# US 12,760,274 B2 · Compositions and methods of stimulating and expanding T cells

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- Number: US 12,760,274 B2
- Document ID: US12760274B2
- Status: Granted
- Kind: B2
- Application number: 17/932,188
- Filed: 2022-09-14
- Published: 2023-04-13
- Granted: 2026-10-13
- Earliest claimed: 2014-10-31
- Assignees: The Trustees of the University of Pennsylvania
- Applicants: The Trustees of the University of Pennsylvania
- Inventors: Yangbing Zhao; Xiaojun Liu; Carl H. June
- Web page: https://patentgrep.com/patent/US12760274B2

## CPC

- C12N 5/0636: {T lymphocytes}
- A01N 1/10: Preservation of living parts
- 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 40/4205: {Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4}
- A61K 40/4211: {CD19 or B4}
- A61K 40/4269: {NY-ESO}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C07K 14/7151: {for tumor necrosis factor [TNF], for lymphotoxin [LT]}
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/30: from tumour cells
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- A61K 2039/505: {comprising antibodies}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C12N 2501/51: B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
- C12N 2510/00: Genetically modified cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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)

## Abstract

The present invention includes a method for expanding a population of electroporated T cells. The method includes electroporating a population of cells comprising T cells with mRNA encoding a chimeric membrane protein comprising an antigen binding domain to a molecule and an intracellular domain of a co-stimulatory molecule, wherein the cultured T cells expand at least 10 fold. The invention further includes an expanded population of T cells, compositions comprising the cells and methods of treatment.

## Claims

### Claim 1

A T cell comprising a nucleic acid encoding a chimeric membrane protein comprising:
an extracellular domain comprising an antigen binding domain comprising an anti-CD3 antibody or fragment thereof;
a transmembrane domain; and
an intracellular domain comprising a fragment of an intracellular domain of CD28 and a fragment of an intracellular domain of 4-1BB;
wherein the intracellular domain does not comprise a CD3-zeta domain;
wherein the chimeric membrane protein is expressed on the surface of the T cell; and
wherein the T cell is capable of expansion to generate a population of expanded T cells upon an interaction between the anti-CD3 antibody or fragment thereof and CD3.

### Claim 2 (depends on 1)

The T cell of claim 1, wherein the anti-CD3 antibody or fragment thereof is a synthetic antibody, human antibody, humanized antibody, single domain antibody, single chain variable fragment, or antigen-binding fragments thereof.

### Claim 3 (depends on 1)

The T cell of claim 1, wherein the chimeric membrane protein further comprises a hinge domain.

### Claim 4 (depends on 1)

The T cell of claim 1, wherein the interaction expands the T cell by at least 10-fold within 7 to 10 days of the interaction.

### Claim 5 (depends on 1)

The T cell of claim 1, wherein the interaction expands the T cell by about 20 fold to about 50 fold.

### Claim 6 (depends on 1)

The T cell of claim 1, wherein the anti-CD3 antibody or fragment thereof is an anti-CD3 single chain variable fragment (scFv), the transmembrane domain is a CD28 transmembrane domain, and the chimeric membrane protein further comprises a CD8 hinge.

### Claim 7 (depends on 1)

The T cell of claim 1, wherein the anti-CD3 antibody or the fragment thereof is OKT3, H5L1, or a fragment thereof.

### Claim 8 (depends on 1)

The T cell of claim 1, wherein the anti-CD3 antibody or the fragment thereof is a scFv of OKT3 or H5L1.

### Claim 9 (depends on 1)

The T cell of claim 1 further comprising a chimeric antigen receptor (CAR).

### Claim 10 (depends on 1)

A population of T cells comprising the T cell of claim 1.

### Claim 11 (depends on 10)

The population of T cells of claim 10, wherein from 54.54% to 69.21% of the T cells express CCR7 protein, CD62L protein, and CD45RO protein.