Title of Invention : NEONATAL FC RECEPTOR BINDING AFFIMERS
Technical Field
[1]
The present invention relates to a polypeptide comprising an FcRn binding AFFIMER® sequence that binds to human FcRn.
[2]
Background Art
[3]
The neonatal Fc receptor (FcRn) binds with high affinity to IgG and albumin through non-overlapping sites at a mildly acidic pH ( e.g., 5.0-6.5); however, it does not bind IgG or albumin at neutral pH. FcRn expression has been detected nearly ubiquitously in a number of tissues, including epithelial cells, endothelial cells, and cells of hematopoietic origin. It facilitates monitoring of IgG and serum albumin turnover, as its expression is upregulated in response to the proinflammatory cytokine, TNF-α and downregulated in response to IFN-γ FcRn has been used therapeutically to shuttle biologics across mucosal surfaces in order to improve drug absorption or distribution.
[4]
Disclosure of Invention
Technical Problem
[5]
The neonatal Fc receptor (FcRn) binds with high affinity to IgG and albumin through non-overlapping sites at a mildly acidic pH (e.g., 5.0-6.5); however, it does not bind IgG or albumin at neutral pH.
[6]
Solution to Problem
[7]
An object of the present invention is to provide a polypeptide comprising an FcRn binding AFFIMER® sequence that binds to human FcRn.
[8]
Another object of the present invention is to provide a pharmaceutical preparations.
[9]
Another object of the present invention is to provide a methods that comprise administering to a subject having an autoimmune disease and/or an inflammatory disease.
[10]
Another object of the present invention is to provide a provide methods of increasing serum half-life of a therapeutic molecule.
[11]
Another object of the present invention is to provide a use of the polynucleotide for targeting FcRn.
[12]
A further object of the present invention is to provide a use of the polynucleotide for increasing serum half-life of a therapeutic molecule.
Advantageous Effects of Invention
[13]
The present disclosure is based on the generation of AFFIMER® polypeptides that bind to human neonatal Fc receptor (FcRn) to extend, in a controlled manner, the serum half-life of any other therapeutic molecules ( e.g., therapeutic AFFIMER® polypeptide, protein, nucleic acid, or drug) to which it is conjugated.
[14]
Brief Description of Drawings
[15]
FIG. 1 Example of LGC01 clones binding in a direct huFcRN ELISA at pH 6.
[16]
FIG. 2 Example of differential binding of LGC01 clones at pH 6 and 7.4 in a direct huFcRN ELISA.
[17]
FIGs. 3A-3C Analytical SEC-HPLC traces of purified FcRn AFFIMER®monomers and AVA04-FcRn binding AFFIMER®fusion.
[18]
FIGs. 4A-4B SDS-PAGE analysis of purified FcRn AFFIMER®monomers and AVA04-FcRn binding AFFIMER®fusion.
[19]
FIGs. 5A-5B FcRn binding ELISA showing the binding activity of purified FcRn AFFIMER®monomers and AVA04-FcRn binding AFFIMER®fusion at pH 6 and 7.
[20]
FIG. 6 FcRn competition ELISA showing the activity of FcRn AFFIMER®monomers and AVA04-FcRn binding AFFIMER®fusion.
[21]
FIG. 7 Flow Cytometry histogram of AFFIMER® clones that have high cell binding affinity at pH 6.0 and various binding affinities at pH 7.4.
[22]
FIG. 8 Confirmation of Affimer's cell binding using hFcRn over-expression CHO single clone cell line (pH　6.0 & pH　7.4).
[23]
FIG. 9 Demonstration of FcRn mediated recycling of the FcRn binding AFFIMER®polypeptides as determined using the human endothelial cell-based recycling assay.
[24]
Best Mode for Carrying out the Invention
[25]
Provided herein, in some aspects, is a half-life extension platform based on AFFIMER® polypeptides that bind ( e.g., competitively or non-competitively) to neonatal Fc receptor (FcRn, such as human FcRn). A range of human FcRn-binding AFFIMER® polypeptides (referred to as anti-human FcRn AFFIMER® polypeptides), with a range of binding affinities, has been developed. These polypeptides have been shown in in vivo pharmacokinetic (PK) studies to extend, in a controlled manner, the serum half-life of any other AFFIMER® polypeptides to which they are conjugated ( e.g., as a single genetic fusion) and can be made, for example, in bacterial cells ( e.g., Escherichia coli). The FcRn-binding AFFIMER® polypeptides provided herein can also be used to extend the half-life of other polypeptides, such as therapeutic proteins.
[26]
In some aspects, the present invention relates to a polypeptide comprising an FcRn binding AFFIMER®sequence that binds to human FcRn with a Kd of 1x10-6M or less at pH 6.0, and (optionally) a Kd for binding human FcRn at pH 7.4 that is at least half a log greater than the Kd for binding at pH 6.0.
[27]
In some embodiments, the FcRn binding AFFIMER®sequence binds to FcRn with a K d of 1x10 -7 M or less at pH 6.0, a K d of 1x10 -8 M or less at pH 6.0, or K d of 1x10 -9 M or less at pH 6.0.
[28]
In some embodiments, the polypeptides at pH 6 bind to human FcRn with a K d that is at least one log less than the K d for binding to human FcRn at pH 7.4, at least 1.5 logs less than the K d for binding to human FcRn at pH 7.4, at least 2 logs less than the K d for binding to human FcRn at pH 7.4, or at least 2.5 log less than the K d for binding to human FcRn at pH 7.4
[29]
In some embodiments, the FcRn binding AFFIMER®sequence binds to FcRn at pH 7.4 with a K d that is at least one log greater than the K d for binding to FcRn at pH 6.0, at least 1.5 logs greater than the K d for binding to FcRn at pH 6, at least 2 logs greater than the K d for binding to FcRn at pH 6, or at least 2.5 log greater than the K d for binding to FcRn at pH 6.
[30]
In some embodiments, the FcRn binding AFFIMER®polypeptide sequence binds to human FcRn and the protein/polypeptide has a circulating half-life in human patients of at least 7 days., preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even 21 days.
[31]
In some embodiments, the polypeptide has a serum half-life in human patients 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.
[32]
In some embodiments, the polypeptide has a serum half-life in human patients of greater than 50%, greater than 60%, greater than 70%, or greater than 80% of the serum half-life of IgG.
[33]
In some embodiments, the polypeptide has a serum half-life in human patients of greater than 50%, greater than 60%, greater than 70%, or greater than 80% of the serum half-life of serum albumin.
[34]
In certain embodiments, the polypeptide does not inhibit binding of human serum albumin to human FcRn.
[35]
In certain embodiments, the polypeptide polypeptide does not inhibit binding of IgG to human FcRn.
[36]
In certain embodiments, binding of the polypeptide to human FcRn facilitates transport of the polypeptide from an apical side to a basal side of an epithelial cell layer.
[37]
[38]
*Another aspect relates to a protein comprising an FcRn binding AFFIMER®polypeptide sequence which binds to human FcRn and facilitates transport of the protein across an epithelial tissue barrier.
[39]
In certain embodiments, the AFFIMER®polypeptide sequence has an amino acid sequence represented in general formula (I)
[40]
FR1-(Xaa) n-FR2-(Xaa) m-FR3 (I),
[41]
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, individually for each occurrence, is an amino acid, n is an integer from 3 to 20, and m is an integer from 3 to 20.
[42]
For instance, FR1 can 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% identity to SEQ ID NO: 1; FR2 has at least 80%, at least 84%, at least 88%, at least 92%, or at least 96% identity to SEQ ID NO: 2; and/or FR3 has at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO: 3. In certain 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.
[43]
In certain embodiments, the AFFIMER®polypeptide sequence has an amino acid sequence wherein (Xaa) n is an amino acid sequence represented in the general formula
[44]
-Xaa-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa-Xaa- (SEQ ID NO: 4)
[45]
wherein Xaa, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6 and Xaa7, individually for each occurrence, is an amino acid residue, with the caveat 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 certain preferred embodiments, at least three, and preferably four of Xaa2, Xaa3, Xaa4, Xaa5, Xaa6 or Xaa7 are selected from His, Lys or Arg.
[46]
In certain embodiments, the AFFIMER®polypeptide sequence has an amino acid sequence wherein (Xaa) n is an amino acid sequence at least 75% identical to the Loop 2 sequence selected from SEQ ID NOs: 6-299 and 1182, and more preferably at least 80%, 85%, 90%, or 95% identical. In certain embodiments, Loop 2 sequence is selected from SEQ ID NOs: 6-299 and 1182.
[47]
In certain embodiments, the AFFIMER®polypeptide sequence has an amino acid sequence wherein (Xaa) m is an amino acid sequence represented in the general formula
[48]
-Xaa-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa- (SEQ ID NO: 5)
[49]
wherein Xaa, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13 and Xaa14, individually for each occurrence, is an amino acid residue, with the caveat that at least three of Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13 and Xaa14 are selected from His, Lys or Arg, and at least an additional two of Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13 and Xaa14 are selected from His, Lys, Arg, Phe, Tyr or Trp.
[50]
In certain embodiments, the AFFIMER®polypeptide sequence has an amino acid sequence wherein (Xaa) m is an amino acid sequence at least 75% identical to the Loop 4 sequence selected from SEQ ID NOs: 300-593 and 1183, and more preferably at least 80%, 85%, 90%, or 95% identical. In certain embnodiments, Loop 4 sequence is selected from SEQ ID NOs: 300-593 and 1183.
[51]
Another aspect relates to a protein comprising an FcRn binding AFFIMER®polypeptide sequence which binds to human FcRn and which is has an amino acid sequence that is at least 75% identical to an AFFIMER®polypeptide sequence selected from SEQ ID NOs: 594-887 and 1184, and more preferably 90%, 85%, 90% or even 95% identical. In certain embnodiments, the FcRn binding AFFIMER®polypeptide sequence which binds to human FcRn and which is has an amino acid sequence that is identical to an AFFIMER®polypeptide sequence selected from SEQ ID NOs: 594-887 and 1184.
[52]
Yet another aspect relates to a protein comprising an FcRn binding AFFIMER®polypeptide sequence which binds to human FcRn and has an amino acid sequence that can be encoded by a nucleic acid having a coding sequence that hybridizes to any one of SEQ ID NOs: 888 to 1181 under stringent conditions of 6X sodium chloride/sodium citrate (SSC) at 45℃ followed by a wash in 0.2X SSC at 65℃.
[53]
Still another aspect relates to a protein comprising (i) an FcRn binding AFFIMER®polypeptide sequence which 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 a therapeutic activity in human patients.
[54]
In some embodiments, the polypeptides further comprise a heterologous polypeptide covalently linked through an amide bond to form a contiguous fusion protein.
[55]
In some embodiments, the heterologous polypeptide comprises a therapeutic polypeptide. In certain embodiments, the therapeutic polypeptide is selected from the group consisting of polypeptide hormones, polypeptide cytokines, polypeptide chemokines, growth factors, hemostasis active polypeptides, enzymes, and toxins. In certain embodiments, the therapeutic polypeptide is selected from the group consisting of receptor traps and receptor ligands. In certain embodiments, the therapeutic polypeptide sequence is selected from the group consisting of angiogenic agents and anti-angiogenic agents. In certain embodiments, the therapeutic polypeptide is a neurotransmitter, and optionally wherein the neurotransmitter is Neuropeptide Y. In certain embodiments, the therapeutic polypeptide is an erythropoiesis-stimulating agent, and optionally wherein the erythropoiesis-stimulating agent is erythropoietin or an erythropoietin mimetic. In certain embodiments, the therapeutic polypeptide is an incretin, and optionally wherein the incretin is 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 certain embodiments, the therapeutic polypeptide is an anticancer immune enhancing agent, such as a checkpoint inhibitor, a costimulatory receptor agonist or an iducer of innate immunity. In certain embodiments, the therapeutic polypeptide is an anti-inflammatory immune inhibiting agent, such as a checkpoint agonist, a costimulatory receptor antagonist or an inhibitor of innate immunity.
[56]
In some embodiments, the polypeptides extend the serum half-life of the heterologous polypeptide in vivo. For example, 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 the AFFIMER® polypeptide.
[57]
In some embodiments, the polypeptides comprise a loop 2 amino acid sequence of any one of SEQ ID NOs: 6-299 and 1182. In some embodiments, the polypeptides comprise a loop 4 amino acid sequence of any one of SEQ ID NOs: 300-593 and 1183.
[58]
In some embodiments, the polypeptides comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of any one of SEQ ID NOs: 594-887 or 1184.
[59]
In some embodiments, the polypeptides are 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 the sequence of any one of SEQ ID NOs: 888-1181.
[60]
Other aspects of the present disclosure provide pharmaceutical preparations, e.g., for therapeutic use in a human patient, comprising any of the AFFIMER® polypeptides described herein, and a pharmaceutically acceptable excipient ( e.g., carrier, buffer, and/or salt, etc.). 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., transepithelial) delivery.

Claims
[Claim 1]
A polypeptide comprising an FcRn binding recombinantly engineered variant of stefin sequence that binds to human FcRn with a K d of 1x10 -6M or less at pH 6.0, and (optionally) a K d for binding human FcRn at pH 7.4 that is at least half a log greater than the Kd for binding at pH 6.0.
[Claim 2]
A protein comprising an FcRn binding recombinantly engineered variant of stefin polypeptide sequence which binds to human FcRn and has a circulating half-life in human patients of at least 7 days.
[Claim 3]
A protein comprising an FcRn binding recombinantly engineered variant of stefin polypeptide sequence which binds to human FcRn and facilitates transport of the protein across an epithelial tissue barrier.
[Claim 4]
A protein comprising (i) an FcRn binding recombinantly engineered variant of stefin polypeptide sequence which binds to human FcRn, and (ii) a heterologous polypeptide covalently associated to the FcRn binding recombinantly engineered variant of stefin polypeptide sequence (optionally as a fusion protein or chemically conjugated) which confers a therapeutic activity in human patients.
[Claim 5]
A protein comprising an FcRn binding recombinantly engineered variant of stefin polypeptide sequence which binds to human FcRn and which is has an amino acid sequence that is at least 75% identical to a recombinantly engineered variant of stefin polypeptide sequence selected from SEQ ID NOs: 594-887 and 1184.
[Claim 6]
A protein comprising an FcRn binding recombinantly engineered variant of stefin polypeptide sequence which binds to human FcRn and has an amino acid sequence that can be encoded by a nucleic acid having a coding sequence that hybridizes to any one of SEQ ID NOs: 888 to 1181 under stringent conditions of 6X sodium chloride/sodium citrate (SSC) at 45℃ followed by a wash in 0.2X SSC at 65℃.
[Claim 7]
The polypeptide of any of the preceding claims, wherein the FcRn binding recombinantly engineered variant of stefin sequence binds to FcRn with a K d of 1x10 -7 M or less at pH 6.0, a K d of 1x10 -8 M or less at pH 6.0, or K d of 1x10 -9 M or less at pH 6.0.
[Claim 8]
The polypeptide of any of the preceding claims, wherein the FcRn binding recombinantly engineered variant of stefin sequence binds to FcRn at pH 7.4 with a K d that is at least one log greater than the K d for binding to FcRn at pH 6.0, at least 1.5 logs greater than the K d for binding to FcRn at pH 6, at least 2 logs greater than the K d for binding to FcRn at pH 6, or at least 2.5 log greater than the K d for binding to FcRn at pH 6.
[Claim 9]
The polypeptide of any one of the preceding claims, wherein the polypeptide has a serum half-life in human patients 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.
[Claim 10]
The polypeptide of any one of the preceding claims, wherein the polypeptide has a serum half-life in human patients of greater than 50%, greater than 60%, greater than 70%, or greater than 80% of the serum half-life of IgG.
[Claim 11]
The polypeptide of any one of the preceding claims, wherein the polypeptide has a serum half-life in human patients of greater than 50%, greater than 60%, greater than 70%, or greater than 80% of the serum half-life of serum albumin.
[Claim 12]
The polypeptide of any one of the preceding claims, wherein the polypeptide does not inhibit binding of human serum albumin to human FcRn.
[Claim 13]
The polypeptide of any one of the preceding claims, wherein the polypeptide does not inhibit binding of IgG to human FcRn.
[Claim 14]
The polypeptide of any one of the preceding claims, wherein binding of the polypeptide to human FcRn facilitates transport of the polypeptide from an apical side to a basal side of an epithelial cell layer.
[Claim 15]
The polypeptide of any one of the preceding claims comprising an amino acid sequence represented in general 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); and Xaa, individually for each occurrence, is an amino acid, n is an integer from 3 to 20, and m is an integer from 3 to 20.
[Claim 16]
The polypeptide of claim 15, wherein: FR1 has 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% identity to SEQ ID NO: 1; FR2 has at least 80%, at least 84%, at least 88%, at least 92%, or at least 96% identity to SEQ ID NO: 2; and/or. FR3 has at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO: 3.
[Claim 17]
The polypeptide of claim 15, wherein: FR1 comprises the amino acid sequence of SEQ ID NO: 1; FR2 comprises the amino acid sequence of SEQ ID NO: 2; and/or FR3 comprises the amino acid sequence of SEQ ID NO: 3.
[Claim 18]
The polypeptide of any one of claims 15-17, wherein (Xaa) n is an amino acid sequence represented in the general formula -Xaa-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa-Xaa- (SEQ ID NO: 4) wherein Xaa, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6 and Xaa7, individually for each occurrence, is an amino acid residue, with the caveat 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.
[Claim 19]
The polypeptide of claim 18, wherein wherein at least three, and preferably four of Xaa2, Xaa3, Xaa4, Xaa5, Xaa6 or Xaa7 are selected from His, Lys or Arg.
[Claim 20]
The polypeptide of any of claims 15-19, wherein (Xaa) n is at least 75% identical to the Loop 2 sequence selected from SEQ ID NOs: 6-299 and 1182.
[Claim 21]
The polypeptide of any one of claims 15-20, wherein (Xaa) m is an amino acid sequence represented in the general formula -Xaa-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12-Xaa13-Xaa14-Xaa- (SEQ ID NO: 5) wherein Xaa, Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13 and Xaa14, individually for each occurrence, is an amino acid residue, with the caveat that at least three of Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13 and Xaa14 are selected from His, Lys or Arg, and at least an additional two of Xaa8, Xaa9, Xaa10, Xaa11, Xaa12, Xaa13 and Xaa14 are selected from His, Lys, Arg, Phe, Tyr or Trp.
[Claim 22]
The polypeptide of any of claims 15-18, wherein (Xaa) m is at least 75% identical to the Loop 4 sequence selected from SEQ ID NOs: 300-593 and 1183.
[Claim 23]
The polypeptide of any one of the preceding claims, wherein the polypeptide includes at least one cysteine, which is (optionally) available for chemical conjugation, and which (optionally) is located at the C-terminal end or the N-terminal end of the polypeptide.
[Claim 24]
The polypeptide of any one of the preceding claims further comprising a heterologous polypeptide covalently linked through an amide bond to form a contiguous fusion protein.
[Claim 25]
The polypeptide of claim 24, wherein the heterologous polypeptide comprises a therapeutic polypeptide.
[Claim 26]
The polypeptide of claim 25, wherein the therapeutic polypeptide is selected from the group consisting of polypeptide hormones, polypeptide cytokines, polypeptide chemokines, growth factors, hemostasis active polypeptides, enzymes, and toxins.
[Claim 27]
The polypeptide of claim 25, wherein the therapeutic polypeptide is selected from the group consisting of receptor traps and receptor ligands.
[Claim 28]
The polypeptide of claim 25, wherein the therapeutic polypeptide sequence is selected from the group consisting of angiogenic agents and anti-angiogenic agents.
[Claim 29]
The polypeptide of claim 25, wherein the therapeutic polypeptide sequence is a neurotransmitter, and optionally wherein the neurotransmitter is Neuropeptide Y.
[Claim 30]
The polypeptide of claim 25, wherein the therapeutic polypeptide sequence is an erythropoiesis-stimulating agent, and optionally wherein the erythropoiesis-stimulating agent is erythropoietin or an erythropoietin mimetic.
[Claim 31]
The polypeptide of claim 25, wherein the therapeutic polypeptide is an incretin, and optionally wherein the incretin is 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).
[Claim 32]
The polypeptide of claim 25, wherein the therapeutic polypeptide is an anticancer immune enhancing agent, such as a checkpoint inhibitor, a costimulatory receptor agonist or an iducer of innate immunity.
[Claim 33]
The polypeptide of claim 25, wherein the therapeutic polypeptide is an anti-inflammatory immune inhibiting agent, such as a checkpoint agonist, a costimulatory receptor antagonist or an inhibitor of innate immunity.
[Claim 34]
A pharmaceutical composition suitable for therapeutic use in a human patient, comprising a polypeptide of any of any one of the preceding claims, and a pharmaceutically acceptable excipient.
[Claim 35]
The pharmaceutical composition of claim 34, wherein the pharmaceutical composition is formulated for pulmonary delivery or topical application.
[Claim 36]
The pharmaceutical composition of claim 35, wherein the pulmonary delivery is intranasal delivery.
[Claim 37]
A polynucleotide comprising a sequence encoding the polypeptide of any of any one of the preceding claims.
[Claim 38]
The polynucleotide of claim 37, wherein the sequence encoding the polypeptide is operably linked to a transcriptional regulatory sequence.
[Claim 39]
The polynucleotide of claim 38, wherein the transcriptional regulatory sequence is selected from the group consisting of promoters and enhancers.
[Claim 40]
The polynucleotide of any of claims 37-39, further comprising an origin of replication, a minichromosome maintenance element (MME), and/or a nuclear localization element.
[Claim 41]
The polynucleotide of any of claims 37-40, further comprising a polyadenylation signal sequence operably linked and transcribed with the sequence encoding the polypeptide.
[Claim 42]
The polynucleotide of any of claims 37-40, wherein the sequence encoding the polypeptide comprises at least one intronic sequence
[Claim 43]
The polynucleotide of any of claims 37-42, further comprising at least one ribosome binding site transcribed with the sequence encoding the polypeptide.
[Claim 44]
The polynucleotide of any of claims 37-43, wherein the polynucleotide is a deoxyribonucleic acid (DNA).
[Claim 45]
The polynucleotide of any of claims 37-43, wherein the polynucleotide is a ribonucleic acid (RNA).
[Claim 46]
A viral vector comprising the polynucleotide of any of claims 37-43.
[Claim 47]
A plasmid or minicircle comprising the polynucleotide any of claims 37-43.
[Claim 48]
A cell comprising the polypeptide of any one of claims 1-33, the polynucleotide of any one of claims 37-45, the viral vector of claim 46, or the plasmid or minicircle of claim 47.
[Claim 49]
A method of increasing serum half-life of a therapeutic molecule, the method comprising conjugating the polypeptide of any one of claims 1-33 to the therapeutic molecule.
[Claim 50]
A polypeptide of any one of claims 1-25 for use in a method for treating an autoimmune disease and/or an inflammatory disease.
[Claim 51]
A polypeptide of any one of claims 1-25 for use in a method for treating cancer.
[Claim 52]
A polypeptide of any one of claims 1-25 for use in a method for treating cardiovascular or metabolic disease or disorder.
[Claim 53]
A method of producing the polypeptide of any one of claims 1-33, the method comprising expressing in a host cell a nucleic acid encoding the polypeptide, and optionally isolating the polypeptide from the host cell.
[Claim 54]
A protein comprising an FcRn binding recombinantly engineered variant of stefin polypeptide sequence which binds to human FcRn and inhibits the binding of human IgG to human FcRn.
[Claim 55]
The protein of claim 54 for use in a method for treating an autoimmune or inflammatory disorder or disease.
[Claim 56]
A pharmaceutical composition suitable for therapeutic use in a human patient, comprising a protein of claim 54, and a pharmaceutically acceptable excipient.
[Claim 57]
The polypeptide of any one of the preceding claims comprising a loop 2 amino acid sequence of any one of SEQ ID NOs: 6-299 and 1182.
[Claim 58]
The polypeptide of any one of the preceding claims comprising a loop 4 amino acid sequence of any one of SEQ ID NOs: 300-593 and 1183.
[Claim 59]
The polypeptide of any one of the preceding claims comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the sequence of any one of SEQ ID NOs: 594-887 or 1184.
[Claim 60]
The polypeptide of any one of the preceding claims 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 the sequence of any one of SEQ ID NOs: 888-1181.
[Claim 61]
A use of the polynucleotide of any one of the preceding claims for targeting FcRn.
[Claim 62]
A use of the polynucleotide of any one of the preceding claims for increasing serum half-life of a therapeutic molecule.