# US 11,028,143 B2 · Enhanced antigen presenting ability of RNA CAR T cells by co-introduction of costimulatory molecules

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- Number: US 11,028,143 B2
- Document ID: US11028143B2
- Status: Granted
- Kind: B2
- Application number: 15/113,044
- Filed: 2015-01-21
- Published: 2016-11-24
- Granted: 2021-06-08
- Earliest claimed: 2014-01-21
- Assignees: Novartis AG; The Trustees of the University of Pennsylvania
- Applicants: Novartis AG; The Trustees of the University of Pennsylvania
- Inventors: Yangbing Zhao; Devon J. Shedlock; Carl H. June; Xiaojun Liu
- Web page: https://patentgrep.com/patent/US11028143B2

## CPC

- C07K 14/70517: {CD8}
- A61K 39/0011: {Cancer antigens}
- A61K 39/02: Bacterial antigens
- A61K 39/12: Viral antigens
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61K 39/3955: {against proteinaceous materials, e.g. enzymes, hormones, lymphokines}
- 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 40/4224: {Molecules with a "CD" designation not provided for elsewhere}
- A61K 40/4255: {Mesothelin [MSLN]}
- A61K 40/4272: {Melan-A/MART}
- C07K 14/54: Interleukins [IL]
- C07K 14/5418: {IL-7}
- C07K 14/5443: {IL-15}
- C07K 14/55: IL-2
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70532: {B7 molecules, e.g. CD80, CD86}
- C07K 14/70575: {NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154 (NGF C07K14/48, TNF C07K14/525)}
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]}
- 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 33/505: {involving T-cells}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 2039/5158: {Antigen-pulsed cells, e.g. T-cells}
- C07K 2317/53: Hinge
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C12N 2501/51: B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
- C12N 2501/515: CD3, T-cell receptor complex
- C12N 2510/00: Genetically modified cells
- C12N 2760/16034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The invention provides T cells comprising nucleic acid sequence encoding a chimeric antigen receptor and a nucleic acid sequence encoding an enhancer of T cell priming, compositions including the T cells, and methods of using the T cells to treat diseases associated with the expression of disease-associated antigens.

## Claims

### Claim 1

A T cell comprising an exogenous nucleic acid, wherein:
(a) the nucleic acid comprises a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a costimulatory signaling domain that is a functional signaling domain from a protein selected from the group consisting of OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a/CD18), ICOS and 4-1BB (CD137), or any combination thereof, and
(b) the nucleic acid comprises a second nucleic acid sequence encoding a polypeptide which enhances T cell priming, or a functional fragment or variant thereof, wherein the polypeptide is selected from the group consisting of CD70, CD86, and CD252,
wherein the polypeptide is expressed at a level that: (1) the T cell enhances the priming of another T cell that does not comprise the exogenous nucleic acid and (2) expression of the polypeptide does not inhibit the cell-killing function of the CAR by more than 40%, provided that
(i) the first and/or second nucleic acid sequence comprises an RNA; or
(ii) the CAR further comprises a second intracellular signaling domain.

### Claim 2 (depends on 1)

The T cell of claim 1, wherein:
(i) the first and second nucleic acid sequences are disposed on a single nucleic acid molecule,
(ii) the first and second nucleic acid sequences are disposed on a single nucleic acid molecule, wherein the single nucleic acid molecule comprises an RNA,
(iii) the first and second nucleic acid sequences are disposed on a single nucleic acid molecule, wherein the single nucleic acid molecule comprises a DNA,
(iv) the first and second nucleic acid sequences are disposed on two or more distinct nucleic acid molecules,
(v) the first and second nucleic acid sequences are disposed on two or more distinct nucleic acid molecules, wherein one or both nucleic acid molecules comprise RNA,
(vi) the first and second nucleic acid sequences are disposed on two or more distinct nucleic acid molecules, wherein one or both nucleic acid molecules comprise DNA, or
(vii) the first and second nucleic acid sequences are disposed on two or more distinct nucleic acid molecules, wherein one nucleic acid molecule comprises an RNA and the other nucleic acid molecule comprises a DNA.

### Claim 3 (depends on 1)

The T cell of claim 1, wherein:
(i) the first nucleic acid sequence comprises an RNA,
(ii) the second nucleic acid sequence comprises an RNA, or
(iii) the first and the second nucleic acid sequences each comprise RNA.

### Claim 4 (depends on 3)

The T cell of claim 3, wherein:
(i) the T cell is transfected to transiently express the first and/or second RNAs,
(ii) the cell does not comprise an exogenous DNA encoding the first or second RNA,
(iii) the first and/or second RNAs are generated by in vitro transcription,
(iv) the first and/or second RNAs are synthetic RNAs, or
(v) the first and/or second RNAs are introduced into the T cell by electroporation.

### Claim 5 (depends on 1)

The T cell of claim 1, wherein the first and/or second nucleic acid sequence comprises DNA or cDNA.

### Claim 6 (depends on 1)

The T cell of claim 1, wherein:
(i) the first and/or second nucleic acid sequence comprises a vector,
(ii) the first and/or second nucleic acid sequence comprises a viral vector,
(iii) the first and/or second nucleic acid sequence comprises a retroviral vector or a lentiviral vector, or
(iv) the T cell is virally transduced to express the first and/or second nucleic acid sequence.

### Claim 7 (depends on 1)

The T cell of claim 1, wherein the extracellular domain of the CAR comprises an antigen-binding domain, wherein:
(i) the antigen-binding domain is a scFv domain,
(ii) the antigen-binding domain binds to an antigen associated with a disease state, wherein the disease state is selected from the group consisting of a proliferative disease, a precancerous condition, a non-cancer indication, a viral infection, and a bacterial infection,
(iii) the antigen-binding domain binds to a tumor antigen, a viral antigen, or a bacterial antigen, or
(iv) the antigen-binding domain binds to a tumor antigen, wherein the tumor antigen is an antigen associated with 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 8 (depends on 1)

The T cell of claim 1, wherein:
(i) the transmembrane domain comprises a transmembrane protein of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154,
(ii) the intracellular signaling domain comprises a functional signaling domain of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, or DAP12,
(iii) the second intracellular signaling domain comprises a costimulatory signaling domain,
(iv) the intracellular signaling domain comprises a CD3zeta domain and the second intracellular signaling domain comprises a 4-1BB domain, or
(v) the extracellular domain is connected to the transmembrane domain by a hinge region.

### Claim 9 (depends on 1)

The T cell of claim 1, wherein:
(i) the T cell has enhanced antigen presentation ability relative to a T cell which lacks the second nucleic acid sequence,
(ii) the T cell has increased efficacy in killing tumor cells or reducing tumor size in a subject with a tumor relative to a T cell comprising only the first nucleic acid sequence, or
(iii) the T cell enhances the priming of T cells with a tumor antigen, a viral antigen, a bacterial antigen.

### Claim 10 (depends on 1)

The T cell of claim 1, wherein the T cell is transfected to transiently express a nucleic acid comprising a third nucleic acid sequence encoding a polypeptide which enhances T cell priming, or a functional fragment or variant thereof, which differs from the polypeptide encoded by the second nucleic acid sequence.

### Claim 11 (depends on 10)

The T cell of claim 10, wherein:
(i) the T cell has increased T cell priming ability relative to a T cell comprising the first nucleic acid sequence and second nucleic acid sequence, but not the third nucleic acid sequence,
(ii) the third nucleic acid sequence comprises an RNA,
(iii) the third nucleic acid sequence comprises an RNA, wherein the T cell is transfected to transiently express the third RNA,
(iv) the third nucleic acid sequence comprises an RNA, wherein the cell does not comprise an exogenous DNA encoding the third RNA, or
(v) the CAR comprises one or more costimulatory signaling domains, and the third nucleic acid sequence comprises DNA.

### Claim 12 (depends on 10)

The T cell of claim 10, wherein the T cell further comprises one or more additional distinct nucleic acid sequences encoding a polypeptide which enhances T cell priming, or a functional fragment or variant thereof, which differs from the polypeptides encoded by the second and third nucleic acid sequences.

### Claim 13 (depends on 12)

The T cell of claim 12, wherein:
(i) the one or more additional nucleic acid sequences comprise RNA, or
(ii) the CAR comprises one or more costimulatory signaling domains, and the one or more additional nucleic acids comprise DNA.

### Claim 14 (depends on 1)

A composition comprising the T cell of claim 1.

### Claim 15 (depends on 14)

The composition of claim 14, further comprising a second therapeutic agent.

### Claim 16

A method of generating a T cell having enhanced anti-tumor activity, the method comprising introducing a nucleic acid in a T cell, wherein:
(a) the nucleic acid comprises a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a costimulatory signaling domain that is a functional signaling domain from a protein selected from the group consisting of OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a/CD18), ICOS and 4-1BB (CD137), or any combination thereof, and
(b) the nucleic acid comprises a second nucleic acid sequence encoding a polypeptide which enhances T cell priming, or a functional fragment or variant thereof, wherein the polypeptide is selected from the group consisting of CD70, CD86, and CD252,
wherein the polypeptide is expressed at a level that: (1) the T cell enhances the priming of another T cell that does not comprise the nucleic acid and (2) expression of the polypeptide does not inhibit the cell-killing function of the CAR by more than 40%,
provided that
(i) the first and/or second nucleic acid sequence comprises an RNA;
or
(ii) the CAR further comprises a second intracellular signaling domain.