# US 12,215,348 B2 · Methods for producing genetically engineered cell compositions and related compositions

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- Number: US 12,215,348 B2
- Document ID: US12215348B2
- Status: Granted
- Kind: B2
- Application number: 18/506,851
- Filed: 2023-11-10
- Published: 2024-03-07
- Granted: 2025-02-04
- Earliest claimed: 2017-08-09
- Assignees: Juno Therapeutics, Inc.
- Applicants: Juno Therapeutics, Inc.
- Inventors: Mirna Mujacic; Ayu Rahardjo
- Web page: https://patentgrep.com/patent/US12215348B2

## CPC

- A61K 39/39558: {against tumor tissues, cells, antigens}
- C12N 5/0636: {T lymphocytes}
- 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 40/4215: {Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30}
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/86: Viral vectors
- G01N 33/56972: {White blood cells}
- A61K 2039/545: {characterised by the dose, timing or administration schedule}
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C12N 2502/1114: T cells
- C12N 2740/13043: viral genome or elements thereof as genetic vector
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- G01N 2333/70514: CD4
- G01N 2333/70517: CD8
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

Provided herein are methods and compositions for generating engineered cells, such as cells expressing a recombinant receptor, including methods involving stimulation and/or engineering of an input composition having a defined ratio of naïve-like CD4+ T cells to naïve-like CD8+ T cells. In particular, the methods can be used to engineer T cells with genetically engineered receptors, such as genetically engineered antigen receptors such as engineered (recombinant) TCRs and chimeric antigen receptors (CARs), or other recombinant chimeric receptors. Features of the methods include producing a more consistent and/or predictable T cell product and/or a product with lower toxicity compared with other methods.

## Claims

### Claim 1

A method of generating a composition of engineered cells, the method comprising:
contacting an input cell composition comprising naïve-like CD4+ T cells and naïve-like CD8+ T cells from a biological sample from a subject with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition;
wherein the naïve-like CD4+ T cells and the naïve-like CD8+ T cells are surface positive for CD45RA, CD27 and/or CCR7 and are surface negative for CD45RO and/or CD62L;
wherein the ratio of naïve-like CD4+ T cells to naïve-like CD8+ T cells present in the input cell composition is between 10:1 and 0.05:1, inclusive; and
wherein the method produces recombinant receptor expressing T cells.

### Claim 2 (depends on 1)

The method of claim 1, further comprising stimulating the cells, prior to, during and/or subsequent to said contacting, wherein stimulating comprises incubating the cells in the presence of one or more stimulating agents, wherein stimulating results in activation and/or proliferation of the cells.

### Claim 3

A method of generating a composition of engineered cells, the method comprising:
(i) stimulating an input cell composition comprising naïve-like CD4+ T cells and naïve-like CD8+ T cells from a biological sample from a subject in the presence of one or more stimulating agents, wherein the naïve-like CD4+ T cells and the naïve-like CD8+ T cells are surface positive for CD45RA, CD27 and/or CCR7 and are surface negative for CD45RO and/or CD62L, wherein the ratio of naïve-like CD4+ T cells to naïve-like CD8+ T cells present in the input cell composition is between 10:1 and 0.05:1, inclusive; and wherein stimulating results in activation of the cells;
(ii) contacting the input cell composition with an agent comprising a nucleic acid molecule encoding a recombinant receptor under conditions to introduce the nucleic acid encoding the recombinant receptor into cells in the composition; and
(iii) stimulating the cells subsequent to said contacting, wherein stimulating comprises incubating the cells in the presence of one or more stimulating agents and wherein stimulating results in proliferation of the cells,
wherein the method produces recombinant receptor expressing T cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the naïve-like CD4+ T cells and the naïve-like CD8+ T cells are surface positive for CD45RA and CCR7.

### Claim 5 (depends on 1)

The method of claim 1, wherein the naïve-like CD4+ T cells and the naïve-like CD8+ T cells are surface positive for CD27 and CCR7.

### Claim 6 (depends on 1)

The method of claim 1, wherein the naïve-like CD4+ T cells and the naïve-like CD8+ T cells are surface positive for CCR7 and surface negative for CD62L.

### Claim 7 (depends on 1)

The method of claim 1, wherein the input cell composition comprises a ratio of naïve-like CD4+ T cells to naïve-like CD8+ T cells of between 0.8:1 and 2.2:1, each inclusive.

### Claim 8 (depends on 4)

The method of claim 4, wherein the input cell composition comprises a ratio of CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells of between 0.8:1 and 2.2:1, each inclusive.

### Claim 9 (depends on 4)

The method of claim 4, wherein the input cell composition comprises a ratio of CCR7+CD45RA+CD4+ cells to CCR7+CD45RA+CD8+ cells of or about 1.4:1, 1.1:1, or 1.0:1.

### Claim 10 (depends on 5)

The method of claim 5, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of between 1.2:1 and 2.4:1, each inclusive.

### Claim 11 (depends on 5)

The method of claim 5, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of or about 2.7:1.

### Claim 12 (depends on 5)

The method of claim 5, wherein the input cell composition comprises a ratio of CD27+CCR7+CD4+ cells to CD27+CCR7+CD8+ cells of or about 1.69:1.

### Claim 13 (depends on 6)

The method of claim 6, wherein the input cell composition comprises a ratio of CD62L-CCR7+CD4+ cells to CD62L-CCR7+CD8+ cells of between 0.8:1 and 2.2:1, each inclusive.

### Claim 14 (depends on 6)

The method of claim 6, wherein the input cell composition comprises a ratio of CD62L-CCR7+CD4+ cells to CD62L-CCR7+CD8+ cells of or about 2.2:1, 2:1, 1.6:1, or 1.66:1.

### Claim 15 (depends on 1)

The method of claim 1, wherein the composition of engineered cells comprises at least 50%, at least 70%, at least 90%, or 100% of cells that express the recombinant receptor.

### Claim 16 (depends on 1)

The method of claim 1, wherein the ratio of recombinant receptor expressing CD4+ T cells to recombinant receptor expressing CD8+ T cells is or is about 8.6:1, 7.6:1, 2.6:1, 2.0:1, 1.6:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, or 1.0:1.

### Claim 17 (depends on 1)

The method of claim 1, wherein the ratio of recombinant receptor expressing CD4+ T cells to recombinant receptor expressing CD8+ T cells is or is about 2.6:1 or 2.0:1.

### Claim 18 (depends on 1)

The method of claim 1, wherein the ratio of recombinant receptor expressing CD27+CCR7+CD4+ T cells to recombinant receptor expressing CD27+CCR7+CD8+ T cells is or is about 8.6:1, 7.6:1, 2.6:1, 2.0:1, 1.6:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, or 1.0:1.

### Claim 19 (depends on 1)

The method of claim 1, wherein the ratio of recombinant receptor expressing CCR7+CD45RA+CD4+ T cells to recombinant receptor expressing CCR7+CD45RA+CD8+ T cells is or is about 8.6:1, 7.6:1, 2.6:1, 2.0:1, 1.6:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, or 1.0:1.

### Claim 20 (depends on 1)

The method of claim 1, wherein the ratio of recombinant receptor expressing CD62L-CCR7+CD4+ T cells to recombinant receptor expressing CD62L-CCR7+CD8+ T cells is or is about 8.6:1, 7.6:1, 2.6:1, 2.0:1, 1.6:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, or 1.0:1.

### Claim 21 (depends on 1)

The method of claim 1, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

### Claim 22 (depends on 1)

The method of claim 1, wherein the biological sample is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.

### Claim 23 (depends on 1)

The method of claim 1, wherein the subject is a human subject.

### Claim 24 (depends on 1)

The method of claim 1, wherein the input cell composition comprises from or from about 1×107 to 5×109 total cells or total T cells, or of viable populations of any of the foregoing.

### Claim 25 (depends on 3)

The method of claim 3, wherein the one or more one stimulating agents is selected from the group consisting of CD3-binding molecules; CD28-binding molecules; recombinant IL-2; recombinant IL-15; and recombinant IL-7, a vaccine comprising an antigen specifically recognized by the antigen receptor, and an anti-idiotype antibody that specifically binds the antigen receptor or combinations thereof.

### Claim 26 (depends on 25)

The method of claim 25, wherein the one or more one stimulating agents comprises CD3-binding molecules and CD28-binding molecules.