# US 12,000,064 B2 · Universal reporter cell assay for specificity test of novel antigen binding moieties > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,000,064 B2 - Document ID: US12000064B2 - Status: Granted - Kind: B2 - Application number: 16/906,778 - Filed: 2020-06-19 - Published: 2020-10-08 - Granted: 2024-06-04 - Earliest claimed: 2017-12-21 - Assignees: Hoffmann-La Roche Inc. - Applicants: Hoffmann-La Roche Inc. - Inventors: Christian Klein; Ekkehard Moessner; Lydia Jasmin Hanisch; Wei Xu; Camille Loise Sophie Delon; Diana Darowski; Christian Jost; Vesna Pulko - Web page: https://patentgrep.com/patent/US12000064B2 ## CPC - C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding - A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells - A61K 40/31: Chimeric antigen receptors [CAR] - A61K 40/4266: {Carcinoembryonic antigen [CEA]} - C07K 14/70539: {MHC-molecules, e.g. HLA-molecules} - C07K 16/18: against material from animals or humans - C07K 16/2887: {against CD20} - C07K 16/30: from tumour cells - C07K 16/3007: {Carcino-embryonic Antigens} - G01N 33/505: {involving T-cells} - G01N 33/575: for cancer - C07K 2317/52: Constant or Fc region; Isotype - 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) - 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) - 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 present invention generally relates to specificity assays using cell cultures, in particular to chimeric antigen receptor (CAR) expressing reporter T (CAR-T) cell assays to test novel antigen binding moieties in different formats. Furthermore, the present invention relates to the use of CAR-T cells, transfected/transduced with an engineered chimeric antigen receptor (CAR) comprising a target antigen binding moiety capable of specific binding to a recognition domain of an antigen binding molecule. The invention also relates to methods and kits for specificity testing of a candidate antigen binding moiety and/or nucleic acid molecules and vectors expressing engineered CARs. ## Claims ### Claim 1 A method for assessing the specificity of a Fab fragment comprising the steps of: a) providing an antigen binding molecule comprising an antigen binding domain and a recognition domain, wherein the antigen binding domain comprises the Fab fragment, wherein the Fab fragment is specific for a target antigen; b) contacting the antigen binding molecule with a target cell comprising the target antigen on the surface; c) contacting the antigen binding molecule with a chimeric antigen receptor (CAR) expressing reporter T (CAR-T) cell wherein the reporter CAR-T cell comprises: i. a CAR capable of specific binding to the recognition domain wherein the CAR is operationally coupled to a response element; ii. a reporter gene under the control of the response element; and d) determining T cell activation by determining the expression of the reporter gene to establish the specificity of the Fab fragment; wherein the recognition domain is an Fc domain. ### Claim 2 (depends on 1) The method of claim 1, wherein the Fc domain is a mutated Fc domain, wherein the mutated Fc domain comprises at least one amino acid substitution compared to the non-mutated parent Fc domain, wherein the CAR is capable of specific binding to the mutated Fc domain but not capable of specific binding to the non-mutated parent Fc domain. ### Claim 3 (depends on 2) The method of claim 2, wherein the mutated Fc domain comprises at least one amino acid mutation at a position selected from the group consisting of L234, L235, 1253, H310, P331, P329 and H435 according to EU numbering, in particular wherein the amino acid mutation is L234A, L235A, I253A, N297A, H310A, P329G and/or H435A. ### Claim 4 (depends on 2) The method of claim 2, wherein the mutated Fc domain comprises the amino acid mutation P329G according to EU numbering. ### Claim 5 (depends on 1) The method of claim 1, wherein the Fab fragment is derived from a phage display library screening. ### Claim 6 (depends on 1) The method according to claim 1, wherein activation of the response element leads to expression of the reporter gene. ### Claim 7 (depends on 1) The method according to claim 1, wherein the reporter gene is coding for green fluorescent protein (GFP) or luciferase. ### Claim 8 (depends on 1) The method according to claim 1, wherein the target antigen is selected from the group consisting of CD20, CEA, HER2, TYRP, EGFR, MCSP, STEAP1, WT1 and FolR1, or a fragment thereof. ### Claim 9 (depends on 1) The method according to claim 1, wherein high level of expression of the reporter gene in the presence of the target cell and low level of expression of the reporter gene in the absence of the target cell is indicative for high specificity of the FAB fragment. ### Claim 10 (depends on 1) The method according to claim 1, wherein high level of expression of the reporter gene in the presence of the target cell and low level of expression of the reporter gene in the absence of the target cell is indicative for high specificity of a T cell bispecific (TCB) antibody comprising the FAB fragment. ### Claim 11 (depends on 1) A method for generating a T cell bispecific (TCB) antibody, wherein the TCB antibody comprises a first antigen binding moiety specific for a target antigen and a second antigen binding moiety capable of specific binding to a T cell activating receptor, wherein the first antigen binding moiety is a Fab fragment selected according to the method of claim 1. ### Claim 12 (depends on 2) The method of claim 2, wherein the mutated Fc domain comprises the amino acid mutation P329G according to EU numbering. ### Claim 13 (depends on 1) The method of claim 1, wherein the target cell is a cancer cell.