# US 12,091,682 B2 · Methods for making, compositions comprising, and methods of using rejuvenated T cells

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- Number: US 12,091,682 B2
- Document ID: US12091682B2
- Status: Granted
- Kind: B2
- Application number: 17/534,341
- Filed: 2021-11-23
- Published: 2022-05-26
- Granted: 2024-09-17
- 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/US12091682B2

## 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 starting population of T cells with reprogramming factors comprising KLF4, OCT3/4, SOX2, and C-MYC for 6 to 13 days, thereby producing a population of partially reprogrammed cells, wherein the partially reprogrammed cells are not transformed into induced pluripotent stem (iPS) cells; and
b) contacting the partially reprogrammed cells with at least one T cell activating compound, thereby producing a population of rejuvenated T cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the starting population of T cells is contacted with the reprogramming factors for a period of time sufficient for at least a portion of the starting population of T cells to express CD3 and at least one marker selected from the group consisting of integrin α6β1, SSEA4, CD9, and CD90.

### Claim 3 (depends on 2)

The method of claim 2, wherein the starting population of T cells is contacted with the reprogramming factors for a period of time sufficient for at least a portion of the contacted starting population of T cells to express CD3, SSEA4, CD9 and CD90.

### Claim 4 (depends on 1)

The method of claim 1, wherein, prior to contacting the starting population of T cells with the reprogramming factors, the starting population of T cells is activated with IL-2 and at least one agent capable of activating the starting population of T cells.

### Claim 5 (depends on 4)

The method of claim 4, wherein the activated T cells are enriched by selection for cells expressing CD137, PD1, or LAG3.

### Claim 6 (depends on 1)

The method of claim 1, wherein the starting population of T cells is comprised of TCRαβ cells, 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 7 (depends on 1)

The method of claim 1, wherein the reprogramming factors are expressed in the partially reprogrammed cells.

### Claim 8 (depends on 7)

The method of claim 7, wherein the reprogramming factors are expressed using a non-integrating viral vector.

### Claim 9 (depends on 8)

The method of claim 8, wherein the reprogramming factors are expressed using a Sendai virus.

### Claim 10 (depends on 7)

The method of claim 7, further comprising inhibiting expression of the reprogramming factors within the partially reprogrammed cells by adding a compound that inhibits expression of the reprogramming factors.

### Claim 11 (depends on 10)

The method of claim 10, wherein the 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 12 (depends on 7)

The method of claim 7, wherein expression of the reprogramming factors are inhibited by contacting the partially reprogrammed cells with an agent that activates T cells.

### Claim 13 (depends on 7)

The method of claim 7, wherein the at least one reprogramming factors are delivered with a nanoparticle.

### Claim 14 (depends on 1)

The method of claim 1, further comprising contacting the starting population of T cells with a cytokine.

### Claim 15 (depends on 1)

The method of claim 1, wherein the at least one T cell activating compound comprises an antibody or an antigen binding fragment thereof that binds CD3 or an antibody or an antigen binding fragment thereof that binds CD28 or both; or wherein the at least one T cell activating compound is a tumor antigen.

### Claim 16 (depends on 1)

The method of claim 1, further comprising engineering the starting population of T cells to express a chimeric antigen receptor (CAR), a T cell Receptor or a hybrid receptor thereof, wherein the starting population of T cells is engineered prior to contacting the starting population of T cells with the reprogramming factors, and wherein the cell surface receptor recognizes a specific antigen on the surface of a target cell.

### Claim 17 (depends on 1)

The method of claim 1, further comprising engineering the partially reprogrammed cells to express a CAR, a T cell Receptor or a hybrid receptor thereof, wherein the partially reprogrammed cells are engineered after contacting the starting population of T cells with the reprogramming factors, and wherein the cell surface receptor recognizes a specific antigen on the surface of a target cell.

### Claim 18 (depends on 1)

The method of claim 1, wherein the rejuvenated T cells comprise an incomplete set of V, D, and J segments of T cell receptor genes.

### Claim 19 (depends on 1)

The method of claim 1, wherein the epigenetic age of the rejuvenated T cells is at least 5% younger than the starting population of T cells prior to reprogramming.

### Claim 20 (depends on 1)

The method of claim 1, wherein the partially reprogrammed cells are capable of expanding at least 25-fold greater than control T cells.

### Claim 21 (depends on 1)

The method of claim 1, wherein contacting the starting population of T cells with the reprogramming factors results in a reduction in CD3 and CD8 expression.

### Claim 22 (depends on 1)

The method of claim 1, further comprising contacting the starting population of T cells with SV40, LIN28, NANOG, or a combination thereof.

### Claim 23 (depends on 1)

The method of claim 1, wherein the starting population of T cells is contacted with the reprogramming factors of (a) for a period of about 7 days before (b) contacting the partially reprogrammed cells with the at least one T cell activating compound.

### Claim 24 (depends on 1)

The method of claim 1, wherein the partially reprogrammed cells are contacted with the at least one T cell activating compound about 7 days after the starting population of T cells is contacted with the reprogramming factors.

### Claim 25 (depends on 1)

The method of claim 1, wherein the starting population of T cells is contacted with the reprogramming factors of (a) for 6 days before (b) contacting the partially reprogrammed cells with the at least one T cell activating compound.

### Claim 26 (depends on 1)

The method of claim 1, wherein the partially reprogrammed cells are contacted with the at least one T cell activating compound 6 days after the starting population of T cells is contacted with the reprogramming factors.

### Claim 27

A method of producing T cells, comprising:
a) culturing a starting population of T cells in a first medium comprising IL-2 and activating the T cells with at least one antibody or an antigen binding fragment thereof specific for CD3 or for CD28, or both; or a tumor antigen, tumor organoid, or tumor cell line;
b) contacting the activated T cells with KLF4, OCT3/4, SOX2, and C-MYC for 6 to 13 days in a second culture medium that does not comprise IL-2, or an antibody or an antigen binding fragment thereof specific for CD3 or CD28; or tumor antigen, tumor organoid, or tumor cell line, thereby producing a population of partially reprogrammed cells, wherein the partially reprogrammed cells are not completely reprogrammed into iPS cells; and
c) replacing the second culture medium with a third culture medium comprising IL-2 and at least one antibody or an antigen binding fragment thereof specific for CD3 and/or CD28, wherein the contacted partially reprogrammed cells are cultured in the third culture medium for at least about 5 days.

### Claim 28 (depends on 27)

The method of claim 27, further comprising expanding the contacted partially reprogrammed cells.

### Claim 29 (depends on 27)

The method of claim 27, wherein the starting population of T cells is comprised of TCRαβ cells, 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 30 (depends on 27)

The method of claim 27, wherein the starting population of T cells is contacted with autologous tumor cells in step (a) and express an activation marker after coculture with the autologous tumor cells, wherein the activation marker is CD137, PD1, or LAG3.

### Claim 31 (depends on 27)

The method of claim 27, wherein the activated T cells are contacted with KLF4, OCT3/4, SOX2 and C-MYC for a period of about 7 days before (c) replacing the second culture medium with a third culture medium comprising IL-2 and at least one antibody specific for CD3 and/or CD28.

### Claim 32 (depends on 27)

The method of claim 27, wherein the activated T cells are contacted with KLF4, OCT3/4 SOX2 and C-MYC for 6 days before (c) replacing the second culture medium with a third culture medium comprising IL-2 and at least one antibody specific for CD3 and/or CD28;
contacting the first cultured cell population with a first culturing medium comprising IL-2 to obtain a second cultured cell population;
contacting the second cultured cell population with a second culturing medium comprising IL-2 and IL-12 to obtain a third cultured cell population; and
contacting the third cultured cell population with a third culturing medium comprising IL-2, IL-12 and IL-18 after contacting with the second culturing medium to obtain a fourth cultured cell population that comprises the modified T cells;
wherein each of the inducing culturing medium, the first culturing medium, the second culturing medium, and the third culturing medium further comprises L-glutamine, streptomycin sulfate, gentamicin sulfate and human platelet lysate.