# US 8,465,937 B2 · Methods for identifying B-clones which bind cell-surface antigens > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,465,937 B2 - Document ID: US8465937B2 - Status: Granted - Kind: B2 - Application number: 13/616,267 - Filed: 2012-09-14 - Published: 2013-04-11 - Granted: 2013-06-18 - Earliest claimed: 2009-02-24 - Assignees: ESBATech, an Alcon Biomedical Research Unit LLC - Applicants: David Urech; Valerie Hulmann-Cottier - Inventors: David Urech; Valerie Hulmann-Cottier - Web page: https://patentgrep.com/patent/US8465937B2 ## CPC - C07K 16/2866: {against receptors for cytokines, lymphokines, interferons} - C07K 16/22: against growth factors {; against growth regulators} - C07K 16/241: {Tumor Necrosis Factors} - C07K 16/28: against receptors, cell surface antigens or cell surface determinants - C07K 16/4241: {against anti-human or anti-animal Ig} - G01N 33/5052: {involving B-cells} - G01N 33/566: using specific carrier or receptor proteins as ligand binding reagents {where possible specific carrier or receptor proteins are classified with their target compounds} - G01N 33/56972: {White blood cells} - C07K 2317/20: characterized by taxonomic origin - C07K 2317/21: from primates, e.g. man - C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered - C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® - C07K 2317/622: Single chain antibody (scFv) - 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) - 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) ## Abstract The invention provides methods for identifying immunobinders, such as scFv antibodies, capable of specifically binding to cell surface antigens, and compositions identified according to said methods. ## Claims ### Claim 1 A method for identifying a B-cell clone that specifically binds to an antigen of interest comprising: (a) providing a plurality of B-cells; (b) contacting the plurality of B-cells with the antigen of interest, wherein the antigen of interest comprises a first sortable label, and staining the plurality of B-cells with anti-IgG antibodies comprising a second sortable label and anti-IgM antibodies comprising a third sortable label; (c) separating from the plurality of B-cells one or more B-cells that can specifically bind to the antigen of interest using a cell sorter, wherein the presence of the first and second sortable label, but not the third sortable label, in a complex is indicative of the binding of a B-cell to an antigen of interest, thereby identifying a B-cell clone that binds to the antigen of interest. ### Claim 2 (depends on 1) The method of claim 1, wherein the plurality of B-cells is provided in a plurality of lymphocytes. ### Claim 3 (depends on 1) The method of claim 1, wherein the plurality of B-cells are stained prior to being contacted with the antigen of interest in step (b). ### Claim 4 (depends on 1) The method of claim 1, wherein the plurality of B-cells are stained after being contacted with the antigen of interest in step (b). ### Claim 5 (depends on 1) The method of claim 1, wherein identification of the B-cell clone comprises clonally isolating the B-cells obtained in step (c), optionally followed by clonal expansion of the clonally isolated cells. ### Claim 6 (depends on 5) The method of claim 5, further comprising isolating a nucleic acid molecule encoding an immunobinder from the B-cell clone that binds to an antigen of interest. ### Claim 7 (depends on 6) The method of claim 6, further comprising producing an immunobinder capable of binding to an antigen of interest, wherein the immunobinder is produced by introducing the nucleic acid sequence into an expression environment such that the encoded immunobinder is produced. ### Claim 8 (depends on 1) The method of claim 1, wherein the one or more separated B-cells are cultivated under suitable conditions so that antibodies are secreted into the culture medium. ### Claim 9 (depends on 8) The method of claim 8, wherein the antibodies are tested for specific binding to the antigen of interest. ### Claim 10 (depends on 8) The method of claim 8, wherein the antibodies are mouse, rabbit, rat, hamster, sheep, chicken, camel, goat, human antibodies. ### Claim 11 (depends on 1) The method of claim 1, wherein the sortable labels are fluorescent labels. ### Claim 12 (depends on 1) The method of claim 1, wherein the antigen of interest is a soluble antigen. ### Claim 13 (depends on 1) The method of claim 1, wherein the B-cells are B-cells from a rabbit. ### Claim 14 (depends on 13) The method of claim 13, wherein the rabbit was immunized with an antigen of interest. ### Claim 15 A method for identifying a B-cell clone that specifically binds to an antigen of interest comprising: (a) immunizing an animal with an antigen of interest; (b) isolating a plurality of B-cells from the immunized animal; (c) contacting the plurality of B-cells with the antigen of interest, wherein the antigen of interest comprises a first sortable label, and staining the plurality of B-cells with anti-IgG antibodies comprising a second sortable label and anti-IgM antibodies comprising a third sortable label; (d) separating from the plurality of B-cells one or more B-cells that can specifically bind to the antigen of interest using a cell sorter, wherein the presence of the first and second sortable label, but not the third sortable label, in a complex is indicative of the binding of a B-cell to an antigen of interest, thereby identifying a B-cell clone that binds to the antigen of interest. ### Claim 16 (depends on 15) The method of claim 15, wherein the sortable labels are fluorescent labels. ### Claim 17 (depends on 15) The method of claim 15, wherein the antigen of interest is a soluble antigen. ### Claim 18 (depends on 15) The method of claim 15, wherein the animal is rabbit.