# US 2022/0162551 A1 · Methods for making, compositions comprising, and methods of using rejuvenated t cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2022/0162551 A1 - Document ID: US20220162551A1 - Status: Published application - Kind: A1 - Application number: 17/534,341 - Filed: 2021-11-23 - Published: 2022-05-26 - Earliest claimed: 2020-11-24 - Assignees: Lyell Immunopharma, Inc. - Applicants: Lyell Immunopharma, Inc. - Inventors: Raul E. Vizcardo Sakoda; Nicholas Restifo; Richard D. Klausner; Yin Huang; Takuya Maeda; Naritaka Tamaoki; Yasuhiro Yamazaki - Web page: https://patentgrep.com/patent/US20220162551A1 ## CPC - C12N 5/0636: {T lymphocytes} - 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 40/11: 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 40/32: T-cell receptors [TCR] - A61K 40/4211: {CD19 or B4} - A61K 40/4269: {NY-ESO} - A61P 35/00: Antineoplastic agents - C07K 14/7051: {T-cell receptor (TcR)-CD3 complex} - C07K 16/2803: {against the immunoglobulin superfamily} - C12N 2501/2302: Interleukin-2 (IL-2) - C12N 2501/51: B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand) - C12N 2501/515: CD3, T-cell receptor complex - C12N 2501/602: Sox-2 - C12N 2501/603: Oct-3/4 - C12N 2501/604: Klf-4 - C12N 2501/606: c-Myc - C12N 2510/00: Genetically modified cells - C12N 2760/18821: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12N 2760/18842: virus or viral particle as vehicle, e.g. encapsulating small organic molecule - C12N 2760/18843: viral genome or elements thereof as genetic vector - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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 present disclosure relates generally to methods of producing rejuvenated T cells, comprising, contacting T cells with at least one reprogramming factor and reactivating the contacted cells; and compositions and methods of using same. The present disclosure also describes cell populations prepared according to methods described herein. The disclosure also provides for methods of treating patients using cell populations prepared by the methods described herein. ## Claims ### Claim 1 A method of producing rejuvenated T cells, comprising a. contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2 and C-MYC, and (ii) optionally SV40, for a period of time sufficient for a T cell derived adherent cell to form and wherein said T cells are not transformed into iPS or totipotent cells; and b. contacting the T cell derived adherent cell with at least one T cell activating compound. ### Claim at position 2 2-4. (canceled) ### Claim 5 (depends on 1) The method of claim 1, wherein the T cells are contacted with the at least one reprogramming factor for a period of time sufficient for at least a portion of the T cells to express CD3 and at least one marker selected from the group consisting of integrin α6β1, SSEA4, CD9, and CD90. ### Claim at position 4 6-7. (canceled) ### Claim 8 (depends on 5) The method of claim 5, wherein the T cells are contacted with the at least one reprogramming factor for a period of time sufficient for at least a portion of said contacted T cells to express CD3, SSEA4, CD9 and CD90. ### Claim 9 (depends on 1) The method of claim 1, wherein, prior to contacting said T cells with the at least one reprogramming factor, the T cells are activated with IL-2 and at least one agent capable of activating said T cells. ### Claim 10 (depends on 1) The method of claim 1, wherein the T cells are TCRαβ cells, TCRγδ cells, CD4+CD8αβ+ double positive cells, CD4+ single positive cell (such as Th1, Th2, Th17, Treg), naïve T cells, central memory T cells, effector memory T cells, or a combination thereof. ### Claim 11 (depends on 9) The method of claim 9, wherein the activated T cells are enriched by selection for cells expressing CD137, PD1, or LAG3. ### Claim at position 9 12-13. (canceled) ### Claim 14 (depends on 1) The method of claim 1, wherein the T cells are contacted with KLF4, OCT3/4, SOX2 and C-MYC. ### Claim 15 (depends on 14) The method of claim 14, wherein the T cells are contacted with KLF4, OCT3/4, SOX2 and C-MYC for at least about 4 to about 10 days. ### Claim 16 (depends on 15) The method of claim 15, wherein the T cells are contacted with KLF4, OCT3/4, SOX2 and C-MYC for at least about 4 to about 7 days. ### Claim 17 (canceled) ### Claim 18 (depends on 1) The method of claim 1, wherein the at least one reprogramming factor is expressed in the T cells. ### Claim 19 (depends on 18) The method of claim 18 wherein the at least one reprogramming factor is expressed using a non-integrating viral vector. ### Claim 20 (depends on 19) The method of claim 19, wherein the at least one reprogramming factor is expressed using a Sendai virus. ### Claim 21 (depends on 18) The method of claim 18, wherein the at least one reprogramming factor is expressed and wherein expression is later inhibited by the addition of a compound that inhibits expression of the at least one reprogramming factor. ### Claim 22 (depends on 21) The method of claim 21, wherein said compound is a small molecule inhibitor that specifically inhibits the expression of one or more of KLF4, OCT3/4, SOX2 and C-MYC expression. ### Claim 23 (depends on 18) The method of claim 18, wherein expression of the at least one reprogramming factor is inhibited by contacting the T cell derived adherent cells with an agent that activates T cells. ### Claim 24 (depends on 18) The method of claim 18, wherein the at least one reprogramming factor is delivered with a nanoparticle. ### Claim 25 (depends on 1) The method of claim 1, further comprising contacting the T cells with a cytokine. ### Claim 26 (depends on 1) The method of claim 1, wherein said at least one T cell activating agent comprises an antibody that binds CD3 or an antibody that binds CD28 or both; or wherein the at least one T cell activating agent is a tumor antigen. ### Claim 27 (depends on 1) The method of claim 1, further comprising engineering the T cells to express a chimeric antigen receptor (CAR), a T cell Receptor or a hybrid receptor thereof, wherein said T cells are engineered prior to contacting said T cells with said at least one reprogramming factor, and wherein said cell surface receptor recognizes a specific antigen on the surface of a target cell. ### Claim 28 (depends on 1) The method of claim 1, further comprising engineering the T cells to express a CAR, a T cell Receptor or a hybrid receptor thereof, wherein said T cells are engineered after contacting said T cells with said at least one reprogramming factors, and wherein said cell surface receptor recognizes a specific antigen on the surface of a target cell. ### Claim at position 25 29-32. (canceled) ### Claim 33 (depends on 1) The method of claim 1, wherein the resulting T cells comprise an incomplete set of V, D, and J segments of T cell receptor genes. ### Claim 34 (canceled) ### Claim 35 (depends on 1) The method of claim 1, wherein the epigenetic age of the resulting T cells is at least 5% younger than the population of T cells prior to reprogramming. ### Claim 36 (depends on 1) The method of claim 1, wherein the partially reprogrammed T cells are capable of expanding at least 25-fold greater than control T cells. ### Claim 37 (depends on 1) The method of claim 1, wherein contacting said T cells with (i) at least one factor selected from the group consisting of KLF4, OCT3/4, SOX2 and C-MYC; and (ii) SV40 results in a reduction in CD3 and CD8 expression. ### Claim 38 A method of producing T cells, comprising a. culturing T cells in a first medium comprising IL-2 and activating said T cells with at least one antibody specific for CD3 or for CD28, or both; or a tumor antigen, tumor organoid, or tumor cell line; b. contacting said activated T cells with KLF4, OCT3/4, SOX2 and C-MYC, for a period of time in a second culture medium that does not comprise IL-2, or an antibody specific for CD3 or CD28; or tumor antigen, tumor organoid, or tumor cell line; wherein said T cells are not completely reprogrammed into iPS or totipotent cells; c. replacing said second culture medium with a third culture medium comprising IL-2 and at least one antibody specific for CD3 and/or CD28; wherein said contacted T cells are cultured in said third culture medium for at least about 5 days. ### Claim 39 (depends on 38) The method according to claim 38, further comprising expanding said contacted T cells. ### Claim 40 (depends on 38) The method according to claim 38, wherein said T cells are TCRαβ cell, TCRγδ cells, CD4+CD8αβ+ double positive cells, CD4+ single positive cells, naïve T cells, central memory T cells, effector memory T cells, or a combination thereof. ### Claim 41 (depends on 38) The method according to claim 38, wherein said contacting further comprises contacting the activated T cells with SV40. ### Claim 42 (depends on 38) The method of claim 38, wherein the T cells express an activation marker after coculture with autologous tumor cells, wherein the activation marker is CD137, PD1, or LAG3. ### Claim 43 A population of T cells whose epigenetic age is at least 5% younger than its chronological age. ### Claim 44 (canceled) ### Claim 45 A population of T cell derived adherent cells, wherein at least 70% of the cells express CD3 and SSEA4. ### Claim at position 39 46-48. (canceled) ### Claim 49 A population of T cell derived adherent cells, wherein at least 30% of the cells express CD9 or CD90. ### Claim 50 (canceled) ### Claim 51 (depends on 1) A population of T cells produced by the method of claim 1. ### Claim at position 43 52-53. (canceled) ### Claim 54 A population of T cells produced by a method comprising a. contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2 and C-MYC, and optionally (ii) SV40 for a period of time sufficient for the T cells to express at least one marker selected from the group consisting of integrin α6β1, SSEA4, CD9, and CD90 and wherein said T cells are not transformed into iPS or totipotent cells; and b. contacting the T cells of (a) with at least one T cell activating agent. ### Claim 55 (depends on 54) The population of T cells of claim 54, wherein the T cells are contacted with the at least one reprogramming factor for a period of time sufficient for at least a portion of the T cells to express CD3 and at least one marker selected from the group consisting of integrin α6β1, SSEA4, CD9, and CD90. ### Claim at position 46 56-90. (canceled) ### Claim 91 (depends on 1) A method of treating a subject in need thereof with a population of T cells produced by then method of claim 1. ### Claim at position 48 92-95. (canceled) ### Claim 96 A method of producing a rejuvenated tumor infiltrating lymphocyte (TIL), comprising: a. contacting a population of TIL with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2 and C-MYC, and optionally SV40, for a period of time sufficient for T cell derived adherent cells to form and wherein said TIL are not transformed into iPS or totipotent cells; and b. contacting the T cell derived adherent cells with at least one T cell activating compound. ### Claim 97 (canceled) ### Claim 98 A method of producing rejuvenated tumor infiltrating lymphocytes (TILs), comprising a. activating a population of TIL with a first tumor antigen, such that the activated TILs express CD137; optionally, enriching the CD137+ TIL; b. contacting the population of activated TILs with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2 and C-MYC, and (ii) optionally SV40, for a period of time sufficient for TIL-derived adherent cells to form; wherein said TILs are not transformed into iPS or totipotent cells. ### Claim 99 (depends on 98) The method according to claim 98, further comprising contacting the TIL-derived adherent cells with at least one T cell activating compound. ### Claim 100 (depends on 99) The method according to claim 99, wherein the at least one T cell activating compound is the first tumor antigen. ### Claim 101 (depends on 100) The method of claim 100, wherein the first tumor antigen is an autologous tumor cell, an autologous tumor cell line, a tumor organoid or an antigen derived from any of the foregoing. ### Claim at position 55 102-104. (canceled) ### Claim 105 (depends on 98) A population of rejuvenated TILs prepared according to the method of claim 98. ### Claim 106 (depends on 105) The population of rejuvenated TILs according to claim 105, wherein at least 50% of the rejuvenated TILs express both CCR7 and CD62L. ### Claim 107 (canceled) ### Claim 108 (depends on 105) The population of rejuvenated TILs according to claim 105, wherein at least 50% of the rejuvenated TILs express both CCR7 and TCF7. ### Claim 109 (canceled) ### Claim 110 (depends on 98) A method of treating cancer in a patient in need thereof with a population of rejuvenated TILs produced by theft method of claim 98. ### Claim at position 62 111-112. (canceled)