# US 11,111,489 B1 · Multiplexed testing of lymphocytes for antigen specificity > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,111,489 B1 - Document ID: US11111489B1 - Status: Granted - Kind: B1 - Application number: 17/142,745 - Filed: 2021-01-06 - Granted: 2021-09-07 - Earliest claimed: 2021-01-06 - Assignees: Think Therapeutics, Inc. - Applicants: Think Therapeutics, Inc. - Inventors: David Gifford; Brandon Carter - Web page: https://patentgrep.com/patent/US11111489B1 ## CPC - C12N 15/1034: {Isolating an individual clone by screening libraries} - C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display} - C12N 15/1089: {Design, preparation, screening or analysis of libraries using computer algorithms} - C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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 Described herein is a method for determining a lymphocyte cell receptor chain sequence specific to a unique antigen, comprising: sorting a plurality of antigens into a plurality of reaction mixtures, wherein the sorting comprises adding a unique antigen of the plurality of antigens to a unique subset of the plurality of reaction mixtures such that two different unique antigens are not added to the unique subset; contacting each reaction with a biological sample comprising a plurality of lymphocytes; separating a target lymphocyte from a subset of the plurality of lymphocytes, wherein the target lymphocyte recognizes the unique antigen; after separating the target lymphocyte, sequencing nucleic acids of the target lymphocyte to obtain the lymphocyte receptor chain sequence, wherein the sequencing is performed by single-cell sequencing; and detecting the unique antigen, wherein the detecting comprises: computing a frequency of lymphocyte cells that express the lymphocyte receptor chain sequence. ## Claims ### Claim 1 A method for determining a lymphocyte cell receptor chain sequence, or a portion thereof, specific for at least two or more unique antigens, the method comprising: sorting a plurality of antigens into a plurality of reaction mixtures, wherein the sorting comprises adding the at least two unique antigens of the plurality of antigens to at least two unique subsets of the plurality of reaction mixtures such that the at least two unique antigens are not added to any two identical subsets of the plurality of reaction mixtures, and wherein the at least two unique subsets are configured to allow a detection of the at least two or more unique antigens that are specific to the lymphocyte cell receptor chain sequence; contacting each reaction mixture of the plurality of reaction mixtures with a biological sample comprising a plurality of lymphocytes; separating a target lymphocyte from a subset of the plurality of lymphocytes, wherein the target lymphocyte recognizes the at least two unique antigens of the plurality of antigens; sequencing nucleic acids of the target lymphocyte to obtain the lymphocyte cell receptor chain sequence; and detecting the at least two or more unique antigens that are specific to the lymphocyte cell receptor chain sequence. ### Claim 2 (depends on 1) The method of claim 1, wherein the lymphocyte is a T cell or a B cell. ### Claim 3 (depends on 1) The method of claim 1, wherein separating the target lymphocyte is performed using multimer sorting. ### Claim 4 (depends on 1) The method of claim 1, wherein the target lymphocyte is a T cell, and wherein separating the T cell is based on a marker selected from the group consisting of CD3, CD4, CD8, CD137, OX40, CD25, PD-L1, CD69, and CD154. ### Claim 5 (depends on 1) The method of claim 1, wherein the lymphocyte cell receptor chain sequence comprises a receptor chain sequence pair, and wherein the receptor chain sequence pair consists of two components of a receptor of the target lymphocyte. ### Claim 6 (depends on 1) The method of claim 1, wherein a number of reaction mixtures comprising the at least two unique subsets is a function of a number of expected antigens that are specific to the lymphocyte cell receptor chain sequence. ### Claim 7 (depends on 1) The method of claim 1, wherein the plurality of reaction mixtures comprises at least one control reaction mixture, and wherein the control reaction mixture does not contain any antigens that are added to the biological sample. ### Claim 8 (depends on 1) The method of claim 1, wherein the target lymphocyte recognizes the at least two unique antigens of the plurality of antigens by binding the at least two unique antigens of the plurality of antigens or by binding two or more molecular complexes comprising the at least two or more unique antigens of the plurality of antigens. ### Claim 9 (depends on 1) The method of claim 1, wherein the detecting further comprises applying, by a processor, a nearest neighbor decoding algorithm configured to determine the at least two or more unique antigens that are specific to the lymphocyte cell receptor chain sequence. ### Claim 10 (depends on 1) The method of claim 1, wherein the detecting further comprises: applying, by a processor, a decoding algorithm, wherein the decoding algorithm is configured to detect the at least two or more unique antigens that are specific to the lymphocyte cell receptor chain sequence when the lymphocyte cell receptor chain sequence is not substantially present in at least one reaction mixture of the at least two unique subsets of the plurality of reaction mixtures. ### Claim 11 (depends on 1) The method of claim 1, further comprising assigning a superimposed code to each antigen of the plurality of antigens, wherein the superimposed code is configured to allow a detection of the at least two or more unique antigens that are specific to the lymphocyte cell receptor chain sequence. ### Claim 12 A method for determining a lymphocyte cell receptor chain sequence, or a portion thereof, specific for at least two or more unique antigens, the method comprising: sorting a plurality of antigens into a plurality of reaction mixtures, wherein the sorting comprises adding the at least two unique antigens of the plurality of antigens to at least two unique subsets of the plurality of reaction mixtures such that the at least two unique antigens are not added to any two identical subsets of the plurality of reaction mixtures, and wherein the at least two unique subsets are configured to allow a detection of the at least two or more unique antigens that are specific to the lymphocyte cell receptor chain sequence; contacting each reaction mixture with a biological sample comprising a plurality of lymphocytes; separating a target lymphocyte from a subset of the plurality of lymphocytes, wherein the target lymphocyte reacts with the at least two unique antigens of the plurality of antigens; sequencing nucleic acids of the target lymphocyte to obtain the lymphocyte cell receptor chain sequence; and applying, using a processor, a nearest neighbor decoding algorithm configured to detect specificity of the lymphocyte cell receptor chain sequence to the at least two or more antigens. ### Claim 13 (depends on 12) The method of claim 12, wherein the lymphocyte is a T cell or a B cell. ### Claim 14 (depends on 12) The method of claim 12, wherein the lymphocyte cell receptor chain sequence comprises a receptor chain sequence pair, and wherein the receptor chain sequence pair consists of two components of a receptor of the target lymphocyte. ### Claim 15 (depends on 12) The method of claim 12, further comprising contacting at least one reaction mixture of the plurality of reaction mixtures with a query antigen.