# US 10,502,745 B2 · Identification of antigen- or ligand-specific binding proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,502,745 B2 - Document ID: US10502745B2 - Status: Granted - Kind: B2 - Application number: 15/418,192 - Filed: 2017-01-27 - Published: 2017-08-24 - Granted: 2019-12-10 - Earliest claimed: 2008-03-05 - Assignees: Agenus Inc. - Applicants: Agenus Inc. - Inventors: Ulf Grawunder; Jorn Stitz - Web page: https://patentgrep.com/patent/US10502745B2 ## CPC - G01N 33/6854: {Immunoglobulins} - C07K 14/43595: {from coelenteratae, e.g. medusae} - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C07K 16/18: against material from animals or humans - C12N 5/0635: {B lymphocytes} - C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5) - C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display} - C12Q 1/6897: involving reporter genes operably linked to promoters - G01N 33/56966: {Animal cells} - G01N 33/56972: {White blood cells} - C07K 2317/21: from primates, e.g. man - C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding - 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) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - 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) ## Abstract The present invention discloses novel methods for the generation, expression and screening of diverse collections of binding proteins such as antibodies or fragments thereof in vertebrate host cells in vitro, for the identification and isolation of ligand- or antigen-specific binding proteins. The methods disclosed herein allow the expression of diverse collections of binding proteins from at least one vector construct, which optionally can give rise to collections of diverse binding proteins upon transfer and expression into vertebrate host cells in situ. ## Claims ### Claim 1 A method of producing at least one stably transduced vertebrate host cell that recombinantly expresses on its cell surface a dimeric T cell receptor (TCR) specific for a desired antigen, the method comprising the steps of: (a) transducing a first library of replication incompetent retroviral expression constructs encoding a plurality of first TCR chain polypeptides or second TCR chain polypeptides into a population of vertebrate host cells that express low or substantially no endogenous TCR such that a first library of stably transduced vertebrate host cells is generated, wherein each member of the first library of stably transduced vertebrate host cells comprises at least one retroviral expression construct from the first library of retroviral expression constructs stably integrated into the host cell genome; (b) transducing the first library of stably transduced vertebrate host cells of step (a) with a second library of replication incompetent retroviral expression constructs encoding a plurality of first TCR chain polypeptides or second TCR chain polypeptides such that a second library of stably transduced vertebrate host cells is generated, wherein (i) the first library of replication incompetent retroviral expression constructs encodes a plurality of first TCR chain polypeptides and the second library of replication incompetent retroviral expression constructs encodes a plurality of second TCR chain polypeptides, or (ii) the first library of replication incompetent retroviral expression constructs encodes a plurality of second TCR chain polypeptides and the second library of replication incompetent retroviral expression constructs encodes a plurality of first TCR chain polypeptides; and wherein each member of the second library of stably transduced vertebrate host cells comprises at least one replication incompetent retroviral expression construct from the first library of retroviral expression constructs stably integrated into the host cell genome and at least one replication incompetent retroviral expression construct from the second library of retroviral expression constructs stably integrated into the host cell genome; (c) allowing expression of the integrated retroviral expression constructs in the second library of stably transduced vertebrate host cells such that dimeric TCRs are expressed on the cell surface of the second library of stably transduced vertebrate host cells, wherein each expressed dimeric TCR comprises a first TCR chain polypeptide and a second TCR chain polypeptide; and (d) selecting from the second library of stably transduced vertebrate host cells at least one stably transduced vertebrate host cell expressing a dimeric TCR specific for the desired antigen. ### Claim 2 (depends on 1) The method of claim 1, wherein the method further comprises identifying from the at least one stably transduced vertebrate host cell selected in step (d) at least one nucleotide sequence encoding the first TCR chain polypeptide or the second TCR chain polypeptide of the dimeric TCR specific for the desired antigen. ### Claim 3 (depends on 1) The method of claim 1, wherein the method further comprises, between steps (c) and (d), enriching the TCR-expressing cells of the second library of stably transduced vertebrate host cells on the basis of their ability to bind to the desired antigen. ### Claim 4 (depends on 3) The method of claim 3, wherein the enrichment is performed by physical separation of cells from a non-binder population using: (i) fluorescence activated cell sorting (FACS); (ii) micromanipulation; or (iii) panning methods to immobilized antigen. ### Claim 5 (depends on 1) The method of claim 1, wherein the method further comprises, between steps (c) and (d), expanding the population of enriched cells in the second library of stably transduced vertebrate host cells. ### Claim 6 (depends on 1) The method of claim 1, wherein the vertebrate host cells are derived from a mammal. ### Claim 7 (depends on 6) The method of claim 6, wherein the mammal is a mouse or a human. ### Claim 8 (depends on 1) The method of claim 1, wherein the vertebrate host cells are precursor lymphocytes. ### Claim 9 (depends on 1) The method of claim 1, wherein the expression of said dimeric TCR in the stably transduced vertebrate host cells is operably linked to at least one antibiotic selection marker. ### Claim 10 (depends on 9) The method of claim 9, wherein the first library and second library of replication incompetent retroviral expression constructs each comprise a different antibiotic selection marker. ### Claim 11 (depends on 9) The method of claim 9, wherein the at least one antibiotic expression marker is operably linked to the nucleotide sequence encoding the first TCR chain polypeptide or the second TCR chain polypeptide using at least one internal ribosomal entry sequence (IRES). ### Claim 12 (depends on 1) The method of claim 1, wherein the expression of said TCR in the stably transduced vertebrate host cells is operably linked to at least one screening marker. ### Claim 13 (depends on 12) The method of claim 12, wherein the first library and second library of replication incompetent retroviral expression constructs each comprise a different screening marker. ### Claim 14 (depends on 12) The method of claim 12, wherein the at least one screening marker is operably linked to the nucleotide sequence encoding the first TCR chain polypeptide or the second TCR chain polypeptide using at least one internal ribosomal entry sequence (IRES). ### Claim 15 (depends on 12) The method of claim 12, wherein the at least one screening marker is a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and blue fluorescent protein (BFP). ### Claim 16 (depends on 12) The method of claim 12, wherein the at least one screening marker is a cell surface marker selected from the group consisting of CD7, CD34, and the low affinity nerve growth factor receptor. ### Claim 17 (depends on 1) The method of claim 1, wherein the multiplicity of infection in step (a) and/or step (b) is less than or equal to 0.1. ### Claim 18 (depends on 1) The method of claim 1, wherein the multiplicity of infection in each of steps (a) and (b) is less than or equal to 0.1.