# US 2016/0354479 A1 · Functionalized polypeptides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2016/0354479 A1 - Document ID: US20160354479A1 - Status: Published application - Kind: A1 - Application number: 15/163,208 - Filed: 2016-05-24 - Published: 2016-12-08 - Earliest claimed: 2008-06-30 - Assignees: ESBATech, an Alcon Biomedical Research Unit LLC - Applicants: ESBATech, an Alcon Biomedical Research Unit LLC - Inventors: David Urech - Web page: https://patentgrep.com/patent/US20160354479A1 ## CPC - C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display} - A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol - A61K 47/65: Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers - A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics - A61P 37/00: Drugs for immunological or allergic disorders - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C07K 16/241: {Tumor Necrosis Factors} - C12N 15/1062: {mRNA-Display, e.g. polypeptide and encoding template are connected covalently} - C07K 2317/40: characterized by post-translational modification - C07K 2317/41: Glycosylation, sialylation, or fucosylation - C07K 2317/622: Single chain antibody (scFv) - C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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 The invention provides functionalized polypeptides, especially therapeutic polypeptides (e.g., scFv), comprising a linker sequence that can be rapidly and specifically functionalized by the addition of one or functional moieties (e.g., PEG) or binding specificities (e.g., an amino acid sequence with a particular binding specificity). Such functionalized polypeptides are advantageous in that they have improved pharmacokinetic properties (e.g., improved in vivo half-life, tissue penetration and tissue residency time) over non-functionalized polypeptides. Methods for the rapid and reproducible generation of functionalized polypeptides are also provided. ## Claims ### Claim 1 A polypeptide comprising two domains connected by an amino acid linker; wherein the linker comprises two cysteines capable of forming an intrachain disulphide bond, and wherein the amino acids between the cysteines in the linker sequence form a loop when the two cysteines are disulphide bonded to one another. ### Claim 2 (depends on 1) The polypeptide of claim 1, wherein the linker comprises the amino acid sequence set forth in SEQ ID No. 1. ### Claim 3 (depends on 1) The polypeptide of claim 1, wherein the linker comprises the amino acid sequence set forth in SEQ ID No. 2. ### Claim 4 (depends on 1) The polypeptide of claim 1, wherein the loop binds to a target molecule. ### Claim 5 (depends on 4) The polypeptide of claim 4, wherein the target molecule is a PK modifier. ### Claim 6 (depends on 5) The polypeptide of claim 5, wherein the PK modifier is serum albumin. ### Claim 7 (depends on 5) The polypeptide of claim 5, wherein the PK modifier is a hyaluronic acid. ### Claim 8 (depends on 1) The polypeptide of claim 1, wherein the linker comprises the amino acid sequence set forth in SEQ ID No. 3. ### Claim 9 (depends on 1) The polypeptide of claim 1, wherein the linker comprises the amino acid sequence set forth in SEQ ID No. 4. ### Claim 10 (depends on 1) The polypeptide of claim 1, wherein the polypeptide is an immunobinder. ### Claim 11 (depends on 10) The polypeptide of claim 10, which is a scFv. ### Claim 12 (depends on 1) The polypeptide of claim 1, comprising the amino acid sequence set forth in SEQ ID No. 6 or 8. ### Claim 13 (depends on 1) A polypeptide of claim 1, wherein at least one cysteine residue in the linker is covalently linked to a functional moiety. ### Claim 14 (depends on 13) The polypeptide of claim 13, wherein the functional moiety is PEG. ### Claim 15 (depends on 1) A polypeptide of claim 1, wherein the two cysteine residues in the linker are covalently linked to the same functional moiety. ### Claim 16 (depends on 15) The polypeptide of claim 15, wherein the functional moiety is PEG. ### Claim 17 (depends on 1) A composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier. ### Claim 18 (depends on 1) An isolated nucleic acid molecule encoding the polypeptide of claim 1. ### Claim 19 (depends on 18) An expression vector comprising the nucleic acid molecule of claim 18. ### Claim 20 (depends on 19) A host cell comprising the expression vector of claim 19. ### Claim 21 A method producing a functionalized polypeptide comprising: (a) providing a library of peptide sequences; (b) identifying from the library at least one peptide sequence that bind to a target molecule; (c) modifying the loop region of a linker-containing polypeptide to comprise at least one peptide sequence identified in step (b), thereby producing a functionalized polypeptide. ### Claim 22 (depends on 21) The method of claim 21, wherein said identifying step is performed using phage display, yeast display, or mRNA display.