# US 2025/0375520 A1 · Production of immune cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0375520 A1 - Document ID: US20250375520A1 - Status: Published application - Kind: A1 - Application number: 18/872,766 - Filed: 2023-06-12 - Published: 2025-12-11 - Earliest claimed: 2022-06-10 - Assignees: Adaptimmune Limited - Applicants: Adaptimmune Limited - Inventors: Garth HAMILTON; Nail FATKHUTDINOV - Web page: https://patentgrep.com/patent/US20250375520A1 ## CPC - A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells - A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00) - A61K 40/32: T-cell receptors [TCR] - A61K 40/42: Cancer antigens - C07K 14/7051: {T-cell receptor (TcR)-CD3 complex} - C12N 5/0636: {T lymphocytes} - C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS} - C12N 5/10: Cells modified by introduction of foreign genetic material - C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein} - C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} - C12N 15/86: Viral vectors - C12N 15/8645: {Adeno-associated virus} - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12N 2502/45: Artificially induced pluripotent stem cells - C12N 2502/99: genetically modified cells - C12N 2506/11: from blood or immune system cells - C12N 2506/45: from artificially induced pluripotent stem cells - C12N 2510/00: Genetically modified cells - C12N 2750/14143: viral genome or elements thereof as genetic vector - 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) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract This invention relates to methods for producing immune cells expressing a therapeutic antigen receptor. An immune cell is provided that comprises a heterologous expression cassette. The heterologous expression cassette comprises (a) a coding sequence for a production TCR, (b) a constitutive promoter operably linked to the coding sequence, (c) a 5′ targeting site, and optionally (d) a 3′ targeting site. An expression construct is then introduced into the immune cell at the site of the heterologous expression cassette. The expression construct comprises a coding sequence for a therapeutic antigen receptor and the therapeutic antigen receptor is then expressed in the immune cell. Methods, reagents for use in the methods and immune cells produced by the methods are provided. ## Claims ### Claim 1 A method for producing an immune cell expressing a therapeutic antigen receptor comprising; (i) providing an immune cell comprising a heterologous expression cassette, wherein the expression cassette comprises; (a) a coding sequence for a production T cell receptor (TCR), (b) a constitutive promoter operably linked to the coding sequence, (c) a targeting site, and (ii) replacing the heterologous expression cassette in the immune cell with an expression construct comprising a coding sequence for a therapeutic antigen receptor, such that the therapeutic antigen receptor is expressed in the immune cell. ### Claim 2 (depends on 1) A method according to claim 1 wherein the therapeutic antigen receptor binds specifically to an MHC displaying a peptide fragment of a target antigen expressed by cells or specifically binds to a target antigen or peptide thereof expressed by cells independently of MHC presentation. ### Claim 3 (depends on 2) A method according to claim 2 wherein the therapeutic antigen receptor binds specifically to an MHC displaying a peptide fragment of a tumour antigen expressed by cancer cells or binds specifically to a tumour antigen or peptide fragment thereof expressed by cancer cells independently of MHC presentation. ### Claim 4 A method according to any one of the preceding claims wherein the immune cell is a T cell. ### Claim 5 A method according to any one of the preceding claims wherein the heterologous expression cassette is replaced in the immune cell by a method comprising; introducing into the immune cell a nucleic acid molecule comprising the expression construct and a 5′ homology arm, wherein the 5′ homology arm is complementary to the 5′ targeting site of the heterologous expression cassette, such that the expression construct replaces the expression cassette in the genome of the immune cell. ### Claim 6 (depends on 5) A method according to claim 5 wherein the method further comprises introducing into the immune cell a vector comprising a nucleic acid that encodes a CRISPR/Cas9 targeting the targeting site. ### Claim 7 A method according to any one of the preceding claims wherein the targeting site is a 5′ targeting site. ### Claim 8 (depends on 7) A method according to claim 7 wherein the cassette further comprises a 3′ targeting site. ### Claim 9 (depends on 8) A method according to claim 8 wherein the heterologous expression cassette is replaced in the immune cell by a method comprising; introducing into the immune cell a nucleic acid molecule, such as a DNA molecule, comprising the expression construct flanked by 5′ and 3′ homology arms, wherein the 5′ and 3′ homology arms are complementary to the 5′ and 3′ targeting sites of the heterologous expression cassette, such that the expression construct replaces the expression cassette in the genome of the immune cell. ### Claim 10 (depends on 9) A method according to claim 9 wherein the method further comprises introducing into the immune cell a vector comprising a nucleic acid that encodes a CRISPR/Cas9 targeting the 5′ and 3′ targeting sites. ### Claim 11 A method according to any one of the preceding claims wherein the therapeutic antigen receptor is a T cell receptor (TCR). ### Claim 12 (depends on 11) A method according to claim 11 wherein the therapeutic TCR specifically binds to cancer cells. ### Claim 13 A method according to any one of the preceding claims wherein the expression cassette comprises 5′ and 3′ targeting sites and said targeting sites comprise a nucleotide sequence from the TCRα chain constant region. ### Claim 14 (depends on 13) A method according to claim 13 wherein the 3′ targeting site is located within the coding sequence for the production TCR. ### Claim 15 A method according to any one of the preceding claims wherein the immune cell comprising the heterologous expression cassette is produced by a method comprising (i) transfecting an iPSC with a nucleic acid comprising a heterologous expression cassette, such that the cassette is integrated into the genome of the iPSC, wherein the expression cassette comprises (a) a coding sequence for a production T cell receptor (TCR), (b) a constitutive promoter operably linked to the coding sequence, and (c) a targeting site, and (ii) differentiating the iPSC into an immune cell comprising the heterologous expression cassette. ### Claim 16 (depends on 15) A method according to claim 15, wherein the targeting site is a 5′ targeting site. ### Claim 17 (depends on 16) A method according to claim 16 wherein the cassette further comprises a 3′ targeting site. ### Claim 18 (depends on 15, 16, 17) A method according to claim 15, 16 or 17 wherein the iPSCs are differentiated into immune cells by a method that comprises; (i) differentiating the iPSCs into mesoderm cells, (ii) differentiating the mesoderm cells into haemogenic endothelial cells, (iii) differentiating the haemogenic endothelial cells into a population of HPCs, (iv) differentiating the HPCs into immune cell progenitors; and (v) maturing the population of progenitor immune cells to produce a population of immune cells. ### Claim 19 (depends on 18) A method according to claim 18 wherein the iPSCs are cultured sequentially in first, second and third mesoderm induction media to induce differentiation into mesoderm cells, the first mesoderm induction medium consisting of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of activin, the second mesoderm induction medium consisting of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of activin, BMP, and FGF, and the third mesoderm induction medium consisting of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of activin, BMP, FGF, and a GSK3 inhibitor. ### Claim 20 (depends on 18, 19) A method according to claim 18 or 19 wherein the mesoderm cells are cultured in an HE induction medium to induce differentiation into HECs; said HE induction medium consisting of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of SCF and VEGF. ### Claim 21 (depends on 18, 19, 20) A method according to any one of claims 18 to 20 wherein the HECs are cultured in an haematopoietic induction medium to induce differentiation into HPCs; said haematopoietic induction medium consisting of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of VEGF, SCF, Thrombopoietin (TPO), Flt3 ligand (FIt3L), IL-3, IL-6, IL-7, IL-11, IGF-1, BMP, FGF, Sonic hedgehog (SHH), erythropoietin (EPO), angiotensin Il, and an angiotensin II type 1 receptor (AT1) antagonist. ### Claim 22 (depends on 18, 19, 20, 21) A method according to any one of claims 18 to 21 wherein the HPCs are cultured in a lymphoid expansion medium to produce the progenitor immune cells; said lymphoid expansion medium consisting of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of SCF, FLT3L, TPO and IL7. ### Claim 23 (depends on 18, 19, 20, 21, 22) A method according to any one of claims 18 to 22 wherein the progenitor immune cells are matured by a method comprising culturing the population of progenitor immune cells in a maturation medium to produce the immune cells; said maturation medium consisting of a chemically defined nutrient medium supplemented with one or more differentiation factors, wherein the one or more differentiation factors consist of SCF, FLT3L, and IL7. ### Claim 24 A method according to any one of the preceding claims comprising concentrating the population of immune cells expressing the therapeutic antigen receptor. ### Claim 25 A method according to according to any one of the preceding claims comprising storing the population of immune cells expressing the therapeutic antigen receptor. ### Claim 26 A method according to any one of the preceding claims comprising formulating the population of immune cells expressing the therapeutic antigen receptor with a pharmaceutically acceptable excipient. ### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) A population of immune cells expressing one or more therapeutic antigen receptors produced by a method according to any one of claims 1 to 26. ### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) A pharmaceutical composition comprising a population of immune cells expressing one or more therapeutic antigen receptors produced by a method according to any one of claims 1 to 26 and a pharmaceutically acceptable excipient. ### Claim 29 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) A population of immune cells expressing one or more therapeutic antigen receptors produced by a method according to any one of claims 1 to 26 for use in a method of treatment. ### Claim 30 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) A population of immune cells expressing one or more therapeutic antigen receptors produced by a method according to any one of claims 1 to 26 for use in a method of treatment of cancer. ### Claim 31 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) A method of treatment of cancer comprising administering a population of immune cells expressing one or more therapeutic antigen receptors produced by a method according to any one of claims 1 to 26 to an individual in need thereof. ### Claim 32 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) Use of a population of immune cells expressing one or more therapeutic antigen receptors produced by a method according to any one of claims 1 to 26 in the manufacture of a medicament for use in treating cancer. ### Claim 33 An immune cell comprising a heterologous expression cassette integrated into the genome thereof, wherein the expression cassette comprises (a) a coding sequence for a production T cell receptor (TCR), (b) a constitutive promoter operably linked to the coding sequence, and (c) a targeting site. ### Claim 34 (depends on 31) An immune cell according to claim 31 wherein the targeting site is a 5′ targeting site. ### Claim 35 (depends on 34) An immune cell according to claim 34 wherein the cassette further comprises a 3′ targeting site. ### Claim 36 An iPSC comprising a heterologous expression cassette integrated into the genome thereof, wherein the expression cassette comprises (a) a coding sequence for a production T cell receptor (TCR), (b) a constitutive promoter operably linked to the coding sequence, and (c) a targeting site. ### Claim 37 (depends on 33) An iPSC according to claim 33 wherein the targeting site is a 5′ targeting site. ### Claim 38 (depends on 37) An iPSC according to claim 37 wherein the cassette further comprises a 3′ targeting site.