# US 2023/0272011 A1 · Serum albumin-binding polypeptides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2023/0272011 A1 - Document ID: US20230272011A1 - Status: Published application - Kind: A1 - Application number: 18/018,208 - Filed: 2021-07-30 - Published: 2023-08-31 - Earliest claimed: 2020-07-30 - Assignees: Avacta Life Sciences Limited - Applicants: Avacta Life Sciences Limited - Inventors: Emma Jenkins; Estelle Adam; Emma Stanley; Amrik Basran; Matthew P. Vincent; Bruno Gomes - Web page: https://patentgrep.com/patent/US20230272011A1 ## CPC - C07K 14/001: {by chemical synthesis} - C07K 16/2818: {against CD28 or CD152} - C07K 14/505: Erythropoietin [EPO] - C07K 14/57545: {Neuropeptide Y} - C07K 14/605: Glucagons - C07K 14/8139: {Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin} - C12N 15/70: Vectors or expression systems specially adapted for E. coli - C07K 14/765: Serum albumin, e.g. HSA - C07K 2319/00: Fusion polypeptide - C12N 2800/101: for bacteria - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract Provided herein, in some embodiments, are recombinantly engineered variant of stefin polypeptides (AFFIMER® polypeptides) that binds to human serum albumin and extends the half-life of the polypeptides. Also provided herein, in some embodiments, are composition containing the polypeptides, methods of using the polypeptides, and methods of producing the polypeptides. ## Claims ### Claim 1 A polypeptide that binds to human serum albumin (HSA) with a Kd of 1×10−6 M or less at pH 7.4, and at pH 6 binds to HSA with a Kd that is at least half a log less than the Kd for binding to HSA at pH 7.4. ### Claim 2 (depends on 1) The polypeptide of claim 1, wherein the polypeptide binds to HSA with a Kd of 1×10−7 M or less at pH 7.4, a Kd of 1×10−8 M or less at pH 7.4, or Kd of 1×10−9 M or less at pH 7.4. ### Claim 3 (depends on 1, 2) The polypeptide of claim 1 or 2, wherein the polypeptide at pH 6 binds to HSA with a Kd that is at least one log less than the Kd for binding to HSA at pH 7.4, at least 1.5 logs less than the Kd for binding to HSA at pH 7.4, at least 2 logs less than the Kd for binding to HSA at pH 7.4, or at least 2.5 log less than the Kd for binding to HSA at pH 7.4 ### Claim 4 (depends on 1, 2, 3) The polypeptide of any one of claim 1-3, 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 5 (depends on 1, 2, 3, 4) The polypeptide of any one of claims 1-4, 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 HSA. ### Claim 6 (depends on 1, 2, 3, 4, 5) The polypeptide of any one of claims 1-5 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 7 (depends on 6) The polypeptide of claim 6, 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 8 (depends on 7) The polypeptide of claim 7, 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 9 (depends on 6) The polypeptide of claim 6, wherein the amino acid sequence is represented in general formula (II) (SEQ ID NO: 166) MIP-Xaa1-GLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKT QVLA-(Xaa)n-Xaa2-TNYYIKVRAGDNKYMHLKVF-Xaa3-Xaa4- Xaa5-(Xaa)m-Xaa6-D-Xaa7-VLTGYQVDKNKDDELTGF, (II) wherein 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; Xaa1 is Gly, Ala, Val, Arg, Lys, Asp, or Glu; Xaa2 is Gly, Ala, Val, Ser or Thr; Xaa3 is Arg, Lys, Asn, Gln, Ser, Thr; Xaa4 is Gly, Ala, Val, Ser or Thr; Xaa5 is Ala, Val, Ile, Leu, Gly or Pro; Xaa6 is Gly, Ala, Val, Asp or Glu; and Xaa7 is Ala, Val, Ile, Leu, Arg or Lys. ### Claim 10 (depends on 9) The polypeptide of claim 9, wherein the amino acid sequence is represented in general formula (III) (SEQ ID NO: 167) MIPRGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKT QVLA-(Xaa)n-STNYYIKVRAGDNKYMHLKVFNGP-(Xaa)m-ADR VLTGYQVDKNKDDELTGF, (III) wherein 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 11 (depends on 6, 7, 8, 9, 10) The polypeptide of any one of claims 6-10, wherein (Xaa)n is represented by formula (IV) (SEQ ID NO: 180) aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9, (IV) wherein aa1 is an amino acid selected from D, G, N, and V; aa2 is an amino acid selected from W, Y, H, and F; aa3 is an amino acid selected from W, Y, G, W, and F; aa4 is an amino acid selected from Q, A, and P; aa5 is an amino acid selected from A, Q, E, R, and S; aa6 is an amino acid selected from K, R, and Y; aa7 is an amino acid selected from W and Q; aa8 is an amino acid selected from P and H; and aa9 is an amino acid selected from H, G, and Q. ### Claim 12 (depends on 6, 7, 8, 9, 10, 11) The polypeptide of any one of claims 6-11, wherein (Xaa)n is 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: 4-55. ### Claim 13 (depends on 12) The polypeptide of claim 12, wherein (Xaa)n is the amino acid sequence of any one of SEQ ID NOS: 4-55. ### Claim 14 (depends on 6, 7, 8, 9, 10, 11, 12, 13) The polypeptide of any one of claims 6-13, wherein (Xaa)n is 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: 22, 24, 26, 35, 40, 41, and 45. ### Claim 15 (depends on 14) The polypeptide of claim 14, wherein (Xaa)n is an amino acid sequence of any one of SEQ ID NOS: 22, 24, 26, 35, 40, 41, and 45. ### Claim 16 (depends on 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) The polypeptide of any one of claims 6-15, wherein (Xaa)m is represented by formula (IV) (SEQ ID NO: 181) aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9, (IV) wherein aa1 is an amino acid selected from Y, F, W, and N; aa2 is an amino acid selected from K, P, H, A, and T; aa3 is an amino acid selected from V, N, G, Q, A, and F; aa4 is an amino acid selected from H, T, Y, W, K, V, and R; aa5 is an amino acid selected from Q, S, G, P, and N; aa6 is an amino acid selected from S, Y, E, L, K, and T; aa7 is an amino acid selected from S, D, V, and K; aa8 is an amino acid selected from G, L, S, P, H, D, and R; and aa9 is an amino acid selected from G, Q, E, and A. ### Claim 17 (depends on 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) The polypeptide of any one of claims 6-16, wherein (Xaa)m is 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: 57-108. ### Claim 18 (depends on 17) The polypeptide of claim 17, wherein (Xaa)m is the amino acid sequence of any one of SEQ ID NOS: 57-108. ### Claim 19 (depends on 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) The polypeptide of any one of claims 6-16, wherein (Xaa)m is 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: 75, 77, 79, 88, 93, 94, and 98. ### Claim 20 (depends on 19) The polypeptide of claim 19, wherein (Xaa)m is an amino acid sequence of any one of SEQ ID NOS: 75, 77, 79, 88, 93, 94, and 98. ### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) The polypeptide of any one of any one of claims 1-20, wherein the polypeptide comprises an amino acid sequence that has at least 70%, at least 80%, or at least 90% identity to an amino acid sequence of any one of SEQ ID NOS: 110-116 and 138. ### Claim 22 (depends on 21) The polypeptide of claim 21, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 110-116 and 138. ### Claim 23 (depends on 6, 7, 8, 9, 10) The polypeptide of any one of claims 6-10, wherein (Xaa)n is represented by formula (IV) aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9 (IV), wherein aa1 is an amino acid with a neutral polar hydrophilic side chain; aa2 is an amino acid with a neutral nonpolar hydrophobic side chain; aa3 is an amino acid with a neutral nonpolar hydrophobic side chain; aa4 is an amino acid with a neutral polar hydrophilic side chain; aa5 is an amino acid with a positively charged polar hydrophilic side chain; aa6 is an amino acid with a positively charged polar hydrophilic side chain; aa7 is an amino acid with a neutral nonpolar hydrophobic side chain; aa8 is an amino acid with a neutral nonpolar hydrophobic side chain; and aa9 is an amino acid with a neutral nonpolar hydrophilic side chain. ### Claim 24 (depends on 6, 7, 8, 9, 10) The polypeptide of any one of claims 6-10, wherein (Xaa)m is represented by formula (IV) aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9 (IV) wherein aa1 is an amino acid with a neutral nonpolar hydrophobic side chain; aa2 is an amino acid with a positively charged polar hydrophilic side chain; aa3 is an amino acid with a neutral nonpolar hydrophobic side chain; aa4 is an amino acid with a positively charged polar hydrophilic side chain; aa5 is an amino acid with a neutral polar hydrophilic side chain; aa6 is an amino acid with a neutral polar hydrophilic side chain; aa7 is an amino acid with a negatively charged polar hydrophilic side chain; aa8 is an amino acid with a positively charged polar hydrophilic side chain; and aa9 is an amino acid with a neutral nonpolar hydrophilic side chain. ### Claim 25 (depends on 23, 24) The polypeptide of claim 23 or 24, wherein the amino acid with the neutral nonpolar hydrophilic side chain is selected from cysteine (C) and glycine (G); the amino acid with the neutral nonpolar hydrophobic side chain is selected from alanine (A), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), tryptophan (W), and valine (V); the amino acid with the neutral polar hydrophilic side chain is selected from asparagine (N), glutamine (Q), serine (S), threonine (T), and tyrosine (Y); the amino acid with the positively charged polar hydrophilic side chain is selected from arginine (R), histidine (H), and lysine (K); and the amino acid with the negatively charged polar hydrophilic side chain is selected from aspartate (D) and glutamate (E). ### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) The polypeptide of any one of claims 1-25, wherein the polypeptide comprises an amino acid sequence that comprises a cysteine. ### Claim 27 (depends on 26) The polypeptide of claim 26, wherein the cysteine is available for chemical conjugation, and optionally wherein the cysteine is located at the C-terminal end or the N-terminal end of the polypeptide, and optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 138. ### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) The polypeptide of any one of claims 1-27 further comprising a heterologous polypeptide covalently linked through an amide bond to form a contiguous fusion protein. ### Claim 29 (depends on 28) The polypeptide of claim 28, wherein the heterologous polypeptide comprises a therapeutic polypeptide. ### Claim 30 (depends on 29) The polypeptide of claim 29, 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 31 (depends on 29) The polypeptide of claim 29, wherein the therapeutic polypeptide is selected from the group consisting of receptor traps and receptor ligands. ### Claim 32 (depends on 29) The polypeptide of claim 29, wherein the therapeutic polypeptide sequence is selected from the group consisting of angiogenic agents and anti-angiogenic agents. ### Claim 33 (depends on 29) The polypeptide of claim 29, wherein the therapeutic polypeptide sequence is a neurotransmitter, and optionally wherein the neurotransmitter is Neuropeptide Y. ### Claim 34 (depends on 29) The polypeptide of claim 29, wherein the therapeutic polypeptide sequence is an erythropoiesis-stimulating agent, and optionally wherein the erythropoiesis-stimulating agent is erythropoietin or an erythropoietin mimetic. ### Claim 35 (depends on 29) The polypeptide of claim 29, 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 36 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) A pharmaceutical composition suitable for therapeutic use in a human patient, comprising a polypeptide of any of claim 1-35, and a pharmaceutically acceptable excipient. ### Claim 37 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) A polynucleotide comprising a sequence encoding the polypeptide of any of claims 1-36. ### Claim 38 (depends on 37) The polynucleotide of claim 37, wherein the sequence encoding the polypeptide is operably linked to a transcriptional regulatory sequence. ### Claim 39 (depends on 38) The polynucleotide of claim 38, wherein the transcriptional regulatory sequence is selected from the group consisting of promoters and enhancers. ### Claim 40 (depends on 38, 39) The polynucleotide of claim 38 or 39, further comprising an origin of replication, a minichromosome maintenance element (MME), and/or a nuclear localization element. ### Claim 41 (depends on 38, 39, 40) The polynucleotide of any of claims 38-40, further comprising a polyadenylation signal sequence operably linked and transcribed with the sequence encoding the polypeptide. ### Claim 42 (depends on 38, 39, 40, 41) The polynucleotide of any of claims 38-41, wherein the sequence encoding the polypeptide comprises at least one intronic sequence. ### Claim 43 (depends on 38, 39, 40, 41, 42) The polynucleotide of any of claims 38-42, further comprising at least one ribosome binding site transcribed with the sequence encoding the polypeptide. ### Claim 44 (depends on 38, 39, 40, 41, 42, 43) The polynucleotide of any of claims 38-43, wherein the polynucleotide is a deoxyribonucleic acid (DNA). ### Claim 45 (depends on 38, 39, 40, 41, 42, 43) The polynucleotide of any of claims 38-43, wherein the polynucleotide is a ribonucleic acid (RNA). ### Claim 46 (depends on 38, 39, 40, 41, 42, 43, 44, 45) A viral vector comprising the polynucleotide of any of claims 38-45. ### Claim 47 (depends on 38, 39, 40, 41, 42, 43, 44, 45, 46) A plasmid or minicircle comprising the polynucleotide of any of claims 38-46. ### Claim 48 A fusion protein comprising a polypeptide of any one of the preceding claims. ### Claim 49 (depends on 48) The fusion protein of claim 48 further comprising a linker. ### Claim 50 (depends on 49) The fusion protein of claim 49, wherein the linker is a rigid linker. ### Claim 51 (depends on 50) The fusion protein of claim 50, wherein the rigid linker comprises the sequence of SEQ ID NO: 161. ### Claim 52 (depends on 49) The fusion protein of claim 49, wherein the linker is a flexible linker. ### Claim 53 (depends on 50) The fusion protein of claim 50, wherein the flexible linker comprises the sequence of SEQ ID NO: 165. ### Claim 54 (depends on 48, 49, 50, 51, 52, 53) The fusion protein of any one of claims 48-53 comprising two polypeptides of any one of the preceding claims. ### Claim 55 (depends on 48, 49, 50, 51, 52, 53, 54) The fusion protein of any one of claims 48-54 further comprising a therapeutic molecule. ### Claim 56 (depends on 55) The fusion protein of claim 55, wherein the therapeutic molecule is a therapeutic polypeptide. ### Claim 57 (depends on 56) The fusion protein of claim 56, wherein the therapeutic polypeptide is selected from hormones, cytokines, chemokines, growth factors, hemostasis active polypeptides, enzymes, and toxins, or is selected from antagonists of hormones, cytokines, chemokines, growth factors, hemostasis active polypeptides, enzymes, and toxins. ### Claim 58 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57) The fusion protein of any one of claims 48-57, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 110. ### Claim 59 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57) The fusion protein of any one of claims 48-57, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 113. ### Claim 60 (depends on 48, 49, 50, 51, 52, 53, 54, 55, 56, 57) The fusion protein of any one of claims 48-57, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 116.