# US 11,028,185 B2 · Using sortases to install click chemistry handles for protein ligation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,028,185 B2 - Document ID: US11028185B2 - Status: Granted - Kind: B2 - Application number: 16/140,384 - Filed: 2018-09-24 - Published: 2019-04-18 - Granted: 2021-06-08 - Earliest claimed: 2011-06-28 - Assignees: Whitehead Institute for Biomedical Research - Applicants: Whitehead Institute for Biomedical Research - Inventors: Hidde L. Ploegh; Martin D. Witte; Nicholas C. Yoder - Web page: https://patentgrep.com/patent/US11028185B2 ## CPC - C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies} - C07K 1/107: by chemical modification of precursor peptides - C07K 1/1072: {by covalent attachment of residues or functional groups} - C07K 16/18: against material from animals or humans - C12N 9/52: derived from bacteria {or Archaea} - C12N 15/62: DNA sequences coding for fusion proteins - C07K 2317/31: multispecific - C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® - C07K 2317/62: comprising only variable region components - C07K 2319/00: Fusion polypeptide - C07K 2319/21: containing a His-tag - 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) ## Abstract Methods and reagents for the installation of click chemistry handles on target proteins are provided, as well as modified proteins comprising click chemistry handles. Further, chimeric proteins, for example, bi-specific antibodies, that comprise two proteins conjugated via click chemistry, as well as methods for their generation and use are disclosed herein. ## Claims ### Claim 1 A bispecific, chimeric antibody comprising a first antibody or antigen-binding antibody fragment comprising a sortase recognition sequence; and a second antibody or antigen-binding antibody fragment comprising a sortase recognition sequence; wherein the first and the second antibody or antibody fragment are conjugated together via click chemistry. ### Claim 2 (depends on 1) The chimeric antibody of claim 1, wherein the first and the second antibody or antibody fragment are conjugated together via a covalent bond at their C-termini (C—C) or at their N-termini (N—N). ### Claim 3 (depends on 1) The chimeric antibody of claim 1, wherein the first and/or the second antibody comprises a single-domain antibody or an antigen-binding fragment thereof. ### Claim 4 (depends on 1) The chimeric antibody of claim 1, wherein the first and/or the second antibody comprises a camelid antibody or an antigen-binding fragment thereof. ### Claim 5 (depends on 1) The chimeric antibody of claim 1, wherein the first and/or the second antibody comprises a single variable domain of a heavy chain antibody (VHH domain) or an antigen-binding fragment thereof. ### Claim 6 (depends on 1) The chimeric antibody of claim 1, wherein the first and/or the second antibody comprises a single-chain variable fragment (scFv) or an antigen-binding fragment thereof. ### Claim 7 (depends on 1) The chimeric antibody of claim 1, wherein the first and/or the second antibody comprises a nanobody or an antigen-binding fragment thereof. ### Claim 8 (depends on 1) The chimeric antibody of claim 1, wherein the first and the second antibody or antigen-binding fragment bind different antigens. ### Claim 9 (depends on 1) The chimeric antibody of claim 1, wherein the first and the second antibody or antigen-binding fragment bind the same antigen. ### Claim 10 (depends on 9) The chimeric antibody of claim 9, wherein the first and the second antibody or antigen-binding fragment bind different epitopes of the same antigen. ### Claim 11 (depends on 1) The chimeric antibody of claim 1, wherein the sortase recognition sequence of the first antibody or antigen-binding antibody fragment comprises the sequence LPXTG (SEQ ID NO: 2), wherein X is any amino acid. ### Claim 12 (depends on 1) The chimeric antibody claim 1, wherein the sortase recognition sequence of the second antibody or antigen-binding antibody fragment comprises the sequence LPXTG (SEQ ID NO: 2), wherein X is any amino acid. ### Claim 13 (depends on 1) The chimeric antibody of claim 1, wherein the sortase recognition sequence of the first antibody or antigen-binding antibody fragment comprises the sequence LPETG (SEQ ID NO: 4). ### Claim 14 (depends on 1) The chimeric antibody of claim 1, wherein the sortase recognition sequence of the second antibody or antigen-binding antibody fragment comprises the sequence LPETG (SEQ ID NO: 4). ### Claim 15 (depends on 1) The chimeric antibody of claim 1, wherein the sortase recognition sequence of the first antibody or antigen-binding antibody fragment is at the C-terminus. ### Claim 16 (depends on 1) The chimeric antibody of claim 1, wherein the sortase recognition sequence of the second antibody or antigen-binding antibody fragment is at the C-terminus. ### Claim 17 (depends on 1) The chimeric antibody of claim 1, wherein the first or the second antibody or antigen-binding antibody fragment comprises an anti-GFP antibody or a fragment thereof. ### Claim 18 (depends on 1) The chimeric antibody of claim 1, wherein the first or the second antibody or antigen-binding antibody fragment comprises an anti-beta-2 microglobulin (anti-β2M) antibody or a fragment thereof. ### Claim 19 (depends on 1) A pharmaceutical composition comprising the chimeric antibody of claim 1. ### Claim 20 (depends on 19) The pharmaceutical composition of claim 19, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.