# US 10,370,651 B2 · Target antigen discovery, phenotypic screens and use thereof for identification of target cell specific target epitopes > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,651 B2 - Document ID: US10370651B2 - Status: Granted - Kind: B2 - Application number: 14/897,892 - Filed: 2014-06-20 - Published: 2016-05-05 - Granted: 2019-08-06 - Earliest claimed: 2013-06-28 - Assignees: X-Body, Inc. - Applicants: X-Body, Inc. - Inventors: Yan Chen; Steven M. Shamah - Web page: https://patentgrep.com/patent/US10370651B2 ## CPC - C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding - C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display} - G01N 33/6878: {in epitope analysis} - C07K 16/28: against receptors, cell surface antigens or cell surface determinants - G01N 2500/04: Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A) - C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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) - 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 methods and compositions for identifying binding polypeptides (e.g., antibodies or antigen binding fragments thereof) that specifically binds to a cell-surface antigen. The methods of the invention generally comprise contacting a variegated nucleic acid-display library of binding polypeptides with a cell-surface antigen displayed on the exterior surface of a cell; and isolating from the library at least one library member that specifically binds to the cell-surface antigen on the exterior surface of the cell. ## Claims ### Claim 1 A method of identifying a binding polypeptide that specifically binds to a cell-surface antigen, the method comprising: (a) contacting a variegated nucleic acid-display library of binding polypeptides with a cell-surface antigen naturally expressed on an exterior surface of a first cell type, wherein the variegated nucleic acid-display library is a DNA-display library, wherein each member of the DNA-display library comprises a binding polypeptide linked through an intervening DNA linker to a DNA coding sequence encoding the binding polypeptide, and wherein the DNA linker comprises a restriction endonuclease site not present in the coding sequence of members of the DNA-display library; (b) washing unbound library members from the first cell type; (c) enzymatically separating bound library members from the first cell type by cleaving the restriction endonuclease site with a suitable restriction endonuclease; and (d) isolating from the library at least one library member that specifically binds to the cell-surface antigen on the exterior surface of the first cell type, thereby identifying a binding polypeptide that specifically binds to the cell surface antigen. ### Claim 2 (depends on 1) The method of claim 1, wherein prior to step (a), an unselected precursor of the variegated nucleic acid-display library of binding polypeptides is contacted with a second cell type that does not express the cell-surface antigen on its exterior surface, and library members that bind to the second cell type are isolated and removed from the unselected precursor of the variegated nucleic acid-display library to produce the variegated nucleic acid-display library used in step (a). ### Claim 3 (depends on 1) The method of claim 1, further comprising: (e) contacting the variegated nucleic acid-display library of binding polypeptides with a second cell type that does not express the cell-surface antigen, and isolating from the library at least one library member that specifically binds to the second cell type; and (f) selecting library members that specifically bind to the first cell type but not to the second cell type, thereby identifying a binding polypeptide that specifically binds to the cell-surface antigen. ### Claim 4 (depends on 1) The method of claim 1, wherein the method further comprises determining the DNA coding sequence of at least a portion of the isolated library members. ### Claim 5 (depends on 4) The method of claim 4, wherein the DNA coding sequence is determined by pyrosequencing. ### Claim 6 (depends on 1) The method of claim 1, where the antigen is a naturally occurring protein, glycan or lipid. ### Claim 7 (depends on 1) The method of claim 1, where the antigen is a glycophosphatidylinositol (GPI) anchored protein. ### Claim 8 (depends on 1) The method of claim 1, wherein the first cell type is a disease-associated variant of a normal cell. ### Claim 9 (depends on 1) The method of claim 1, wherein the first cell type is a tumor cell. ### Claim 10 (depends on 1) The method of claim 1, wherein the binding polypeptide is an antibody or fragment thereof. ### Claim 11 (depends on 1) The method of claim 1, wherein the binding polypeptide is an antibody VH or VL domain. ### Claim 12 (depends on 1) The method of claim 1, wherein the first cell type is a live cell. ### Claim 13 (depends on 3) The method of claim 3, wherein the first and second cell types are live cells.