# US 2025/0085290 A1 · Human fibronectin type iii protein scaffolds > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0085290 A1 - Document ID: US20250085290A1 - Status: Published application - Kind: A1 - Application number: 18/580,205 - Filed: 2022-07-19 - Published: 2025-03-13 - Earliest claimed: 2021-07-19 - Assignees: ARO Biotherapeutics Company - Applicants: Aro Biotherapeutics Company - Inventors: Ronald V. Swanson; Zhanna Druzina; Yao Xin; Karyn O’Neil - Web page: https://patentgrep.com/patent/US20250085290A1 ## CPC - G01N 33/6845: {Methods of identifying protein-protein interactions in protein mixtures} - C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - 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) ## Abstract Protein scaffolds and scaffold libraries based on a fibronectin type III (FN3) domain with an alternative binding surface design, isolated nucleic acids encoding the protein scaffolds, vectors, host cells, methods of making thereof, and uses as therapeutic molecules for treatment and diagnosis of diseases and disorders. ## Claims ### Claim 1 A library comprising a plurality of fibronectin type III module (FN3) domains having a diversified C-CD-D-F-FG-G alternative surface comprising a diversified C beta-strand, a CD loop, a D beta-strand, an F beta-strand, an FG loop and a G beta-strand, wherein the polypeptides comprise an amino acid sequence of: (SEQ ID NO: 44) MLSPPSNLRVTDVISTSVTLSWKPPAPITGYXVXYXEXXXXGEW KXVXVPGSETSYTVTGLKPGTEYXFXVXAVNGAXXGXPSQXVXV TT or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 44, wherein each X is, independently, any amino acid. ### Claim 2 (depends on 1) The library of claim 1, wherein each X is, independently, any amino acid, except a methionine or a cysteine. ### Claim 3 (depends on 1) The library of claim 1, wherein the polypeptides comprise at least one mutated amino acid residue as compared to SEQ ID NO: 24 in one or more of, or each of, the C beta-strand, the CD loop, the D beta-strand, the F beta-strand, the FG loop, or the G beta-strand to form the FN3 domain library having the diversified C-CD-D-F-FG-G alternative surface. ### Claim 4 (depends on 1) The library of claim 1, wherein the plurality of polypeptides have one or more mutations at a position that corresponds to positions 32, 34, 36, 38, 39, 40, 41, 46, 48, 68, 70, 72, 78, 79, 81, 85, and/or 87 of SEQ ID NO: 24. ### Claim 5 (depends on 1) The library of claim 1, wherein the diversified C beta-strand has an amino acid sequence of TGYXVXYXE (SEQ ID NO: 45), wherein each X is, independently, any amino acid except a methionine or a cysteine. ### Claim 6 (depends on 1) The library of claim 1, wherein the diversified CD loop has an amino acid sequence of XXXXGE (SEQ ID NO: 46), wherein each X is, independently, any amino acid except a methionine or a cysteine. ### Claim 7 (depends on 1) The library of claim 1, wherein the diversified D beta-strand has an amino acid sequence of WKXVXVP (SEQ ID NO: 47), wherein each X is, independently, any amino acid except a methionine or a cysteine. ### Claim 8 (depends on 1) The library of claim 1, wherein the diversified F beta-strand has an amino acid sequence of TEYXFXVXAV (SEQ ID NO: 48), wherein each X is, independently, any amino acid except a methionine or a cysteine. ### Claim 9 (depends on 1) The library of claim 1, wherein the diversified FG loop has an amino acid sequence of NGAXXG (SEQ ID NO: 49), wherein each X is, independently, any amino acid except a methionine or a cysteine. ### Claim 10 (depends on 1) The library of claim 1, wherein the diversified G beta-strand has an amino acid sequence XPSQXVXVTT (SEQ ID NO: 50), wherein each X is, independently, any amino acid except a methionine or a cysteine. ### Claim 11 (depends on 1) The library of claim 1, wherein the library comprises an amino acid sequence having an amino acid sequence that is at least, or about, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or is identical to a sequence selected from the group consisting of SEQ ID NOs: 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, 37, 38, 39, 40, 41, 42, and 43. ### Claim 12 (depends on 1) The library of claim 1, wherein: the diversified C beta-strand has an amino acid sequence of TGYXVXYXE (SEQ ID NO: 45), wherein each X is, independently, any amino acid except a methionine or a cysteine; the diversified CD loop has an amino acid sequence of XXXXGE (SEQ ID NO: 46), wherein each X is, independently, any amino acid except a methionine or a cysteine; the diversified D beta-strand has an amino acid sequence of WKXVXVP (SEQ ID NO: 47), wherein each X is, independently, any amino acid except a methionine or a cysteine; the diversified F beta-strand has an amino acid sequence of TEYXFXVXAV (SEQ ID NO: 48), wherein each X is, independently, any amino acid except a methionine or a cysteine; the diversified FG loop has an amino acid sequence of NGAXXG (SEQ ID NO: 49), wherein each X is, independently, any amino acid except a methionine or a cysteine; and wherein the diversified G beta-strand has an amino acid sequence XPSQXVXVTT (SEQ ID NO: 50), wherein each X is, independently, any amino acid except a methionine or a cysteine. ### Claim 13 (depends on 1) A method of producing the library of claim 1, the method comprising expressing a polynucleotide encoding the plurality of polypeptides. ### Claim 14 A method of making a library of fibronectin module of type III (FN3) domains having a diversified C-CD-F-FG-G alternative surface comprising a diversified one or more, or each of, C beta-strand, a CD loop, an F beta-strand, an FG loop and G-beta strand, comprising a. providing a reference FN3 domain polypeptide having an amino acid sequence at least 80% identical to that of SEQ ID NO: 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, 37, 38, 39, 40, 41, 42, 43 or 44; and b. introducing diversity into the reference FN3 domain polypeptide by mutating at least one residue in the C beta-strand, CD loop region, F beta-strand region, FG loop region, or G-beta strand region to form the FN3 domain library having the diversified C-CD-F-FG-G alternative surface, wherein: the diversified C beta-strand has an amino acid sequence of TGYXVXYXE (SEQ ID NO: 45), wherein each X is, independently, any amino acid except a methionine or a cysteine; the diversified CD loop has an amino acid sequence of XXXXGE (SEQ ID NO: 46), wherein each X is, independently, any amino acid except a methionine or a cysteine; the diversified D beta-strand has an amino acid sequence of WKXVXVP (SEQ ID NO: 47), wherein each X is, independently, any amino acid except a methionine or a cysteine; the diversified F beta-strand has an amino acid sequence of TEYXFXVXAV (SEQ ID NO: 48), wherein each X is, independently, is any amino acid except a methionine or a cysteine; the diversified FG loop has an amino acid sequence of NGAXXG (SEQ ID NO: 49), wherein each X is, independently, any amino acid except a methionine or a cysteine; and the diversified G beta-strand has an amino acid sequence XPSQXVXVTT (SEQ ID NO: 50), wherein each X is, independently, any amino acid except a methionine or a cysteine. ### Claim 15 (canceled) ### Claim 16 (depends on 1) A method of obtaining a polypeptide comprising a fibronectin type III module (FN3) domain having a diversified C-CD-D-F-FG-G alternative surface that binds or specifically binds to a target molecule, comprising contacting the library of claim 1 with the target molecule and isolating the polypeptide that binds or specifically binds to the target molecule. ### Claim 17 A polypeptide having an amino acid sequence that is at least, or about, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or is identical to a sequence selected from the group consisting of SEQ ID NOS: 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, 37, 38, 39, 40, 41, 42, and 43. ### Claim 18 A polypeptide comprising an amino acid sequence of: (SEQ ID NO: 44) MLSPPSNLRVTDVTSTSVTLSWKPPAPITGYXVXYXEXXXXGEW KXVXVPGSETSYTVTGLKPGTEYXFXVXAVNGAXXGXPSQXVXV TT wherein each X is, independently, any amino acid. ### Claim 19 (depends on 18) The polypeptide of claim 18, wherein each X is, independently, any amino acid, except methionine or cysteine. ### Claim 20 A polypeptide comprising an amino acid sequence of: (SEQ ID NO: 74) LSPPSNLRVTDVTSTSVTLSWKPPAPITGYXVXYXEXXXXGEWK XVXVPGSETSYTVTGLKPGTEYXFXVXAVNGAXXGXPSQXVXVI T wherein each X is, independently, any amino acid. ### Claim 21 (depends on 20) The polypeptide of claim 20, wherein each X is, independently, any amino acid, except methionine or cysteine. ### Claim at position 22 -25. (canceled)