the polymer). A polypeptide may be modified (e.g., naturally or non-naturally) as provided herein, for example, via disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. A polypeptide may in some instances contain at least one analog of an amino acid (e.g., including a non-natural amino acid) and / or other modifications.

[0078] Amino acids (also called amino acid residues) participate in the peptide bonds of polypeptides. In general, the abbreviations used herein to designate amino acids are based on the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission (see Non-Patent Document 7). For example, Met, Ile, Leu, Ala and Gly represent the "residues" of methionine, isoleucine, leucine, alanine and glycine, respectively. Residues are radicals derived from the corresponding α-amino acids by removing the OH moiety of the carboxyl group and the H moiety of the α-amino group. The amino acid side chain is the portion of the amino acid excluding the --CH(NH2)COOH moiety, as defined in the literature [KD Kopple, "Peptides and Amino Acids" WA Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33].

[0079] As used herein, an amino acid, in some embodiments, is a naturally occurring amino acid found, for example, in proteins, or a naturally occurring anabolic or catabolic product of such an amino acid that contains amino and carboxyl groups. Exemplary amino acid side chains include side chains selected from the side chains 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, as well as the amino acids and amino acid analogs identified as components of peptidylglycan bacterial cell walls.

[0080] Amino acids with basic side chains include Arg, Lys, and His. Amino acids with acidic side chains include Glu and Asp. Amino acids with neutral polar side chains include Ser, Thr, Asn, Gln, Cys, and Tyr. Amino acids with neutral nonpolar side chains include Gly, Ala, Val, Ile, Leu, Met, Pro, Trp, and Phe. Amino acids with nonpolar aliphatic side chains include Gly, Ala, Val, Ile, and Leu. Amino acids with hydrophobic side chains include Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp. Amino acids with small hydrophobic side chains include Ala and Val. Amino acids with aromatic side chains include Tyr, Trp, and Phe.

[0081] The term amino acid includes analogs, derivatives, and congeners of any particular amino acid referred to herein. For example, AFFIMER® polypeptides (especially when produced by chemical synthesis) may contain amino acid analogs such as cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid. Other naturally occurring amino acid metabolites or precursors having suitable side chains herein will be recognized by those of skill in the art and are within the scope of the present disclosure.

[0082] The present application also includes (D) and (L) stereoisomers of amino acids, if the structure of the amino acid allows for such stereoisomeric forms. The amino acids and sequences of amino acids in the present application are represented by the appropriate symbols (D), (L) or (DL), and further, where the sequence is not indicated, the amino acid or residue may have the sequence (D), (L) or (DL). It will be understood that some structures of the compounds of the present disclosure include asymmetric carbon atoms. It should therefore be understood that isomers arising from such asymmetry are included within the scope of the present disclosure. Such isomers may be obtained in substantially pure form by classical separation techniques and by stereoregulated synthesis. For the purposes of the present disclosure, unless expressly indicated otherwise, a named amino acid should be construed as including both the (D) or (L) stereoisomers.

[0083] In the context of two or more nucleic acids or polypeptides, percent identity refers to two or more sequences or subsequences that, when compared and aligned (if necessary, with breaks) for maximum correspondence, are identical (matching / 100% identity) or have a specified percentage (e.g., at least 70% identity) of identical nucleotides or amino acid residues, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software used to obtain amino acid or nucleotide sequence alignment are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin package, and variations thereof. In some embodiments, two nucleic acids or polypeptides of the present disclosure are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as determined using a sequence comparison algorithm or by visual inspection. In some embodiments, the identity exists over a region of the amino acid sequence 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 integer value therebetween. In some embodiments, the identity exists over a region of more than 60-80 residues, e.g., at least about 80-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., the coding regions of the target proteins or antibodies. In some embodiments, the identity exists over a region of the nucleotide sequence 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 integer value therebetween. In some embodiments, the identity exists over a region greater than 60-80 bases, e.g., at least about 80-100 bases or more, and in some embodiments, the sequences are compared. The sequence is substantially identical over the entire length of the nucleotide sequence encoding the desired protein, for example.

[0084] Conservative amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue with a similar side chain.Families of amino acid residues with similar side chains include 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), non-polar 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), as generally defined in the art.For example, the substitution of phenylalanine for tyrosine is a conservative substitution. In general, conservative substitutions in the sequences of the polypeptides, soluble proteins and / or antibodies of the present disclosure do not eliminate binding of the polypeptide, soluble protein or antibody comprising said amino acid sequence to a target binding site. Methods for identifying conservative substitutions of amino acids that do not eliminate binding are well known in the art.

[0085] It will be understood that an isolated molecule (e.g., a polypeptide (e.g., a soluble protein, antibody, etc.), a polynucleotide (e.g., a vector), a cell or other composition) herein is in a form not found in nature. An isolated molecule, for example, has been purified to a degree not possible in nature.

[0086] In some embodiments, an isolated molecule (e.g., a polypeptide (e.g., a soluble protein, antibody, etc.), polynucleotide (e.g., a vector), cell or other composition) is substantially pure, which refers to an isolated molecule that is at least 50% pure (e.g., free from 50% of the contaminants associated with an unpurified form of the molecule), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0087] Conjugates Containing Polypeptide Fusions The term conjugate (used interchangeably with the term link) in this application refers to two or more molecules (e.g., polypeptides and / or chemical moieties) joining together to form another molecule. Thus, one molecule (e.g., an anti-FcRn AFFIMER® polypeptide) conjugated to another molecule (e.g., another AFFIMER® polypeptide, a drug molecule, or other therapeutic protein or nucleic acid) forms a conjugate. The binding of two or more molecules may be, for example, via non-covalent or covalent bonds. For example, an anti-FcRn AFFIMER® polypeptide is directly or indirectly linked to an FcRn affimer. For example, an anti-FcRn AFFIMER® polypeptide is directly or indirectly linked to a chemical moiety or another polypeptide (e.g., a heterologous polypeptide) to form a conjugate as provided herein. Non-limiting examples of conjugates include chemical conjugates (e.g., linked by "click" chemistry or another chemical reaction) and fusions (two molecules linked by adjacent peptide bonds). In some embodiments, the conjugate is a fusion polypeptide, e.g., a fusion protein. In some embodiments, the anti-FcRn AFFIMER® polypeptide is conjugated to two or more other molecules. For example, a dual (or multiple) mode of action drug conjugate may be conjugated to the anti-FcRn AFFIMER® polypeptide of the present disclosure. Such dual mode of action drug conjugates include tumor microenvironment-activated conjugate (TMAC) platform conjugates (see, e.g., avacta.com / therapeutics / tmac-affimer-drug-conjugates).

[0088] A fusion polypeptide (e.g., a fusion protein) is a polypeptide that includes at least two domains (e.g., protein domains) encoded by a polynucleotide that includes the nucleotide sequences of at least two separate molecules (e.g., two genes). In some embodiments, the polypeptide includes a heterologous polypeptide that is covalently linked (to the amino acids of the polypeptide) via an amide bond to form a contiguous fusion polypeptide (e.g., a fusion protein). In some embodiments, the heterologous polypeptide includes a therapeutic polypeptide. In some embodiments, the anti-FcRn AFFIMER® polypeptide is conjugated to the heterologous polypeptide via a peptide bond contiguous to the C-terminus or N-terminus of the anti-human FcRn AFFIMER® polypeptide.

[0089] A linker is a molecule inserted between a first polypeptide (e.g., as an AFFIMER® polypeptide) and a second polypeptide (e.g., another AFFIMER® polypeptide, an Fc domain, a ligand binding domain, etc.). A linker can be any molecule, such as one or more nucleotides, amino acids, or chemical functional groups. In some embodiments, the linker is a peptide linker (e.g., two or more amino acids). The linker should not adversely affect the expression, secretion, or biological activity of the polypeptide. In some embodiments, the linker is not antigenic and does not elicit an immune response. An immune response includes responses from the innate immune system and / or the adaptive immune system. Thus, the immune response can be a cell-mediated response and / or a humoral immune response. The immune response can be, for example, a T cell response, a B cell response, a natural killer (NK) cell response, a monocyte response, and / or a macrophage response. Other cellular responses are contemplated herein.

[0090] In some embodiments, the linker does not code for a protein.

[0091] In some embodiments, the conjugate comprises an AFFIMER® polypeptide linked to a therapeutic or diagnostic molecule, hi some embodiments, the conjugate comprises an AFFIMER® polypeptide linked to another protein, nucleic acid, drug, or other small molecule or macromolecule.

[0092] To conjugate a molecule to the AFFIMER® polypeptide of the present disclosure, any conjugation method may be used, or easily adapted, for example, the methods described in the literature [Non-Patent Documents 8-11].

[0093] treatment In some embodiments, the AFFIMER® polypeptide is linked to a therapeutic molecule, which may be used herein, for example, to prevent and / or treat a disease in a subject, e.g., a human subject or other animal subject.

[0094] In some embodiments, the therapeutic molecule is for the treatment of an autoimmune disease (a condition in which a subject's immune system mistakenly attacks the body's own immune system). Non-limiting examples of autoimmune diseases include myasthenia gravis, pemphigus vulgaris, neuromyelitis optica, Guillain-Barre syndrome, rheumatoid arthritis, systemic lupus erythematosus (lupus), idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, antiphospholipid syndrome (APS), autoimmune urticarial ulcers, chronic inflammatory demyelinating polyneuropathy, and chronic inflammatory demyelinating polyneuropathy. polyneuropathy (CIDP), psoriasis, Goodpasture's syndrome, Graves' disease, inflammatory bowel disease, Crohn's disease, Sjorgren's syndrome, hemolytic anemia, neutropenia, paraneoplastic cerebellar degeneration, paraproteinemic polyneuropathies, primary biliary cirrhosis, stiff person syndrome, vitiligo, warm idiopathic haemolytic anaemia, multiple sclerosis, and type 1 diabetes. Autoimmune diseases include thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, and celiac disease. Other autoimmune diseases are contemplated herein.

[0095] In some embodiments, the therapeutic molecule is for treating cancer. Non-limiting examples of cancer include skin cancer (e.g., melanoma or non-melanoma, e.g., basal cell or squamous cell), lung cancer, prostate cancer, breast cancer, colorectal cancer, renal (renal) cancer, bladder cancer, non-Hodgkin's lymphoma, thyroid cancer, endometrial cancer, exocrine cancer, and pancreatic cancer. Other cancers are contemplated herein.

[0096] In some embodiments, the therapeutic molecule is for the treatment of an inflammatory disease or disorder (a disease, disorder or condition characterized by inflammation of body tissues or having an inflammatory component), including local inflammatory responses and systemic inflammation. Non-limiting examples of inflammatory disorders include transplant rejection, including skin graft rejection; chronic inflammatory disorders of the joints, including arthritis, rheumatoid arthritis, osteoarthritis, and bone diseases associated with increased bone resorption; inflammatory bowel diseases, such as ileitis, ulcerative colitis, Barrett's syndrome, and Crohn's disease; inflammatory lung disorders, such as asthma, adult respiratory distress syndrome, and chronic obstructive airway disease; corneal dystrophy, trachoma, onchocerciasis, uveitis, sympathetic ophthalmia, and other conditions. inflammatory disorders of the eye, including ophthalmitis and endophthalmitis; chronic inflammatory disorders of the gums, including gingivitis and periodontitis; tuberculosis; inflammatory diseases of the kidneys, including leprosy, uremic complications, glomerulonephritis and nephrosis; inflammatory disorders of the skin, including sclerodermatitis, psoriasis and eczema;chronic demyelinating diseases of the nervous system, multiple sclerosis, AIDS-related neurodegeneration and Alzheimer's disease, infectious meningitis, encephalomyelitis, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and inflammatory diseases of the central nervous system, including viral or autoimmune encephalitis; autoimmune disorders, immune-complex vasculitis, systemic lupus and erythematodes; systemic lupus and systemic lupus erythematodes (SLE); and cardiomyopathy, ischemic heart disease, hypercholesterolemia, and chronic myocardial infarction. inflammatory diseases of the heart, such as the heart disease hypercholesterolemia, atherosclerosis; and various other diseases with a significant inflammatory component, including preeclampsia; chronic liver failure, brain and spinal cord trauma. Additionally, there may be systemic inflammation of the body, exemplified as gram-positive or gram-negative shock, hemorrhagic or anaphylactic shock, or shock caused by cancer chemotherapy in response to proinflammatory cytokines, e.g., shock associated with proinflammatory cytokines. Such shock may be caused, for example, by chemotherapeutic agents used in cancer chemotherapy; In some embodiments, the therapeutic molecule is for the treatment of a cardiovascular disease or disorder, including, but not limited to, abnormal heart rhythm or arrhythmias, aorta disease and Marfan syndrome, congenital heart disease, coronary artery disease (e.g., narrowing of the arteries), deep vein thrombosis and pulmonary embolism, heart attack, heart failure, heart muscle disease (e.g., cardiomyopathy), heart valve disease, pericardial disease, peripheral vascular disease, rheumatic heart disease, stroke, and vascular disease (e.g., vascular disease).

[0097] In some embodiments, the therapeutic molecule is for the treatment of a metabolic disease or disorder. Examples of metabolic disorders include: glycogen storage disorders (also known as glycogenosis or dextrinosis), which include disorders affecting carbohydrate metabolism; fat oxidation disorders, which affect fat metabolism and the metabolism of fat components; and mitochondrial disorders, which affect mitochondria. Examples of glycogen storage disorders (GSDs) include at least GSD type I (glucose-6-phosphatase deficiency; von Gierke's disease); GSD type II (acid maltase deficiency; Pompe's disease); GSD type III (glycogen debrancher deficiency); Cori's disease or Forbe's disease; GSD type IV (glycogen branching enzyme deficiency; Andersen disease); GSD type V (muscle glycogen phosphorylase deficiency; McArdle's disease). GSD type VI (hepatic phosphorylase deficiency; Hers' disease); GSD type VII (muscle phosphofructokinase deficiency; Tarui's disease); GSD type IX (phosphorylase kinase deficiency); and GSD type XI (glucose transporter deficiency; Fanconi-Bickel disease). Examples of fatty acid metabolism deficiencies include at least coenzyme A dehydrogenase deficiency; other coenzyme A enzyme deficiencies; carnitine-related disorders; or lipid storage disorders. Examples of coenzyme A dehydrogenase deficiencies include at least very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCAD); long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency (LCHAD); medium-chain acyl-coenzyme A dehydrogenase deficiency (MCAD); short-chain acyl-coenzyme A dehydrogenase deficiency (SCAD); and short-chain L-3-hydroxyacyl-coA dehydrogenase deficiency (SCHAD).Examples of other coenzyme A enzyme deficiencies include at least 2,4 dienoyl-CoA reductase deficiency, 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, and malonyl-CoA decarboxylase deficiency. Examples of carnitine-related deficiencies include at least primary carnitine deficiency, carnitine acylcarnitine translocase deficiency, carnitine palmitoyltransferase deficiency (CPT), and carnitine palmitoyltransferase II deficiency (CPT). Examples of lipid storage diseases include acid lipase disease, Wolman disease, cholesteryl ester storage disease, Gaucher disease, Niemann-Pick disease, Fabry disease, Farber's disease, gangliosidoses, Krabbe disease, and metachromatic leukodystrophy. Other fatty acid metabolism diseases include at least mitochondrial trifunctional protein deficiency, electron transfer flavoprotein (ETF) dehydrogenase deficiency (GAII&MADD), Tangier disease, and acute fatty liver of pregnancy.Examples of mitochondrial diseases include at least progressive external ophthalmoplegia (PEO); diabetes mellitus and deafness (DAD); Leber hereditary optic neuropathy (LHON); mitochondrial encephalomyopathy; lactic acidosis; and stroke-like syndrome (MELAS); myoclonus epilepsy; and ragged-red fibers (MERRF); Leigh syndrome; subacute sclerosing encephalopathy; Neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP); Kearns-Sayre syndrome. Myoneurogenic gastrointestinal encephalopathy (MNG)

[0098] The term treatment refers to the process of alleviating at least one symptom associated with a disease, as known in the art. The symptom may be a physical, mental, or pathological manifestation of the disease. Symptoms associated with various diseases are known. To treat or prevent a particular condition, the conjugates (e.g., anti-human FcRn AFFIMER® polypeptides linked to a therapeutic molecule) as provided herein should be administered in an effective amount, which may be any amount used to treat or prevent the condition. Thus, in some embodiments, the effective amount is the amount used to alleviate symptoms associated with the particular disease being treated. For example, methods are known for measuring the effective amount of various therapeutic molecules.

[0099] A subject can be any animal (eg, a mammal), including, but not limited to, humans, non-human primates, dogs, cats, and rodents. "Patient" refers to a human subject.

[0100] In some embodiments, the anti-human FcRn AFFIMER® polypeptide is linked to an agonist of a particular molecule of interest (e.g., a receptor). In other embodiments, the anti-human FcRn AFFIMER® polypeptide is linked to an antagonist of a particular molecule of interest. In this application, an agonist refers to a molecule that binds to and activates another molecule to generate a biological response. In contrast, an antagonist blocks the action of an agonist, and an inverse agonist causes an action opposite to that of an agonist. Thus, in this application, an antagonist refers to a molecule that binds to and inactivates or prevents the activation of another molecule.

[0101] In some embodiments, AFFIMER® polypeptides are considered to be "pharmaceutical acceptable" and in some embodiments are formulated with pharmaceutical acceptable excipients. A molecule or other substance / agent is considered to be pharmaceutical acceptable if it is approved or can be approved by a federal or state regulatory agency or is listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, including humans. An excipient may be any inert (non-active), non-toxic agent administered with an AFFIMER® polypeptide. Non-limiting examples of excipients include buffers (e.g., sterile saline), salts, carriers, preservatives, fillers, and colorants.

[0102] Therapeutic molecules for use herein include, for example, those recognized in the official United States Pharmacopoeia, the official United States Homeopathic Pharmacopoeia, the official National Formulary, or supplements thereof, including, but not limited to, small molecule chemicals / drugs, polynucleotides (e.g., RNA interference molecules, e.g., miRNA, siRNA, shRNA, and antisense RNA), and polypeptides (e.g., antibodies). As provided herein, classes of therapeutic molecules for use include, but are not limited to, recombinant proteins, antibodies, cytotoxic agents, antimetabolites, alkylating agents, antibiotics, growth factors (e.g., erythropoietin, granulocyte colony stimulating factor), and the like. granulocyte-macrophage colony-stimulating factor (G-CSF), keratinocyte growth factor (K-CSF), cytokines, chemokines, interferons (e.g., interferon alpha, interferon beta, interferon gamma), blood factors (e.g., factor VIII, factor Vila, factor IX, thrombin, antithrombin), mitotic inhibitors, toxins, apoptotic agents (e.g., DNA alkylating agents), local isomerase inhibitors, endoplasmic reticulum stress inducers, platinum compounds, antimetabolites, vinca alkaloids, taxanes, epothilones, enzyme inhibitors , receptor antagonists, tyrosine kinase inhibitors, radiosensitizers, chemotherapy combination therapies, receptor traps, receptor ligands, angiogenic agents, antiangiogenic agents, anticoagulant and thrombolytic agents (e.g., tissue plasminogen activator, hirudin, protein C), neurotransmitters, erythropoiesis stimulators, insulin, growth hormones (e.g., human growth hormone (hGH), follicle stimulating hirumone), metabolic hormones (e.g., incretins), recombinant IL-1 receptor antagonists, and bispecific T cell engaging molecules (BITEs®).

[0103] Specific examples of therapeutic molecules to which an anti-human FcRn AFFIMER® polypeptide may be linked (e.g., to increase the half-life of the molecule) include fibroblast growth factor 21 (FGF21), insulin, insulin receptor peptide, GIP (glucose-dependent insulinotropic polypeptide), bone morphogenetic protein 9 (BMP9), amylin, peptide YY (PYY3-36), pancreatic polypeptide (PP), interleukin 21 (IL-21), glucagon-like peptide 1 (GLP-1), plectasin, progranulin, osteocalcin (OCN), apelin, GLP-1, exendin 4, adiponectin, IL-1Ra (interleukin 1 receptor antagonist), VIP (vasoactive intestinal peptide), PACAP (pituitary adenylate cyclase-activating polypeptide), leptin, INGAP (islet neogenesis associated protein), BMP (bone morphogenetic protein), and osteocalcin (OCN).

[0104] antibody In some embodiments, the heterologous polypeptide to which the anti-human FcRn AFFIMER® polypeptide is linked is an antibody (e.g., a variable region of an antibody). Thus, in some embodiments, the present disclosure provides an AFFIMER® polypeptide antibody fusion protein. In some embodiments, the AFFIMER® polypeptide antibody fusion protein comprises, for example, a full-length antibody comprising at least one AFFIMER® polypeptide sequence pendant to the C-terminus or N-terminus of at least one VH and / or VL chain of the antibody (at least one chain of the incorporated antibody forms a fusion protein with the AFFIMER® polypeptide). In some embodiments, the AFFIMER® polypeptide antibody fusion protein comprises at least one AFFIMER® polypeptide and an antigen-binding site or variable region of an antibody fragment.

[0105] An antibody is an immunoglobulin molecule that recognizes and specifically binds to a target, such as a polypeptide (e.g., a peptide or protein), a polynucleotide, a carbohydrate, a lipid, or any combination thereof, via at least one antigen-binding site, which in some embodiments is within the variable region of the immunoglobulin molecule. Antibodies include polyclonal antibodies, monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) antibodies (provided that the fragments are formatted to contain an Fc or other FcγIII binding domain), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigen-binding site of an antibody (formatted to contain an Fc or other FcγIII binding domain), and any other modified immunoglobulin molecule containing an antigen-binding site, so long as the antibody exhibits the desired biological activity.

[0106] An antibody may be any one of five major classes of immunoglobulins based on the congestion of heavy chain constant domains designated as alpha, delta, epsilon, gamma and mu: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

[0107] The variable region of an antibody may be only the variable region of an antibody light chain, or only the variable region of an antibody heavy chain, or a combination thereof. In general, each variable region of a heavy chain and a light chain consists of four framework regions (FR) and three complementarity determining regions (CDRs) (also known as hypervariable regions). The CDRs in each chain are held together by the framework regions in close proximity and contribute to the formation of the antigen-binding site of the antibody together with the CDRs of the other chain. There are at least two CDR determination techniques: (1) an approach based on interspecies sequence variability (Non-Patent Document 12), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Non-Patent Document 13). A combination of these two approaches may also be used in the art for CDR determination.

[0108] A humanized antibody is a form of a non-human (e.g., murine) antibody that is a particular immunoglobulin chain, chimeric immunoglobulin, or fragment thereof that contains minimal non-human sequences. Typically, a humanized antibody is a human immunoglobulin in which residues of the CDRs are replaced by residues of the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that has the desired specificity, affinity, and / or binding capacity. In some examples, Fv framework region residues of the human immunoglobulin are replaced by the corresponding residues in the antibody of the non-human species. Humanized antibodies can be further modified by substitution of additional residues in the Fv framework regions and / or within the replaced non-human residues to improve and optimize the antibody specificity, affinity, and / or binding capacity. A humanized antibody may comprise a variable domain that contains all or substantially all of the CDRs corresponding to a non-human immunoglobulin, but all or substantially all of the framework regions are of human immunoglobulin sequences. In some embodiments, the variable domain comprises framework regions of human immunoglobulin sequences. In some embodiments, the variable domains comprise framework regions of human immunoglobulin consensus sequences. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Humanized antibodies are generally considered to be distinct from chimeric antibodies.

[0109] An epitope (also referred to as an antigenic determinant) is a portion of an antigen that can be recognized and specifically bound by a particular antibody, particularly an AFFIMER® polypeptide, or other specific binding domain. When the antigen is a polypeptide, epitopes may be formed from both contiguous amino acids and non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically maintained during protein denaturation, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturation. Epitopes typically include at least 3, and more usually at least 5, 6, 7 or 8-10 amino acids in a unique spatial configuration.

[0110] The term "specifically binds to" or "specific for" refers to an identifiable and reproducible interaction, such as the binding of a target with an AFFIMER® polypeptide, antibody or other binding partner, which determines the presence of said target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an AFFIMER® polypeptide that specifically binds to one target is an AFFIMER® polypeptide that binds with higher affinity, avidity (if formatted as a multimer), more readily, and / or with greater duration to this target than it binds to other targets.

[0111] Non-limiting examples of antibodies that can be conjugated to the FcRn-HSA AFFIMER® polypeptides of the present disclosure include 3F8, 8H9, abagovomab, abciximab, abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, and the like. pentetate, amatuximab, anatumomab mafenatox, andecaliximab, anetumab ravtansine, anifrolumab, anrukinzumab (IMA-638), apolizumab, apurutumab ixadotin ixadotin, arcitumomab, ascrinvacumab, acelizumab, atezolizumab, atidortoxumab, atinumab, atorolimumab, avelumab, azintuxizumab vedotin, bapineuzumab, basiliximab, bavituximab, BCD-100, bectumomab, begelomab, belantamab mafodotin, belimumab, bemarituzumabbemarituzumab, benralizumab, berlimatoxumab, bermekimab, bersanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, birtamimab, bivatuzumab mertansine, bleselumab, blinatumomab, brontuzumab (blontuvetmab), brosozumab, bococizumab, brazikumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontictuzumab, burosumab, cabiralizumab, camidanlumab tesirine, camrelizumab, canakinumab, cantuzumab mertansine, cantuzumab vedotin ravtansine, caplacizumab, capromab pendetide, carlumab, carotuximab, catumaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab, cemiplimab, cergutuzumab amnaleukinamunaleukin, certolizumab pegol, cetrelimab, cetuximab, cibisatamab, cirmutuzumab, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, cofetuzumab pelidotin, coltuximab ravtansine, conatumumab, concizumab, cosfroviximab, CR6261, crenezumab, crizanlizumab, crotedumab, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, derlotuximab biotin, detumomab, detumomab, dezamizumab, dinutuximab, diridavumab, domagrozumab, dormantiximabaritox, dostarlimab, drozitumab, DS-8201, durigotumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab (eldelumab), elezanumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab vedotin, enavatuzumab, enfortumab vedotin, enlimomab pegol pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomabcituxetan, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etigilimab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, fala faralimomab, faricimab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, flotetuzumab (flotetuzumab), fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frovocimab, frunevetmab, fulranumab, futuximab, galcanezumab, galiximab, gancotamab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gemtuzumab ozogamicinozogamicin, gevokizumab, gilvetmab, gimsilumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gosuranemab, guselkumab, ianalumab, ibalizumab IBI308, ibritumomab tiuxetan tiuxetan, icrucumab, idarucizumab, ifabotuzumab, igovomab, iradatuzumab vedotin, IMAB362, imalumab, imaprelimab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, indusatumab vedotin, inebilizumab, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, iomab-b, ipilimumab, iratumumab, isatuximab, iscalimab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, lacnotuzumab, ladiratuzumab vedotinvedotin, lampalizumab, lanadelumab, landogrozumab, laprituximab emtansine, larcaviximab, lebrikizumab, lemalesomab, lendalizumab, lenvervimab, lenzilumab, lerdelimumab, leronlimab, lesofavumab, letolizumab, lexatumumab, libivirumab, rifastuzumab vedotin vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, lodelcizumab, lokivetmab, loncastuximab tesirine, lorvotuzumab mertansine, losatuxizumab vedotin, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, lupartumab amadotinamadotin, lutikizumab, mapatumumab, margetuximab, marstacimab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mirikizumab, mirvetuximab soravtansine, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, mosunetuzumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab emtansine emtansine, narnatumab, natalizumab, navicixizumab, navivumab, naxitamab, nebacumab, necitumumab, nemolizumab, NEOD001, nerelimomab, nesvacumab nesvacumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, oleclumab, orendalizumab (olendalizumab), olokizumab, omalizumab, omburtamab, OMS721, onartuzumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab monatoxmonatox, oregovomab, orticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pancomab, panobacumab nobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pdr001, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin vedotin, pintumomab, placulumab, plozalizumab, pogalizumab, polatuzumab vedotinvedotin, ponezumab, porgaviximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO140, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, lanevetoma ranevetmab, ranibizumab, ravagalimab, ravulizumab, raxibacumab, refanezumab, regavirumab, relatlimab, remtolumab, reslizumab, rilotumumab, rinucumab, risankizumab, rituximab, rivabazumab pegol pegol, rmab, robatumumab, roledumab, romilkimab, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab tesirine, rovelizumab, rozanolixizumab, ruplizumab, SA237, sacituzumab govitecan, samalizumab, samrotamab vedotin vedotin, sarilumab, satralizumab, satumomab pendetidependetide, secukinumab, selicrelumab, seribantumab, setoxaximab, setrusumab, sevirumab, SGN-CD19A, SHP647, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirtratumab vedotin, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tabalumab, tacatuzumab tetraxetan tetraxetan, tadocizumab, talacotuzumab, talizumab, tamtuvetmab, tanezumab, taplitumomab paptox, tarextumab, tavolimab, tefibazumab, telimomab aritox, telisotuzumab vedotinvedotin, tenatumomab, teneliximab, teplizumab, tepoditamab, teprotumumab, tesidolumab, tetulomab, tezepelumab, TGN1412, tibulizumab, tigatuzumab, tildrakizumab, timigutuzumab, timolumab, tiragotumab, tislelizumab, tisotumab vedotin, TNX-650, tocilizumab, tomuzotuximab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukinin celmoleukin, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab talirine, vanalimab, vandortuzumab vedotinvedotin, vantictumab, vanucizumab, vapaliximab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, vobarilizumab, volociximab, vonlerolizumab, vopratelimab, borsetuzumab These include: mafodotin, votumumab, bunakizumab, xentuzumab, XMAB-5574, zalutumumab, zanolimumab, zatuximab, zenocutuzumab, ziralimu- mab, zolbetuximab (IMAB362, claudiximab), and zolimomab aritox.

[0112] Other therapeutic molecules Non-limiting examples of cytokines include IL-2, IL-12, TNF-α, IFNα, IFNβ, IFNγ, IL-10, IL-15, IL-24, GM-CSF, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, LIF, CD80, B70, TNFβ, LT-β, ​​CD-40 ligand, Fas-ligand, TGF -β, IL-1α and IL-1β.

[0113] Non-limiting examples of chemokines include IL-8, GROα, GROβ, GROγ, ENA-78, LDGF-PBP, GCP-2, PF4, Mig, IP-10, SDF-1α / β, BUNZO / STRC33, I-TAC, BLC / BCA-1, MIP-1α, MIP-1β, mDC, TECK, TARC, RANTES, HCC-1, HCC-4, DC-CK1, MIP-3α, MIP-3β, MCP-1-5, eotaxin, eotaxin-2, I-309, MPIF-1, 6Ckine, CTACK, MEC, lymphotactin, and fractalkine. Non-limiting examples of DNA alkylating agents include nitrogen mustards, such as mechlorethamine, cyclophosphamide (ifosfamide, trofosfamide), chlorambucil (melphalan, prednimustine), bendamustine, uramustine, and estramustine; nitrosoureas, such as carmustine (bcnu), lomustine (semustine), fotemustine, nimustine, ranimustine, and streptozocin; alkylsulfonates, such as busulfan (mannosulfan, treosulfan); aziridines, such as carboquone, thiotepa, triaziquone, triethylenemelamine; hydrazines (procarbazine); triazines, such as dacarbazine and temozolomide; altretamine and mitobronitol.

[0114] Non-limiting examples of local isomerase I inhibitors include camptothecin derivatives including CPT-11 (irinotecan), SN-38, APC, NPC, camptothecin, topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lurtotecan, rubitecan, siratecan, dimatecan, diflomotecan, extatecan, BN-80927, DX-8951f, and MAG-CPT, as described in Non-Patent Documents 15 and 16. protoberberine alkaloids and derivatives thereof including berberrubine and coralyne described in Non-Patent Document 17; phenanthroline derivatives including benzo[i]phenanthridine, nitidine and fagaronine described in Non-Patent Document 17; terbenzimidazole and derivatives thereof described in Non-Patent Document 18; and anthracycline derivatives including doxorubicin, daunorubicin and mitoxantrone described in Non-Patent Documents 19 to 21. Local isomerase II inhibitors include, but are not limited to, etoposide and teniposide. Dual topographic isomerase I and II inhibitors include, but are not limited to, sinatoprine and other naphthacenediones, DACA and other acridine-4-carboxamides, intoplicin and other benzopyridoindoles, tas-103 and other 7h-indeno[2,1-c]quinolin-7-ones, pyrazoloacridines, XR 11576 and other benzophenazines, XR 5944 and other dimeric compounds, 7-oxo-7H-dibenz[f,ij]isoquinolines and 7-oxo-7H-benzo[e]perimidine and anthracenyl amino acid conjugates (described in Non-Patent Document 22). Some agents, including but not limited to the anthracyclines (aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, barbicin, zorubicin) and the anthracenediones (mitoxantrone and pixantrone) inhibit local isomerase II and have DNA intercalating activity.

[0115] Non-limiting examples of endoplasmic reticulum stress inducers include dimethylcelecoxib (DMC), nelfinavir, celecoxib, and boron radiosensitizers (ie, Velcade (bortezomib)).

[0116] Non-limiting examples of platinum-based compounds include carboplatin, cisplatin, nedaplatin, oxaliplatin, triplatin tetranitrate, satraplatin, aroplatin, lobaplatin, and JM-216. (See Non-Patent Document 23 and generally Non-Patent Document 24.)

[0117] Non-limiting examples of metabolic antagonists include folates, e.g., dihydrofolate reductase inhibitors, e.g., aminopterin, methotrexate, and pemetrexed; thymidylate synthase inhibitors, e.g., raltitrexed, pemetrexed; purines, e.g., adenosine deaminase inhibitors, e.g., pentostatin, thiopurines, e.g., thioguanine and mercaptopurine, halogenated / ribonucleotide reductase inhibitors, e.g., thiopurines, e.g., thioguanine; or pyrimidines, e.g., cytosine / cytidine; hypomethylating agents, e.g., azacytidine and decitabine, DNA polymerase inhibitors, e.g., cytarabine, ribonucleotide reductase inhibitors, e.g., gemcitabine, or thymine / thymidine; thymidylate synthase inhibitors, e.g., fluorouracil (5-FU). Equivalents to 5-FU include its prodrugs, analogues and derivatives, such as 5'deoxy 5-fluorouridine (doxifluridine), 1-tetrahydrofuranyl-5-fluorouracil (FTORAFUR®), capecitabine (XELODA®), SI (MBMS-247616, consisting of tegafur and two modulators, 5-chloro-2,4-dihydroxypyridine and potassium oxonate), raltitrexed (TOMUDEX®), non-latrexed (Thymitaq, AG337), LY231514 and ZD9331 (e.g., as described in Non-Patent Document 25).

[0118] Non-limiting examples of vinca alkaloids include vinblastine, vincristine, vinflunine, vindesine, and vinorelbine.

[0119] Non-limiting examples of taxanes include docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel.An example of an epothilone is iabepilone.

[0120] Non-limiting examples of enzyme inhibitors include farnesyltransferase inhibitors (tipifamib); CDK inhibitors (alvocidib, seliciclib); proteasome inhibitors (bortezomib); phosphodiesterase inhibitors (anagrelide, rolipram); IMP dehydrogenase inhibitors (thiazopurine); lipoxygenase inhibitors (masoprocol); examples of receptor antagonists include, but are not limited to, ERAs (atrasentan); retinoid X receptor (bexarotene); and sex steroids (testolactone).

[0121] Non-limiting examples of tyrosine kinase inhibitors include inhibitors against ErbB; HER1 / EGFR (erlotinib, gefitinib, lapatinib, vandetanib, sunitinib, neratinib); HER2 / neu (lapatinib, neratinib); RTK class III: C kit (axitinib, sunitinib, sorafenib), FLT3 (lestaurtinib), PDGFR (axitinib, sunitinib, sorafenib); VEGFR (vandetanib, semaxanib, cediranib, axitinib, sorafenib); bcr-abl (imatinib, nilotinib, dasatinib); Src (bosutinib) and Janus kinase 2 (lestaurtinib). Non-limiting examples of chemotherapeutic agents include amsacrine, trabectedin, retinoids (alitretinoin, tretinoin), arsenic trioxide, asparagine depleting agents (asparaginase / pegaspargase), celecoxib, demecolcine, elesclomol, elsamitrucin, etoglucide, lonidamine, lucantone, mitoguazone, mitotane, oblimersen, temsirolimus, and vorinostat.

[0122] Non-limiting examples of therapeutic molecules that can be linked to the AFFIMER® polypeptides of the present disclosure include flomoxef; fortimicins; gentamicins; glucosulfone solasulfone; gramicidin S; gramicidins; grepafloxacin; guamecycline; hetacillin; isepamicin; josamycin; kanamycins; flomoxef; fortimicins; gentamicins; glucosulfone solasulfone; gramicidin S; gramicidins; grepafloxacin; guamecycline; hetacillin; isepamicin; josamycin; kanamycins; bacitracin; bambermycin; biapenem; brodimoprim; butirosin; capreomycin; carbenicillin; carbomycin; carumonam; cefadroxil; cefamandole; cefatrizine; Cefbuperazone;Cefclidin;Cefdinir;Cefditoren;Cefepime;Cefetameth;Cefixime;Cefmenoxime;Cefminox;Cladribine;Apalcillin;Apicycline;Apramycin;Arbekacin;Aspoxicillin;Aztreonam;Cefodizime;Cefonicid;Cefoperazone;Ceforanide;Cefotaxime;Cefotetan;Cefotiam;Cefozopran;Cefpimizole;Cefpiramide;Cefpirome;Cefprozil;Cefroxadine;Cefteram;Ceftibuten;Cefzonam;Cephalexin;Cephaloglycin; Cephalosporin C;Cephradine;Chloramphenicol;Chlortetracycline;Clinafloxacin;Clindamycin;Chromocycline;Colistin;Ciclacillin;Dapsone;Demeclocycline;Diathymosulfone;Dibekacin;Dihydrostreptomycin;6-Mercaptopurine;Thioguanine;Capecitabine;Docetaxel;Etoposide;Gemcitabine;Topotecan;Vinorelbine;Vincristine;Vinblastine;Teniposide;Melphalan;Methotrexate;2-p-Sulfanilinylethanolamine;4,4'-Sulfinyldianiline;4-Sulfanilamidosalicylic acid;Butorphanol;Nalbuphine;Streptozocin;Doxorubicin;Daunorubicin;Plicamycin;Idarubicin;Mitomycin C;Pentostatin;Mitoxantrone; Cytarabine;Fludarabine phosphate;Butorpanol;Nalbuphine;Streptozocin;Doxorubicin;Daunorubicin;Plicamycin;Idarubicin;Mitomycin C;Pentostatin;Mitoxantrone;Cytarabine;Fludarabine phosphate;Acediasulfone;Acetosulfone;Amikacin;Amphotericin B;Ampicillin;Atorvastatin;Enalapril;Ranitidine;Ciprofloxacin;Pravastatin;Clarithromycin;Cyclosporin; Famotidine;Leuprolide;Acyclovir;Paclitaxel;Azithromycin;Lamivudine;Budesonide;Albuterol;Indinavir;Metformin;Alendronate;Nizatidine;Zidovudine;Carboplatin;Metoprolol;Amoxicillin;Diclofenac;Lidinopril;Ceftriaxone;Captopril;Salmeterol;Xinafoate;Imipenem;Cilastatin;Benazepril;Cefaclor; Ceftazidime;Morphine;Dopamine;Bialamycol;Fluvastatin;Phenamidine;2-Ethylhydrazine Podophyllinic Acid;Acriflavine;Chlorazodine;Arsphenamine;Amicarbamide;Aminoquinuride;Quinapril;Oxymorphone;Buprenorphine;Floxuridine;Dirithromycin;Doxycycline;Enoxacin;Enviomycin;Epicillin;Erythromycin;Leucomycins;Lincomycin;Lomefloxacin;Lusensomycin;Limecycline;Meclocycline;Meropenem;Methacycline;Micronomycin;Midecamycins;Minocycline;Moxalactam;Mupirocin;Nadi Floxacin;Natamycin;Neomycin;Netilmicin;Norfloxacin;Oleandomycin;Oxytetracycline;p-Sulfanylbenzylamine;Panipenem;Paromomycin;Pazufloxacin;Penicillin N;Pipacycline;Pipemidic acid;Polymyxin;Primycin;Quinacillin;Ribostamycin;Rifamide;Rifampin;Rifamycin SV;Rifapentine;Rifaximin;Ristocetin;Ritipenem;Rokitamycin;Rolitetracycline;Rosaramycin;Roxithromycin;Salazosulfadimidine;Sancycline;Sisomycin;Sparfloxacin;Spectinomycin;Spiramicin;Streptomycin;S; Kujisulfone;Sulfachrysoidine;Sulfaloxic acid;Sulfamidocrysoidine;Sulfanilic acid;Sulfoxone;Teicoplanin;Temafloxacin;Temocillin;Tetroxoprim;Thiamphenicol;Thiazole sulfone;Thiostrepton;Ticarcillin;Tigemonam;Tobramycin;Tosufloxacin;Trimethoprim;Trospectomycin;Trovafloxacin;Tuberactinomycin;Vancomycin;Azaserine; Candicidins;Chlorphenesin;Dermostatins;Filipin;Fungichromin;Mepartricin;Nystatin;Oligomycins;Perimycin A;Tubercidin;6-Azauridine;6-Diazo-5-oxo-L-norleucine;Aclacinomycins;Ancitabine;Anthramycin;Azacitadine;Azaserine;Bleomycins;Ethyl bismuth acetate;Ethylidene dicumarol;Iloprost;Lamifiban;Taprostene;Thiochromarol;Tirofiban;Amiprilose;Bucillamine;Gusperimus;Gentisic acid;Glucametacin;Glycol salicylate;Meclofenamic acid;Mefenamic acid;Mesalamine;Niflumic acid;Olsalazine;Oxaceprol; S-enosylmethionine;Salicylic acid;Salsalate;Sulfasalazine;Tolfenamic acid;Carbicin;Carzinophilin A;Chlorozotocin;Chromomycins;Denopterin;Doxifluridine;Edatrexate;Efronitine;Elliptinium;Enocitabine;Epirubicin;Mannomustine;Menogaril;Mitobronitol;Mitolactol;Mopidamol;Mycophenolic acid;Nogalamycin;Olivomycins;Peplomycin;Pirarubicin;Piritrexim;Prednimustine;Procarbazine;Pteropterin;Proma Isin;Ranimustine;Streptonigrin;Tiamifrin;Mycophenolic acid;Procodazole;Romurtide;Sirolimus (rapamycin);Tacrolimus;Butethamine;Fenalcomine;Hydroxytetracaine;Nepine;Orthocaine;Piridocaine;Salicyl alcohol;3-amino-4 hydroxybutyric acid;Aceclofenac;Aluminoprofen;Amfenac;Bromfenac;Bromosaligenin;Bumadizone;Carprofen;Diclofenac;Diflunisal;Ditazole;Enfenamic acid;Etodolac;Etofenamate;. Includes fendosal; feprazinol; flufenamic acid; tomudex (N-[[5-[[(1,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]methylamino]-2-thienyl]carbonyl]-L-glutamic acid), trimetrexate; tubercidin; ubenimex; vindesine; zorubicin; argatroban; coumetalol and dicoumarol.

[0123] Non-limiting examples of cytotoxic factors include diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, lysine A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), diacin protein, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, mitogillin, restrictocin, phenomycin, and enomycin.

[0124] Non-limiting examples of neurotransmitters include arginine, aspartate, glutamate, gamma aminobutyric acid, glycine, D-serine, acetylcholine, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), serotonin (5-hydroxytryptamine), histamine, phenethylamine, N-methylphenethylamine, tyramine, octopamine, synephrine, tryptamine, N-methyltryptamine, anandamide, 2-arachidonoyl glycerol, 2-arachidonyl glyceryl ether, N-arachidonoyl dopamine, virodamine, adenosine, adenosine triphosphate, bradykinin, and corticotropin. Releasing hormone, urocortin, galanin, galanin-like peptide, gastrin, cholecystokinin, adrenocorticotropic hormone, proopiomelanocortin, melanocyte-stimulating hormone, vasopressin, oxytocin, neurophysin I, neurophysin II, neuromedin U, neuropeptide B, neuropeptide S, neuropeptide Y, pancreatic polypeptide, peptide YY, enkephalin; dynorphin, endorphin, endomorphin, nociceptin / orphanin FQ, orexin A, orexin B, kisspeptin, neuropeptide FF, prolactin-releasing peptide, pyroglutamylated RFamide peptide , secretin, motilin, glucagon, glucagon-like peptide 1, glucagon-like peptide 2, vasoactive intestinal peptide, growth hormone-releasing hormone, pituitary adenylate cyclase-activating peptide, somatostatin, neurokinin A, neurokinin B, substance P, neuropeptide K, agouti-related peptide, N-acetylaspartylglutamate, cocaine- and amphetamine-regulated transcript, bombesin, gastrin-releasing peptide, gonadotropin-releasing hormone, melanin-concentrating hormone, nitric oxide, carbon monoxide, and hydrogen sulfide.

[0125] Non-limiting examples of metabolic hormones, e.g., incretins (which stimulate a decrease in blood glucose levels), include glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP) and their analogs, e.g., dulaglutide (TRULICITY®), exenatide (BYETTA®), liraglutide (VICTOZA®), and exenatide extended release (BYDUREON®).

[0126] Pharmaceutical Compositions / Dosage Forms The present disclosure also provides a pharmaceutical composition comprising an anti-human FcRn AFFIMER® polypeptide ("AFFIMER® polypeptide") described herein and a pharma- ceutically acceptable vehicle. In some embodiments, the pharmaceutical composition is used in immunotherapy. In some embodiments, the pharmaceutical composition is used in immuno-oncology. In some embodiments, the composition is used to inhibit tumor growth. In some embodiments, the pharmaceutical composition is used to inhibit tumor growth in a subject (e.g., a human patient). In some embodiments, the composition is used to treat cancer. In some embodiments, the pharmaceutical composition is used to treat cancer, an inflammatory disorder, a cardiovascular disorder, a metabolic disorder, or an autoimmune disorder in a subject (e.g., a human patient).

[0127] Dosage forms are prepared by combining purified AFFIMER® polypeptides of the present disclosure with a pharma- ceutically acceptable vehicle (e.g., a carrier or excipient) for storage and use. Those of skill in the art would generally consider pharma- ceutically acceptable carriers, excipients, and / or stabilizers as inactive ingredients of a dosage form or pharmaceutical composition.

[0128] In some embodiments, the AFFIMER® polypeptides described herein are stored in lyophilized and / or lyophilized form. In some embodiments, a formulation comprising an AFFIMER® polypeptide described herein is lyophilized.

[0129] Suitable pharma- ceutically acceptable vehicles include, but are not limited to, non-toxic buffers, such as phosphates, citrates, and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives, such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, tetrahydrofuran ... Examples of suitable surfactants include: glycine, glutamine, asparagine, histidine, arginine, or lysine; glycerol, glycerol, 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, arginine, or lysine; carbohydrates, such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counter-ions, such as sodium; metal complexes, such as Zn-protein complexes; and non-ionic surfactants, such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of PHarmacy, 22nd Edition, 2012, PHarmaceutical Press, London.)

[0130] The pharmaceutical compositions of the present disclosure may be administered in any number of ways for local or systemic treatment: topically, by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary, including by nebulizer, intrabronchial and intranasal, and by inhalation or inhalation of powders or aerosols; orally; or parenterally, including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intraspinal or intraventricular).

[0131] In some embodiments, the composition is formulated for local delivery such that when applied to the skin, for example, the AFFIMER® polypeptide penetrates the skin (crosses epithelial and mucosal barriers) and acts systemically.

[0132] The therapeutic dosage form may be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in aqueous or non-aqueous media, or suppositories. In solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier. Common refined ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These may be used to form a solid preform composition containing a homogenous mixture of the compounds of the present disclosure, or a non-toxic pharma-ceutically acceptable salt thereof. The solid preform composition is then subdivided into unit dosage forms of the type described above. The tablets, pills, etc. of the formulation or composition may be coated or otherwise compounded to provide a dosage form offering the advantage of extended action. For example, the tablet or pill may include an inner composition coated with an outer component. Additionally, the two components may be separated by an enteric layer that serves to resist disintegration and allows the inner component to pass intact through the stomach or be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

[0133] The AFFIMER® polypeptides described herein may be entrapped in microcapsules, for example prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, as described in Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, 2012, Pharmaceutical Press, London.

[0134] In some embodiments, the pharmaceutical dosage form comprises an AFFIMER® polypeptide of the present disclosure complexed with liposomes. Methods for producing liposomes are known to those skilled in the art. For example, some liposomes can be produced by reverse phase evaporation with a liquid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to produce liposomes with the desired diameter.

[0135] In some embodiments, sustained release formulations may be produced that include the AFFIMER® polypeptides described herein. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the AFFIMER® polypeptide, where the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT. TM(injectable microspheres composed of poly(lactic acid-co-glycolic acid) and leuprorelin acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0136] For the treatment of disease, the appropriate dosage of the AFFIMER® polypeptides of the present disclosure will vary depending on the type of disease being treated, the severity and course of the disease, the responsiveness of the disease, whether the AFFIMER® polypeptide is being administered for therapeutic or prophylactic purposes, previous treatments, the patient's medical history, etc., all at the discretion of the treating physician. AFFIMER® polypeptides may be administered once or over a series of treatments lasting from several days to several months, or until the disease is ameliorated or a reduction in the disease state is achieved (e.g., reduction in tumor size). Optimal dosing schedules may be calculated from measurements of drug accumulation in the patient's body, which will vary depending on the relative potency of the individual agents. The administering physician may determine the optimal dosage, method of administration, and repetition ratio. In some embodiments, the dosage is 0.01 mg to 100 mg / kg body weight, 0.1 mg to 100 mg / kg body weight, 1 mg to 100 mg / kg body weight, 1 mg to 100 mg / kg body weight, 1 mg to 80 mg / kg body weight to 10 mg to 100 mg / kg body weight, 10 mg to 75 mg / kg body weight, or 10 mg to 50 mg / kg body weight. In some embodiments, the dosage of the AFFIMER® polypeptide is about 0.1 mg to about 20 mg / kg body weight. In some embodiments, the dosage of the AFFIMER® polypeptide is about 0.1 mg / kg body weight. In some embodiments, the dosage of the AFFIMER® polypeptide is about 0.25 mg / kg body weight. In some embodiments, the dosage of the AFFIMER® polypeptide is about 0.5 mg / kg body weight. In some embodiments, the dosage of the AFFIMER® polypeptide is about 1 mg / kg body weight. In some embodiments, the dosage of the AFFIMER® polypeptide is about 1.5 mg / kg body weight. In some embodiments, the dosage of the AFFIMER® polypeptide is about 2 mg / kg body weight. In some embodiments, the dosage of the AFFIMER® polypeptide is about 2.5 mg / kg body weight. In some embodiments, the dosage of the AFFIMER® polypeptide is about 5 mg / kg body weight. In some embodiments, the dosage of the AFFIMER® polypeptide is about 7.5 mg / kg body weight. In some embodiments, the AFFIMER® polypeptide is administered at a dose of about 100 mg / kg body weight. The dosage of MER® polypeptide is about 10 mg / kg body weight. In some embodiments, the dosage of AFFIMER® polypeptide is about 12.5 mg / kg body weight. In some embodiments, the dosage of AFFIMER® polypeptide is about 15 mg / kg body weight. In some embodiments, dosages may be provided one or more times daily, weekly, monthly, or yearly. In some embodiments, AFFIMER® polypeptide is provided once every week, once every two weeks, once every three weeks, or once every four weeks.

[0137] In some embodiments, the AFFIMER® polypeptide may be administered at an initial higher "loading" dose, followed by one or more lower doses. In some embodiments, the frequency of administration may also vary. In some embodiments, the administration regimen may include an initial dose followed by additional doses (or "maintenance" doses) once a week, once every two weeks, once every three weeks, or once a month. For example, the administration regimen may include an initial loading dose followed by weekly maintenance doses, e.g., half the initial dose. Alternatively, the administration regimen may include a loading dose followed by maintenance doses, e.g., half the initial dose, every two weeks. Alternatively, the administration regimen may include three initial doses for three weeks, followed by maintenance doses of the same amount, e.g., every two weeks.

[0138] As known to those skilled in the art, administration of any therapeutic agent can lead to side effects and / or toxicity. In some cases, said side effects and / or toxicity are too significant at therapeutically effective doses, precluding administration of a particular agent. In some cases, drug therapy needs to be discontinued and other agents may be tried. However, multiple agents in the same therapeutic class often exhibit similar side effects and / or toxicity, which means that patients either discontinue treatment or suffer from unpleasant side effects associated with the therapeutic agent, if possible.

[0139] In some embodiments, the dosing schedule may be limited to a specified number of doses or "cycles." In some embodiments, the AFFIMER® polypeptide is administered for 3, 4, 5, 6, 7, 8 or more cycles. For example, the AFFIMER® polypeptide is administered every 2 weeks for 6 cycles, the AFFIMER® polypeptide is administered every 3 weeks for 6 cycles, the AFFIMER® polypeptide is administered every 2 weeks for 4 cycles, the AFFIMER® polypeptide is administered every 3 weeks for 4 cycles, etc. The dosing schedule may be determined by one of skill in the art and may be subsequently modified.

[0140] Thus, the present disclosure provides a method of administering a polypeptide or agent described herein to a subject, including an intermittent dosing strategy for administration of one or more agents, which can reduce side effects and / or toxicity associated with administration of an AFFIMER® polypeptide. In some embodiments, a method of treating cancer in a human subject includes administering to the subject a therapeutically effective amount of an AFFIMER® polypeptide together with a therapeutically effective dose of a therapeutic agent, where one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy includes administering an initial dose of an AFFIMER® polypeptide to the subject and administering a subsequent dose of an AFFIMER® polypeptide about once every two weeks. In some embodiments, the intermittent dosing strategy includes administering an initial dose of an AFFIMER® polypeptide to the subject and administering a subsequent dose of an AFFIMER® polypeptide about once every three weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of AFFIMER® polypeptide to the subject and administering subsequent doses of AFFIMER® polypeptide about once every four weeks. In some embodiments, the AFFIMER® polypeptide is administered using an intermittent dosing strategy, and the therapeutic agent is administered weekly.

[0141] Polynucleotides A polynucleotide (also referred to as a nucleic acid) is a polymer of nucleotides of any length and may contain 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. In some embodiments, the polynucleotides of the present application encode a polypeptide, such as an anti-human FcRn AFFIMER® polypeptide. As is known in the art, the order of deoxyribonucleotides in a polynucleotide determines the order of amino acids in the encoded polypeptide (e.g., protein).

[0142] A polynucleotide sequence may be any sequence of deoxyribonucleotides and / or ribonucleotides and may be single-stranded, double-stranded, or partially double-stranded. The length of a polynucleotide may vary and is not limited. Thus, a polynucleotide may contain, for example, 2 to 1,000,000 nucleotides. In some embodiments, a polynucleotide has a length of 100 to 100,000, 100 to 10,000, 100 to 1,000, 100 to 500, 200 to 100,000, 200 to 10,000, 200 to 1,000, or 200 to 500 nucleotides.

[0143] In this application, vector refers to a vehicle for delivering molecules to cells. In some embodiments, the vector is an expression vector that includes a promoter (e.g., inducible or constitutive) operably linked to a polynucleotide sequence encoding a polypeptide. Non-limiting examples of vectors include viral vectors (e.g., adenovirus vectors, adeno-associated virus vectors, and retrovirus vectors), naked DNA or RNA expression vectors, plasmids, cosmids, phage vectors, DNA and / or RNA expression vectors associated with cationic condensing agents, and DNA and / or RNA expression vectors encapsulated in liposomes. The vector may be transfected into cells using, for example, transfection methods such as calcium phosphate DNA coprecipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retrovirus infection, or gene gun technology (gene gun method).

[0144] Gene transfer An alternative approach to the delivery of therapeutic anti-human FcRn AFFIMER® polypeptides is to rely on the body itself to produce the therapeutic polypeptide. Numerous clinical studies have illustrated the utility of in vivo gene delivery to cells using various delivery systems. In vivo gene delivery seeks to administer to patients the nucleotide sequence of an anti-human FcRn AFFIMER® polypeptide, rather than the anti-human FcRn AFFIMER® polypeptide itself. This allows the patient's body to produce the anti-human FcRn AFFIMER® polypeptide it needs over an extended period of time, and secrete it systemically or locally depending on the site of production. Gene-based nucleotides encoding anti-human FcRn AFFIMER® polypeptides can present a labor- and cost-effective alternative to the traditional production, purification, and administration of polypeptide versions of anti-human FcRn AFFIMER® polypeptides. Numerous antibody expression platforms have been pursued to accommodate the delivery of polynucleotide anti-human FcRn AFFIMER® polypeptides in vivo: these include viral vectors, naked DNA, and RNA. The use of gene transfer with polynucleotides encoding anti-human FcRn AFFIMER® polypeptides can reduce product and production costs, as well as reduce costs and frequency of drug administration. Overall, extended in vivo production of therapeutic anti-human FcRn AFFIMER® polypeptides by expression of polynucleotides encoding anti-human FcRn AFFIMER® polypeptides can contribute to (i) broad therapeutic or prophylactic application of anti-human FcRn AFFIMER® polypeptides under cost-sensitive conditions, (ii) improved accessibility to treatments in both developed and developing countries, and (iii) more effective and less expensive treatment methods. In addition to in vivo gene transfer, cells can be harvested from a host (or donor) that can be engineered with polynucleotides encoding anti-human FcRn AFFIMER® polypeptides to produce anti-human FcRn AFFIMER® polypeptides and re-administered to patients. You may harvest.

[0145] Tumors provide a site for the delivery of targeted anti-human FcRn AFFIMER® polypeptide-encoding polynucleotides via intravenous or direct injection / electroporation. Indeed, intratumoral expression of anti-human FcRn AFFIMER® polypeptide-encoding polynucleotides allows for local production of therapeutic anti-human FcRn AFFIMER® polypeptides, eliminating the need for high systemic anti-human FcRn AFFIMER® polypeptide levels that may otherwise be required to penetrate and affect solid tumors. For example, see the following documents (Non-Patent Documents 26 and 27).

[0146] The success of gene therapy has been driven primarily by improvements in viral and non-viral gene transfer vectors. A range of physical and chemical non-viral methods are used to deliver DNA and mRNA to mammalian cells, many of which have been developed as clinical-stage techniques for gene therapy both in vitro and in vivo, and are readily adapted to the delivery of polynucleotides encoding the anti-human FcRn AFFIMER® polypeptides of the present disclosure. By way of example, cationic liposome technology may be used, based on the ability of amphipathic lipids with positively charged head groups and hydrophobic lipid tails to bind negatively charged DNA or RNA and form particles that typically enter cells by endocytosis. Some cationic liposomes also contain neutral co-lipids, which are believed to improve uptake of liposomes by mammalian cells. See, for example, the following documents (Non-Patent Documents 28-31).

[0147] Similarly, other multications, such as poly-l-lysine and polyethyleneimine, may be used to deliver polynucleotides encoding anti-human FcRn AFFIMER® polypeptides. These multications complex with nucleic acids through charge interactions and aid in condensation of DNA or RNA into nanoparticles, which are then substrates for endosome-mediated uptake. Many of these cationic nucleic acid complexing technologies, including complexes with plasmid DNA, oligodeoxynucleotides, and various forms of synthetic RNA, have been developed as potential clinical products. Modified (and unmodified or "naked") DNA and RNA have also been demonstrated to successfully mediate gene transfer in many situations, and may also be used as a system for delivery of polynucleotides encoding anti-human FcRn AFFIMER® polypeptides. This includes the use of plasmid DNA by direct intramuscular injection and intratumoral injection of plasmid DNA. See, for example, the following documents (Non-Patent Documents 32-36).

[0148] Viral vectors are currently used as delivery vehicles in a vast number of preclinical and clinical gene therapy trials and the first approved targeted gene therapies. See Gene Therapy Clinical Trials Worldwide 2017 (abedia.com / wiley / ). The main driver is the exceptional gene delivery efficiency, which reflects natural evolutionary development, making viral vector systems attractive for gene delivery because viruses have evolved the ability to cross cell membranes upon infection and deliver nucleic acids, such as polynucleotides encoding anti-human FcRn AFFIMER® polypeptides, to target cells. The field of viral vector-mediated antibody gene delivery pioneered by the adenovirus system has made considerable progress in the past decades. The numerous administration routes, preclinical models, and disease indications that have been successfully evaluated have provided a full display of antibody gene delivery capabilities, which allows those skilled in the art to easily identify and tailor antibody gene delivery systems and techniques for in vivo delivery of polynucleotide constructs encoding anti-human FcRn AFFIMER® polypeptides. In the context of vectored intratumoral polynucleotides encoding anti-human FcRn AFFIMER® polypeptide gene delivery, oncolytic viruses have a clear advantage since they can specifically target tumor cells, enhance anti-human FcRn AFFIMER® polypeptide expression, and amplify therapeutic responses, e.g., responses to anti-human FcRn AFFIMER® polypeptides.

[0149] In vivo gene transfer of polynucleotides encoding anti-human FcRn AFFIMER® polypeptides may also be performed by the use of non-viral vectors, e.g., expression plasmids. Non-viral vectors are easily produced and do not appear to induce a specific immune response. Muscle tissue is most often used as the target tissue for transfection because it is well vascularized, easily accessible, and muscle cells are long-lived cells. Intramuscular injection of naked plasmid DNA transfects a proportion of muscle cells. Using this approach, plasmid DNA encoding cytokines and cytokine / IgG1 chimeric proteins has been introduced in vivo, which has had a positive impact on the outcome of (autoimmune) diseases.

[0150] In some cases, transfection efficiency is enhanced via so-called intravascular delivery, which achieves increased gene delivery and expression levels by inducing a short-term, transient high pressure in the veins. Special blood pressure cuffs exist that can temporarily increase vascular pressure to facilitate local absorption and may be adapted for use in human patients for this type of gene delivery. See, for example, Non-Patent Document 37.

[0151] Increased efficiency may also be obtained by other techniques, for example techniques that improve the transfer of nucleic acids by the use of chemical carriers - cationic polymers or lipids - or by physical approaches - gene gun transfer or electroporation. See the following documents (Non-Patent Documents 38 and 39). Electroporation is considered to be an interesting technique, especially for non-viral gene transfer. See the following documents (Non-Patent Documents 40 and 41). Together with electroporation, pulsed current is applied to a local tissue site to improve cell permeability and generate gene transfer across the cell membrane. Studies have demonstrated that in vivo gene transfer can be at least 10 to 100 times more efficient with electroporation than in the absence of electroporation. See, for example, the following documents (Non-Patent Documents 42 to 45).

[0152] The encoded anti-human FcRn AFFIMER® polypeptide can be delivered by a wide variety of gene delivery systems commonly used in gene therapy, including viral, non-viral, or physical therapies. See, for example, the following documents (Non-Patent Documents 46 and 47). Discussions of methods and compositions for use in gene therapy include the following documents (Non-Patent Documents 48-51), and the reference cited therein, Patent Document 2, also provides a discussion of a wide variety of gene delivery methods and compositions. Delivery routes include, for example, systemic administration and localized administration.

[0153] An effective gene transfer approach must be targeted to specific tissues / cells of need, and the transgene expression generated must be at a level appropriate for the specific application field. The promoter, as the key cis-acting element in vector genome design, can determine not only the cell specificity but also the overall expression strength.

[0154] In some embodiments, viral vectors are used to deliver the nucleic acid encoding the anti-human FcRn AFFIMER® polypeptide of the present disclosure. Non-limiting examples of viral vectors include adenoviral vectors, adeno-associated virus (AAV) vectors, and retroviral vectors. In other embodiments, non-viral vectors are used to deliver the nucleic acid encoding the anti-human FcRn AFFIMER® polypeptide of the present disclosure. Non-limiting examples of non-viral vectors include plasmid vectors (e.g., plasmid DNA (pDNA) delivered via hydrodynamic-based transfection or electroporation), minicircle DNA, and RNA-mediated gene delivery (e.g., delivery of messenger RNA (mRNA) encoding the anti-human FcRn AFFIMER® polypeptide of the present disclosure).

[0155] Exemplary nucleic acids or polynucleotides for the encoded anti-human FcRn AFFIMER® polypeptides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (including LNA, LNA having a β-D-ribo sequence, a-LNA (a partial stereoisomer of LNA) having an aL-ribo sequence, 2'-amino-LNA having a 2'-amino functional group, and 2'-amino-a-LNA having a 2'-amino functional group), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids or combinations thereof.

[0156] mRNA presents a new platform for antibody gene transfer that can be adapted by those skilled in the art for the delivery of polynucleotide constructs encoding the anti-human FcRn AFFIMER® polypeptides of the present disclosure. Although current results vary considerably, it appears that in some cases, mRNA constructs may compete with viral vectors for serum mAb titers generated. Levels were in the therapeutically relevant range within hours of mRNA administration, with a notable transfer of kinetics compared to DNA. The use of lipid nanoparticles (LNPs) for mRNA transfection can provide significant advantages in scope of application in some embodiments over physical methods typically required for DNA.

[0157] Nucleic acids encoding anti-human FcRn AFFIMER® polypeptides may be delivered, for example, intravenously, intramuscularly, or intratumorally (eg, by injection, electroporation, or other means).

[0158] Nucleic acids encoding anti-human FcRn AFFIMER® polypeptides may be formulated, for example, in lipid nanoparticles or liposomes (e.g., cationic lipid nanoparticles or liposomes), biodegradable microspheres, or other nano- or microparticles. Other lipid-based (e.g., PEG-lipids) and polymer-based formulations and delivery vehicles are contemplated herein.

[0159] Working Example Example 1. Selection of AFFIMER® Process overview Phage selection Biopanning for captured humans (HFcRn) Solution selection of biotinylated FcRn Two rounds of selection on FcRn Calculated size and enrichment monitored by polyclonal phage ELISA Primary Screening Monoclonal crude extract ELISA for FcRn captured at pH 6 Secondary Screening ELISA for FcRn at pH 6.0 and 7.4

[0160] Common methods Selection of huFcRn-binding phages from the AFFIMER® library was performed using approximately 6×10 10 Diversity size of the library spiked at approximately 1 x 10 12 This was done using phage as described below.

[0161] Peptides of the present disclosure, e.g., huFcRn binding components, were identified, e.g., by selection from a library of AFFIMER® polypeptides having two random loops of the same general, but not exclusive, length of nine amino acids.

[0162] As described above, the huFcRn-binding peptides of the present disclosure were identified by selection from a phage display library containing a 9 amino acid long random loop sequence shown in the invariant AFFIMER® framework backbone based on the sequence for SQT. Such selection processes are generally known. According to such processes, a phage suspension was incubated with a target antigen (biotinylated antigen captured on streptavidin beads or non-biotinylated antigen captured on a plate). Unbound phages were then washed away, and bound phages were then eluted by incubating the antigen with low pH, high pH, ​​or trypsin. The released pH-neutralized or trypsin-inactivated phages were then used to infect E. coli to obtain a first-round phage preparation. The cycle is repeated, e.g., two or three times, and stringent conditions can be increased in subsequent selection rounds to enrich for targeted phages, e.g., by increasing the number of washing steps, decreasing antigen concentration, and pre-selection onto blocked streptavidin beads or wells coated with a blocking reagent.

[0163] The antigens used in this application are human FcRn (BPS#71285) and biotinylated human FcRn (BPS#71283). Following selection by successive phage amplification rounds, huFcRn-binding clones were confirmed by crude extract ELISA as described below.

[0164] Following phage selection, individual bacterial clones containing the phagemid vector were harvested in titration plates in a 96-well cell culture format. Soluble AFFIMER® in crude cell extracts were prepared from lysis of bacterial cells overexpressing AFFIMER® with a C-terminal myc tag and used in the primary screening ELISA. These AFFIMER® polypeptides in the extracts were screened for binding to antigen at pH 6 and pH 7.4, and the ELISA was developed using a one-step Ultra TMB-ELISA substrate (Thermo Scientific) to detect AFFIMER® bound to antigen immobilized on the plate with an HRP-labeled anti-myc tag antibody (Abcam # ab1261). The screens were also performed against non-target or related target molecules captured on the plate, such as blocking molecules, neutravidin or b-2 microglobulin (Sigma # M4890). The non-target and target binding data were compared to confirm library members that specifically bound to the target.

[0165] Example 2. huFcRn binding ELISA analysis at pH 6 Binding of AFFIMER® to HuFcRn was assessed by enzyme-linked immunosorbent assay (ELISA) using a 384-well plate format. HuFcRn (BPS Bioscience) was coated onto plates at 5 μg / ml in 40 mM MES, pH 6. Plates were washed three times with 100 μl of wash buffer (PBS, 0.05% Tween 20, pH 6) using a plate washer and saturated with MES pH 6, 5% casein (Sigmab) at room temperature (25±1° C.) for 60 min. Plates were washed as before. AFFIMER® and negative control (mAb anti-hFcRn (clone ADM31), negative control) were then diluted in duplicate and loaded onto the plates for 90 min at room temperature (25±1° C.). Plates were washed three times as before. Biotinylated polyclonal antibody anti-cystatin (R&D Systems) was then diluted in dilution buffer (1% casein, 0.05% Tween 20, and 8 mM MES, pH 6) and incubated for 60 min at room temperature (25 ± 1 °C). Plates were washed three times as described above and streptavidin-HRP (N200, thermo-Fisher) was incubated for 30 min at room temperature (25 ± 1 °C). Plates were washed and substrate (TMB, Thermo-Scientific) was added to the plate for 8 ± 1 min. The reaction was stopped using an acidic solution and plates were read at 450-630 nm. EC50s were then calculated using an interpolated nonlinear 4-parameter standard curve (Table 4).

[0166] Example 3. huFcRn binding ELISA analysis at pH 7.4 Binding of AFFIMER® to HuFcRn was assessed by enzyme-linked immunosorbent assay (ELISA) using a 384-well plate format. HuFcRn (BPS Bioscience) was coated onto plates at 5 μg / ml in PBS, pH 7.4. Plates were washed three times with 100 μl of wash buffer (PBS, 0.05% Tween 20, pH 7.4) using a plate washer and saturated with MES pH 7.4, 5% casein (Sigmab) for 60 min at room temperature (25±1° C.). Plates were washed as before. AFFIMER® and controls (mAb anti-hFcRn (clone ADM31), blank) were then diluted in duplicate and loaded onto the plates for 90 min at room temperature (25±1° C.). Plates were washed three times as before. Biotinylated polyclonal antibody anti-cystatin (R&D Systems) was then diluted in dilution buffer (1% casein, 0.05% Tween 20, and 8 mM MES, pH 7.4) and incubated for 60 min at room temperature (25 ± 1 °C). The plate was washed three times as described above and streptavidin-HRP (N200, thermo-Fisher) was incubated for 30 min at room temperature (25 ± 1 °C). The plate was washed and substrate (TMB, Thermo-Scientific) was added to the plate for 8 ± 1 min. The reaction was stopped using an acidic solution and the plate was read at 450-630 nm. EC50 was then calculated using an interpolated nonlinear 4-parameter standard curve and the results are shown in Table 4 below. [Table 4]

[0167] In the present invention, LGC01 may be used interchangeably with FcRn, for example, LGC01-15 refers to FcRn-15.

[0168] Example 4: AFFIMER® Expression and Purification All AFFIMER® constructs expressed in E. coli were cloned with a C-terminal hexa-HIS tag (HHHHHH (SEQ ID NO: 1185)) to simplify protein purification by immobilized metal affinity chromatography resin (IMAC resin). If necessary, an additional peptide sequence may be added between the AFFIMER® and a detection HIS tag, such as MYC (EQKLISEEDL (SEQ ID NO: 1186)) or a TEV protease cleavage site (ENLYFQ(G / S) (SEQ ID NO: 1187)) to allow removal of the tag. The AFFIMER® analyzed in FIG. 4A has MYC (EQKLISEEDL (SEQ ID NO: 1186)) and a TEV protease cleavage site (ENLYFQ(G / S) (SEQ ID NO: 1187)), and the AFFIMER® analyzed in FIG. 4B does not have MYC (EQKLISEEDL (SEQ ID NO: 1186)) and a TEV protease cleavage site (ENLYFQ(G / S) (SEQ ID NO: 1187)). AFFIMER® protein was expressed from E. coli and purified using IMAC, a second stage purification to remove endotoxins, using cation exchange (HiTrap, Cytiva) and size exclusion chromatography (SEC; Cytiva) with CHT (ceramic hydroxyapatite, BioRad) type I resin or a Triton 114× washing step (Sigmab). AFFIMER® monomer purification from E. coli was performed by transforming the expression plasmid pD861 (Atum) into BL21 E. coli cells (Millipore) using the manufacturer's protocol. The entire transformed cell mixture was spread onto an LB agar plate containing 50 μg / ml kanamycin (AppliChem) and grown overnight at 37°C. The next day, the lawn of transformed E. coli was transferred to a sterile flask of 1x TB medium (Melford) and 50 μg / ml kanamycin and grown at 30°C with shaking at 250 rpm. The cells reached an optical density OD of approximately 0.8-1.0. 600Once the ELISA yield reached 10 mM, expression was induced with 10 mM rhamnose (AlfaAesar). The culture was then grown at 37 °C for an additional 5 hours. The resulting cell pellet was centrifuged to lyse and harvest the cells. AFFIMER® purification was performed using batch-bound affinity purification of His-tagged proteins. Specifically, nickel agarose affinity resin (Super-NiNTA500; Generon) was used. The resin was washed with NPI20 buffer (50 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole) and bound proteins were eluted with 5 column volumes (CV) of NPI400 buffer. The eluted proteins were eluted with CHT type I resin in running buffer 10 mM sodium phosphate pH 6.4-6.5 buffer with the addition of 2 M NaCl in a linear gradient and buffer exchange for the second stage purification (SEQ ID NOs: 628, 631, 713 and 1184). Alternatively, a second stage purification using cation exchange was performed using a SP HP ion exchange column (Cytiva) in running buffer 50 mM MES pH 6.2 for clone FcRn-125 including a 0.1% Triton 114x (Sigmab) wash step and the protein was eluted with a 1M NaCl linear gradient (SEQ ID NO: 718). A third stage polishing purification was performed with preparative SEC performed using a HiLoad 26 / 600 Superdex 75 pg (Cytiva) running in PBS 1x buffer. Expression and purity of the clones were analyzed using SEC-HPLC (Figures 3A-3C) with an Acclaim SEC-300 column (Thermo) using a PBS 1x mobile phase. Protein yield was estimated using a Nanodrop (Thermo) A280 read and the final product was eluted with a Novex HPLC column at 200 volts. TM 20×Bolt TMSDS-PAGE Bolt Bis-Tris+ 4-12% gels (Thermo) (Figure 4A) and 20% SDS-PAGE precast gels (Komabiotech) (Figure 4B) were run in MES SDS running buffer (Thermo) and heated in reducing buffer at 95°C for 5 min. Protein bands on the gels were stained with Quick Commassie (Generon), PageRuler prestained protein molecular weight markers (Thermo) (Figure 4A) and Precision plus protein TM Dual color standards (Bio-rad) (Figure 4B) were run on the gel to estimate the molecular weight of the fusion proteins from the three-step purification. Endotoxin levels of the final protein batches were measured using the LAL test on an Endosafe® Nexgen MCS system (Charles River), which was between 1 and 0.1 EU / mg for all protein batches.

[0169] Example 5. huFcRn Binding ELISA Analysis at pH 6 for AFFIMER® Characterization Binding of AFFIMER® to HuFcRn was assessed by enzyme-linked immunosorbent assay (ELISA) using a 384-well plate format. HuFcRn (BPS Bioscience) was coated onto plates at 5 μg / ml in 40 mM MES, pH 6. Plates were washed three times with 100 μl of wash buffer (PBS, 0.05% Tween 20, pH 6) using a plate washer and saturated with MES pH 6, 5% casein (Sigmab) for 60 min at room temperature (25±1° C.). Plates were washed as before. AFFIMER® and negative control (mAb anti-hFcRn (clone ADM31), negative control) were then diluted in duplicate and loaded onto the plates for 90 min at room temperature (25±1° C.). Plates were washed three times as before. Biotinylated polyclonal antibody anti-cystatin (R&D Systems) was then diluted in dilution buffer (1% casein, 0.05% Tween 20, and 8 mM MES, pH 6) and incubated for 60 min at room temperature (25 ± 1 °C). Plates were washed three times as described above and streptavidin-HRP (N200, thermo-Fisher) was incubated for 30 min at room temperature (25 ± 1 °C). Plates were washed and substrate (TMB, Thermo-Scientific) was added to the plate for 8 ± 1 min. The reaction was stopped using an acidic solution and plates were read at 450-630 nm. EC was then calculated using an interpolated nonlinear 4-parameter standard curve. 50 was calculated (Figures 5A-5B and Table 5A).

[0170] A slightly optimized ELISA method was developed to quantitatively compare the affinity for FcRn at pH 6.0 and pH 7.4. After testing temperature and time to find the optimal conditions, the binding affinity of AFFIMER® was measured (Figure 5B).

[0171] Example 6. huFcRn Binding ELISA Analysis at pH 7.4 for AFFIMER® Characterization Binding of AFFIMER® to HuFcRn was assessed by enzyme-linked immunosorbent assay (ELISA) using a 384-well plate format. HuFcRn (BPS Bioscience) was coated onto plates at 5 μg / ml in PBS, pH 7.4. Plates were washed three times with 100 μl of wash buffer (PBS, 0.05% Tween 20, pH 7.4) using a plate washer and saturated with MES pH 7.4, 5% casein (Sigmab) for 60 min at room temperature (25±1° C.). Plates were washed as before. AFFIMER® and controls (mAb anti-hFcRn (clone ADM31), blank) were then diluted in duplicate and loaded onto the plates for 90 min at room temperature (25±1° C.). Plates were washed three times as before. Biotinylated polyclonal antibody anti-cystatin (R&D Systems) was then diluted in dilution buffer (1% casein, 0.05% Tween 20, and 8 mM MES, pH 7.4) and incubated for 60 min at room temperature (25 ± 1 °C). Plates were washed three times as described above and streptavidin-HRP (N200, thermo-Fisher) was incubated for 30 min at room temperature (25 ± 1 °C). Plates were washed and substrate (TMB, Thermo-Scientific) was added to the plate for 8 ± 1 min. The reaction was stopped using an acidic solution and plates were read at 450-630 nm. EC50s were then calculated using an interpolated nonlinear 4-parameter standard curve (Figures 5A-5B and Table 5A). A slightly optimized ELISA method was developed to quantitatively compare the affinity for FcRn at pH 6.0 and pH 7.4. After testing temperature and time to find the optimal conditions, the binding affinity of AFFIMER® was measured (Figure 5B).

[0172] The FcRn AFFIMER® most suitable for FcRn cell recirculation is advantageous when the difference in binding affinity at pH 6.0 and pH 7.4 is large, and the ratio of the measured EC50 at pH 6.0 and pH 7.4 is calculated in Tables 5A-B. [Table 5A] [Table 5B]

[0173] Example 7. BLI-Based FcRn AFFIMER® Screening A BLI (biolayer interference)-based binding assay was performed for AFFIMER® screening, in which the affinity for FcRn changes with pH. hFcRn with a His tag was immobilized on a Ni-NTA biosensor. Then, hFcRn and Ni that was not bound to hFcRn from the AFFIMER® candidate group were analyzed. 2+ was blocked with His-SQT-gly at high concentration, where there was no reactivity. The AFFIMER® candidate group, diluted to the same concentration, was then reacted with hFcRn. All affimers were analyzed at pH 6.0 and pH 7.4, and K d was measured using a 1:1 binding model. The results of the octet kinetic analysis at pH 6.0 and 7.4 are shown in Table 6 below. [Table 6]

[0174] Example 8. FcRn competitive ELISA To assess whether AFFIMER® competes with IgG1, a competitive ELISA (huIgG1 / huFcRn) was performed. Briefly, huIgG1 isotype control (BioXcell) was coated on plates overnight at 5 μg / ml in 40 mM MES, pH 6. Plates were then saturated with 40 mM MES, 5% casein, pH 6. Meanwhile, huFcRn (His-tagged molecule, BPS) was pre-incubated with dilutions of FcRn-binding AFFIMER® and its controls (human IgG1 and HuSA). After saturation, plates were washed with pH 6 PBS, 0.05% Tween and the mixture was added to the plate and incubated for at least 1 hour. Plates were then washed as before and detection monoclonal antibody, anti-B2M HRP (Biolegend), was added and incubated for at least 1 hour. After a final wash, the reaction was developed with TMB (Pierce), the plates were read at 450 nm on a plate reader, and the absorbance was plotted versus the log of AFFIMER® and control concentrations using a four parameter fit. Figure 6 shows that FcRn-binding AFFIMER® does not compete with huIgG1.

[0175] Example 9. FcRn cell binding protocol 1 μl of 100 μM AFFIMER® was placed in a 96-well V-bottom plate and 200 μl of CHO-K1-FcRn (resuspended in wash buffer (PBS pH 6.0 or pH 7.4 + 2% FBS)) was added to 1 × 10 6 The cells were added to the above at a concentration of 1000 cells / ml and incubated at room temperature for 20 minutes. 200 μl of washing buffer was added, and the product was centrifuged at 1000 rpm for 3 minutes at 4°C to remove the supernatant (3 times). Anti-cystatin monoclonal Ab (Novus, NBP2-79882AF488) (conjugated to AF488) was diluted in washing buffer and added at 2 × 10 50.2 μl of anti-cystatin monoclonal Ab was added per cell and reacted at 4° C. for 1 hour. 200 μl of washing buffer was added, and the product was centrifuged at 1000 rpm at 4° C. for 3 minutes to remove the supernatant (3 times). The product was resuspended in 200 μl of washing buffer, and the value was measured using a flow cytometer.

[0176] In Figures 7 and 8, cell binding of Affimer was confirmed using hFcRn-overexpressing CHO monoclonal cell lines (pH 6.0 and pH 7.4).

[0177] Example 10: Screening of leading FcRn-binding AFFIMER® polypeptides for receptor-mediated recirculation in a human endothelial cell-based recirculation assay 7.5 × 10 stably expressing HA-hFcRn-EGFP 5 Endothelial cell line (HMEC1) was seeded per well in 24-well plates (Costar) and cultured for 2 days in growth medium. Cells were washed twice and starved for 1 h with Hank's Balanced Salt Solution (HBSS) (Thermofisher). 800 nM of hIgG1 or AFFIMER® polypeptides were then diluted in 125 μl of HBSS (pH 7.4) and added to the cells followed by 4 h of culture. The medium was removed and cells were washed with ice-cold HBSS (pH 7.4) followed by addition of fresh warmed HBSS (pH 7.4) or growth medium supplemented with MEM free amino acids (Thermofisher) without FCS. Samples were collected after 4 h of cell culture. Wells with absorbed samples and residuals were then lysed before collection. Total protein lysates were obtained using RIPA lysis buffer (ThermoFisher) supplemented with complete protease inhibitor tablets (Roche). The mixture was incubated with the cells (220 μl) on ice, shaken for 10 min, and then centrifuged at 10,000×g for 15 min to remove cell debris. Rescued AFFIMER® polypeptides and controls were quantitatively analyzed by quantitative ELISA anti-cystatin (see Example 11) or anti-human IgG ( FIG. 9 ).

[0178] Example 11. AFFIMER® Quantitative Analysis by ELISA Followed by HERA Analysis 96-well plates (Corning Costar, 3590) were coated with 1 μg / ml anti-His MAB050 diluted in coating buffer (carbonate / non-carbonate). 50 μl of each sample was coated at 4° C. for 16 hours (+ / - 2 h). Plates were washed two more times with 150 μl of wash buffer (1× PBS + 0.05% Tween) and blocked with 100 μl of 1× PBS + 5% casein blocking buffer at room temperature (RT) for 90 minutes (+ / - 15 minutes). HERA samples were then added to the plate and diluted 1:1 in six steps in dilution buffer (PBS + 1% casein + 0.01% Tween) and matching AFFIMER® polypeptides were used as standards for each variant (3.5 nM-0.0017 nM). HERA samples were incubated at RT for 90 minutes (+ / - 15 minutes). Plates were washed three times with wash buffer. Binding was determined by BAF1470 1:1000 at 0.05 mg / mL and polystreptavidin-HRP at 1 mg / ml. 1:5000. The two antibodies were preincubated in small volumes for 20 min, then diluted in dilution buffer and added to the plate in a 50 μl volume and incubated at RT for 90 min (+ / - 15 min). The plate was washed 3 times and binding was visualized by adding 50 μl of RT TMB to each well. The reaction was stopped (after 20-30 min) by adding 50 μl of 1 M HCl. Absorbance was read at 450 nm and 620 nm. Control IgG1 was quantified using a similar protocol using a goat polyclonal anti-human Fc for capture and an alkaline phosphatase-conjugated polyclonal antibody anti-huIgGFc for detection.

[0179] All references, patents, and patent applications disclosed in this application are incorporated by reference with respect to the subject matter cited, in some cases including the entire document.

[0180] The indefinite articles "a" and "an," as used in the specification and the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0181] Also, unless expressly indicated to the contrary, it should be understood that in any method claimed herein that includes one or more steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0182] In the claims and the specification, all transition phrases such as "comprises," "includes," "has," "has," "containing," "accompanying," "possessing," "comprises," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transition phrases "comprising" and "essentially consisting of" are closed or semi-closed transition phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedure, Section 2111.03.

[0183] The terms "about" and "substantially" preceding a numerical value mean ±10% of the cited numerical value.

[0184] Where a range of values ​​is provided, each value therebetween, including the upper and lower ends of the range, is specifically contemplated and described herein.

Claims

1. For human FcRn, 1 × 10 at pH 6.0 －６ K below M ｄ 1. A polypeptide comprising an engineered FcRn-binding variant of the stefin sequence, which binds at A polypeptide comprising an engineered FcRn-binding variant of a stefin sequence comprising an amino acid sequence selected from SEQ ID NOs: 605, 609, 611, 641, 681, 702 and 769.

2. 1. A protein comprising a recombinantly engineered FcRn-binding variant of a stefin polypeptide sequence that binds to human FcRn and facilitates transport of the protein across an epithelial tissue barrier, A protein comprising an amino acid sequence selected from SEQ ID NOs: 605, 608, 609, 611, 628, 631, 641, 681, 702, 713, 718, 769 and 1184.

3. 1. A protein comprising: (i) an engineered FcRn-binding variant of a stefin polypeptide sequence that binds to human FcRn; and (ii) a heterologous polypeptide covalently associated to the engineered FcRn-binding variant of the stefin polypeptide sequence, said protein conferring therapeutic activity in a human patient, A protein comprising an amino acid sequence selected from SEQ ID NOs: 605, 608, 609, 611, 628, 631, 641, 681, 702, 713, 718, 769 and 1184.

4. A protein comprising an FcRn-binding recombinant engineered variant of a stefin polypeptide sequence having an amino acid sequence encoded by a nucleic acid having a coding sequence that hybridizes to any one of SEQ ID NOs: 899, 902, 903, 905, 922, 925, 935, 975, 996, 1007, 1012, and 1063 under stringent conditions of 6× sodium chloride / sodium citrate (SSC) at 45° C., followed by a wash with 0.2× SSC at 65° C.

5. Recombinantly engineered FcRn-binding mutants of the stefin sequence were incubated at 1 × 10 －７ K below M ｄ The polypeptide according to any one of claims 1 to 4, which binds to FcRn at

6. The engineered FcRn-binding mutant of the stefin sequence had a K ｄ K at least 1 log greater than ｄ The polypeptide according to any one of claims 1 to 5, which binds to FcRn at

7. The polypeptide of any one of claims 1 to 6, having a serum half-life of more than 10 hours in a human patient.

8. The polypeptide of any one of claims 1 to 7, having a serum half-life in a human patient that is greater than 50% of the serum half-life of IgG.

9. The polypeptide of any one of claims 1 to 8, having a serum half-life in a human patient that is greater than 50% of the serum half-life of serum albumin.

10. The polypeptide according to any one of claims 1 to 9, which does not inhibit the binding of human serum albumin to human FcRn.

11. The polypeptide according to any one of claims 1 to 10, which does not inhibit the binding of IgG to human FcRn.

12. The polypeptide of any one of claims 1 to 11, wherein binding of the polypeptide to human FcRn promotes transport of the polypeptide from apical to basal sides of an epithelial cell layer.

13. The polypeptide of any one of claims 1 to 12, comprising at least one cysteine ​​located at the C-terminus or N-terminus of the polypeptide.

14. The polypeptide of any one of claims 1 to 13, further comprising a heterologous polypeptide covalently linked via an amide bond to form a contiguous fusion protein.

15. The polypeptide of claim 14 , wherein the heterologous polypeptide comprises a therapeutic polypeptide.

16. 16. The polypeptide of claim 15, wherein the therapeutic polypeptide is selected from the group consisting of polypeptide hormones, cytokine polypeptides, chemokine polypeptides, growth factors, hemostatically active polypeptides, enzymes, and toxins.

17. The polypeptide of claim 15, wherein the therapeutic polypeptide is selected from the group consisting of a receptor trap and a receptor ligand.

18. 16. The polypeptide of claim 15, wherein the therapeutic polypeptide sequence is selected from the group consisting of angiogenic agents and anti-angiogenic agents.

19. The polypeptide of claim 15, wherein the therapeutic polypeptide sequence is a neurotransmitter.

20. The polypeptide of claim 15, wherein the therapeutic polypeptide sequence is an erythropoiesis stimulator.

21. The polypeptide of claim 15 , wherein the therapeutic polypeptide is an incretin.

22. The polypeptide of claim 15, wherein the therapeutic polypeptide is an anti-cancer immune enhancer.

23. The polypeptide of claim 15, wherein the therapeutic polypeptide is an anti-inflammatory immunosuppressant.

24. A pharmaceutical composition adapted for therapeutic use in a human patient comprising a polypeptide according to any one of claims 1 to 23 and a pharma- ceutically acceptable excipient.

25. 25. The pharmaceutical composition of claim 24, formulated for pulmonary delivery or topical application.

26. 26. The pharmaceutical composition of claim 25, wherein the pulmonary delivery is intranasal delivery.

27. A polynucleotide comprising a sequence encoding the polypeptide according to any one of claims 1 to 26.

28. 28. The polynucleotide of claim 27, wherein the sequence encoding the polypeptide is operably linked to a transcriptional regulatory sequence.

29. 29. The polynucleotide of claim 28, wherein the transcriptional regulatory sequence is selected from the group consisting of a promoter and an enhancer.

30. The polynucleotide of any one of claims 27 to 29, further comprising an origin of replication, a minichromosome maintenance element (MME), and / or a nuclear localization element.

31. The polynucleotide of any one of claims 27 to 30, further comprising a polyadenylation signal sequence operably linked to and transcribed together with the sequence encoding the polypeptide.

32. A polynucleotide according to any one of claims 27 to 30, wherein the sequence encoding the polypeptide comprises at least one intron sequence.

33. 33. The polynucleotide of any one of claims 27 to 32, further comprising at least one ribosome binding site transcribed together with the sequence encoding the polypeptide.

34. The polynucleotide of any one of claims 27 to 33, which is a deoxyribonucleic acid (DNA).

35. The polynucleotide of any one of claims 27 to 33, which is a ribonucleic acid (RNA).

36. A viral vector comprising the polynucleotide of any one of claims 27 to 33.

37. A plasmid or minicircle comprising a polynucleotide according to any one of claims 27 to 33.

38. A cell comprising a polypeptide according to any one of claims 1 to 23, a polynucleotide according to any one of claims 27 to 35, a viral vector according to claim 36, or a plasmid or minicircle according to claim 37.

39. A method for increasing the serum half-life of a therapeutic molecule, comprising conjugating a polypeptide according to any one of claims 1 to 23 to said therapeutic molecule.

40. A pharmaceutical composition for treating an autoimmune disease and / or an inflammatory disease, comprising a polypeptide according to any one of claims 1 to 15.

41. A pharmaceutical composition for treating cancer, comprising the polypeptide according to any one of claims 1 to 15.

42. A pharmaceutical composition for the treatment of a cardiovascular or metabolic disease or disorder comprising a polypeptide according to any one of claims 1 to 15.

43. An in vitro method for producing a polypeptide according to any one of claims 1 to 23, comprising expressing a nucleic acid encoding said polypeptide in a host cell.

44. 1. A protein comprising an engineered FcRn-binding variant of a stefin polypeptide sequence that binds to human FcRn and inhibits binding of human IgG to human FcRn, A protein comprising an amino acid sequence selected from SEQ ID NOs: 628, 631, 713 and 718.

45. 45. A pharmaceutical composition for treating an autoimmune or inflammatory disorder or disease comprising the protein of claim 44.

46. 45. A pharmaceutical composition adapted for therapeutic use in a human patient comprising the protein of claim 44 and a pharma- ceutically acceptable excipient.

47. 4. The polypeptide of claim 2 or 3, encoded by any one of the nucleic acid sequences of SEQ ID NOs: 899, 902, 903, 905, 922, 925, 935, 975, 996, 1007, 1012 and 1063.

48. An FcRn targeting agent comprising the polynucleotide of any one of claims 27 to 35.

49. An agent for increasing the serum half-life of a therapeutic molecule, comprising a polynucleotide according to any one of claims 27 to 35.

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