# US 12,215,137 B2 · Compositions and methods for T cell delivery of therapeutic molecules

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- Number: US 12,215,137 B2
- Document ID: US12215137B2
- Status: Granted
- Kind: B2
- Application number: 17/038,676
- Filed: 2020-09-30
- Published: 2021-01-14
- Granted: 2025-02-04
- Earliest claimed: 2015-01-31
- Assignees: The Trustees of the University of Pennsylvania
- Applicants: The Trustees of the University of Pennsylania
- Inventors: Yangbing Zhao; Carl H. June; Xiaojun Liu
- Web page: https://patentgrep.com/patent/US12215137B2

## CPC

- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- A61K 39/39558: {against tumor tissues, cells, antigens}
- 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 40/4205: {Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4}
- A61K 40/4211: {CD19 or B4}
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- C07K 14/70521: {CD28, CD152}
- C07K 14/71: for growth factors; for growth regulators
- C07K 14/7155: {for interleukins [IL]}
- C07K 16/2818: {against CD28 or CD152}
- C07K 16/2827: {against B7 molecules, e.g. CD80, CD86}
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C12N 5/0636: {T lymphocytes}
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- C07K 2317/31: multispecific
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/02: containing a signal sequence
- C07K 2319/10: containing a tag for extracellular membrane crossing, e.g. TAT or VP22
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- C12N 2501/515: CD3, T-cell receptor complex
- C12N 2501/599: with CD designations not provided for elsewhere
- C12N 2510/00: Genetically modified cells
- 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)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention includes compositions and methods for modifying a T cell with a nucleic acid encoding a switch molecule comprising an extracellular domain comprising a membrane receptor or fragment thereof and an intracellular domain comprising a signaling receptor or fragment thereof. In one aspect, a method comprises introducing a nucleic acid encoding a switch molecule and a nucleic acid encoding a soluble fusion protein and/or a nucleic acid encoding a bispecific antibody into a population of cells comprising T cells, wherein the T cells transiently expresses the switch molecule and soluble fusion protein or bispecific antibody. In other aspect, compositions of T cells and methods of treating a disease or condition, such as cancer or an autoimmune disease, are also included.

## Claims

### Claim 1

A method for enhancing T cell antitumor activity in a subject in need thereof, the method comprising parenterally administering to the subject a composition comprising modified CAR T cells comprising a nucleic acid sequence encoding a switch molecule and a nucleic acid sequence encoding a CAR comprising an antigen binding domain that targets a tumor antigen, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain;
wherein the switch molecule comprises:
(a) an extracellular domain of a transforming growth factor-beta receptor (TGF-beta-R), an intracellular domain of a signaling receptor selected from the group consisting of interleukin-12 receptor (IL-12R), CD3, CD28, CD137, CD27, ICOS, and OX40, and a transmembrane domain derived from the same molecule as the extracellular domain or the same molecule as the intracellular domain;
(b) an extracellular domain of a programmed cell death 1 (PD1) receptor, an intracellular domain of a signaling receptor selected from the group consisting of interleukin-12 receptor (IL-12R), CD3, CD28, CD137, CD27, ICOS, and OX40, and a transmembrane domain derived from the same molecule as the extracellular domain or the same molecule as the intracellular domain;
(c) an extracellular domain of a programmed cell death ligand 1 (PDL1) receptor, an intracellular domain of a signaling receptor selected from the group consisting of interleukin-12 receptor (IL-12R), CD3, CD28, CD137, CD27, ICOS, and OX40, and a transmembrane domain derived from the same molecule as the extracellular domain or the same molecule as the intracellular domain; or
(d) an extracellular domain of an interferon-gamma receptor (IFN-gamma) receptor, an intracellular domain of a signaling receptor selected from the group consisting of interleukin-12 receptor (IL-12R), CD3, CD28, CD137, CD27, ICOS, and OX40, and a transmembrane domain derived from the same molecule as the extracellular domain or the same molecule as the intracellular domain;
wherein the modified CAR T cells home to a target tumor site; and
wherein when the modified CAR T cells express the switch molecule, interaction of the extracellular domain of the switch molecule with TGFβ, PD1, PDL1, or IFN-γ in the tumor microenvironment converts an inhibitory signal of the TGFβ receptor, the PD1 receptor, the PDL1 receptor, or the IFN-γ receptor into an IL-12R, CD3, CD28, CD137, CD27, ICOS, or OX40 stimulatory signal thereby enhancing the antitumor activities of the modified CAR T cells.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the switch molecule comprises the extracellular domain of TGF-beta-R.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the signaling receptor is IL-12R.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the nucleic acid comprises in vitro transcribed RNA or synthetic RNA.

### Claim 5 (depends on 1)

The method according to claim 1, wherein enhancing the antitumor activities of the modified CAR T cells comprises inducing a secretion of an activation factor at the target tumor site, and wherein the activation factor is a soluble cytokine selected from the group consisting of IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, TNF, TGF, IFN, and functional fragments and variants thereof.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the tumor target site is selected from the group consisting of brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, skin cancer, thymoma, sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, uterine cancer, and combinations thereof.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the modified CAR T cells home to a solid tumor site.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the modified CAR T cells home to a cancer expressing a tumor antigen selected from the group consisting of p53, Ras, beta-Catenin, CDK4, alpha-Actinin-4, Tyrosinase, TRP1/gp75, TRP2, gp100, Melan-A/MART1, Gangliosides, PSMA, HER2, WT1, EphA3, EGFR, CD19, CD20, MAGE, BAGE, GAGE, NY-ESO-1, Telomerase, Survivin, and any combination thereof.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the modified CAR T cells express a nucleic acid encoding TGF-beta-IL-12 switch molecule and a nucleic acid encoding a CD19 CAR or a HER2 CAR.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the modified CAR T cells are activated.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the modified CAR T cells induce lysis of cells and/or tissues at the tumor target site.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the modified CAR T cells are cryopreserved.

### Claim 13 (depends on 1)

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

### Claim 14 (depends on 1)

The method according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the CAR targets a tumor antigen selected from the group consisting of TRP1/gp75, TRP2, gp100, Melan-A/MART1, Gangliosides, PSMA, HER2, WT1, EphA3, EGFR, CD19, CD20, MAGE, and NY-ESO-1.

### Claim 16 (depends on 1)

The method according to claim 1, wherein the switch molecule is expressed transiently.

### Claim 17 (depends on 1)

The method according to claim 1, wherein the composition is administered to the subject more than once.

### Claim 18 (depends on 1)

The method according to claim 1, wherein the modified CART cells are autologous or allogeneic to the subject.

### Claim 19 (depends on 1)

The method according to claim 1, wherein the antitumor activity is for a cancer selected from the group consisting of brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, skin cancer, thymoma, sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, uterine cancer, and combinations thereof.

### Claim 20 (depends on 1)

The method according to claim 1, wherein the transmembrane domain is a CD28 transmembrane domain.

### Claim 21

A method for enhancing T cell antitumor activity in a subject in need thereof, the method comprising parenterally administering to the subject a composition comprising modified T cells comprising a nucleic acid sequence encoding a switch molecule and a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that targets a tumor antigen,
wherein the tumor antigen is selected from the group consisting of p53, Ras, beta-Catenin, CDK4, alpha-Actinin-4, Tyrosinase, TRP1/gp75, TRP2, gp100, Melan-A/MART1, Gangliosides, PSMA, HER2, WT1, EphA3, EGFR, CD19, CD20, MAGE, BAGE, GAGE, NY-ESO-1, telomerase, and survivin;
wherein the switch molecule comprises:
(a) an extracellular domain of a transforming growth factor-beta receptor (TGF-beta-R), an intracellular domain of a signaling receptor selected from the group consisting of interleukin-12 receptor (IL-12R), CD3, CD28, CD137, CD27, ICOS, and OX40, and a transmembrane domain derived from the same molecule as the extracellular domain; or the same molecule as the intracellular domain;
(b) an extracellular domain of a programmed cell death 1 (PD1) receptor, an intracellular domain of a signaling receptor selected from the group consisting of interleukin-12 receptor (IL-12R), CD3, CD28, CD137, CD27, ICOS, and OX40, and a transmembrane domain derived from the same molecule as the extracellular domain, or the same molecule as the intracellular domain;
(c) an extracellular domain of a programmed cell death ligand 1 (PDL1) receptor, an intracellular domain of a signaling receptor selected from the group consisting of interleukin-12 receptor (IL-12R), CD3, CD28, CD137, CD27, ICOS, and OX40, and a transmembrane domain derived from the same molecule as the extracellular domain, or the same molecule as the intracellular domain; or
(d) an extracellular domain of an interferon-gamma receptor (IFN-gamma) receptor, an intracellular domain of a signaling receptor selected from the group consisting of interleukin-12 receptor (IL-12R), CD3, CD28, CD137, CD27, ICOS, and OX40, and a transmembrane domain derived from the same molecule as the extracellular domain, or the same molecule as the intracellular domain;
wherein the modified T cells home to a target tumor site; and
wherein when the modified T cells express the switch molecule, interaction of the extracellular domain of the switch molecule with TGFβ, PD1, PDL1, or IFN-γ in the tumor site converts the inhibitory signal of the TGFβ receptor, the PD1 receptor, the PDL1 receptor, or the IFN-γ receptor into the IL-12R, the CD3, the CD28, the CD137, the CD27, the ICOS, or the OX40 stimulatory signal, thereby enhancing antitumor activities of the modified T cells.