# US 2022/0033775 A1 · Expansion of tils utilizing akt pathways inhibitors

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- Number: US 2022/0033775 A1
- Document ID: US20220033775A1
- Status: Published application
- Kind: A1
- Application number: 17/290,639
- Filed: 2019-11-04
- Published: 2022-02-03
- Earliest claimed: 2018-11-05
- Assignees: Iovance Biotherapeutics, Inc.
- Applicants: Iovance Biotherapeutics, Inc.
- Inventors: Cecile Chartier-Courtaud; Maria Fardis
- Web page: https://patentgrep.com/patent/US20220033775A1

## CPC

- A61K 38/2013: {IL-2}
- 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 40/42: Cancer antigens
- C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]}
- C12N 2501/2302: Interleukin-2 (IL-2)
- C12N 2501/2315: Interleukin-15 (IL-15)
- C12N 2501/2321: Interleukin-21 (IL-21)
- C12N 2501/515: CD3, T-cell receptor complex
- C12N 2501/727: Kinases (EC 2.7.)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2502/11: blood or immune system cells
- 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)

## Abstract

In some embodiments, methods of expanding tumor infiltrating lymphocytes using AKT pathway inhibitors and methods of using the expanded tumor infiltrating lymphocytes in the treatment of human diseases, including cancers, are disclosed.

## Claims

### Claim 1

A method for preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy, the method comprising:
(a) treating a tumor fragment with a first cell culture medium comprising interleukin 2 (IL-2), OKT-3 antibody, and one or more AKT pathway inhibitor (AKTi) to provide TILs;
(b) expanding the TILs with a second cell culture medium comprising cell culture media, irradiated feeder cells, OKT-3 antibody, and IL-2 to provide an expanded number of TILs; and,
(c) optionally cryopreserving the expanded number of TILs.

### Claim 2 (depends on 1)

The method of claim 1 wherein the one or more AKT pathway inhibitor is an AKT kinase inhibitor.

### Claim 3 (depends on 2)

The method of claim 2, wherein the AKT kinase inhibitor is selected from the group consisting of afuresertib, uprosertib, ipatasertib, AT7867, AT13148, and pharmaceutically acceptable salts, solvates, hydrates, cocrystals, or prodrugs thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein the one or more AKT pathway inhibitor is an mTOR inhibitor.

### Claim 5 (depends on 4)

The method of claim 4, wherein the mTOR inhibitor is AZD8055 or pharmaceutically acceptable salts, solvates, hydrates, cocrystals, or prodrugs thereof.

### Claim 6 (depends on 1)

The method of claim 1, wherein the one or more AKT pathway inhibitor is a PI3K inhibitor.

### Claim 7 (depends on 6)

The method of claim 6, wherein the PI3K inhibitor is LY294002 or pharmaceutically acceptable salts, solvates, hydrates, cocrystals, or prodrugs thereof.

### Claim 8 (depends on 1)

The method of claim 1, wherein the one or more AKT pathway inhibitor comprises two AKT pathway inhibitors each targeting different AKT pathway components.

### Claim 9 (depends on 8)

The method of claim 8 wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets PI3K.

### Claim 10 (depends on 8)

The method of claim 8 wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets mTOR.

### Claim 11 (depends on 8)

The method of claim 8 wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets PDK1.

### Claim 12 (depends on 8)

The method of claim 8 wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets CHK1.

### Claim 13 (depends on 8)

The method of claim 8 wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets Raf1.

### Claim 14 (depends on 8)

The method of claim 8 wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets one or more of FOXO1, FOXO3A, and FOXO4.

### Claim 15 (depends on 8)

The method of claim 8 wherein one AKT pathway inhibitor targets AKT and a second AKT pathway inhibitor targets GSK3.

### Claim 16 (depends on 1)

The method of claim 1, wherein the tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy are enriched for TILs expressing one or more selected from the group consisting of CD8, CD27, CD 28, IL7R, CCR9, SELL (L-selectin or CD62L), FCER1G, SATB1, LEF1, and KLF2.

### Claim 17 (depends on 1)

The method of claim 1, wherein the tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy are enriched for TTLs expressing memory subset T-cell surface markers.

### Claim 18 (depends on 1)

The method of claim 1, wherein the wherein the tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy are enriched for TTLs expressing young TIL phenotype.

### Claim 19 (depends on 1)

The method of claim 1, wherein the wherein the tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy are enriched for TILs with long telomeres as compared to TILs expanded without AKT pathway inhibitors.

### Claim 20 (depends on 1)

The method of claim 1, wherein in step (a) the tumor fragment is cryopreserved.

### Claim 21

A method for treating a subject with cancer, the method comprising administering expanded tumor infiltrating lymphocytes (TILs) comprising:
(a) obtaining a first population of TILs from a tumor obtained from the subject;
(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 and one or more AKT pathway inhibitor to produce a second population of TILs, wherein the first expansion is performed in a closed system comprising a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3-14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50-fold greater in number than the first population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system;
(c) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, OKT-3, and antigen presenting cells (APCs) and culturing to produce a third population of TILs, wherein the second expansion is performed for about 7-14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and/or central memory T cells relative to the second population of TILs, wherein the second expansion is performed in the closed system in a second closed container providing a second gas-permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system;
(d) harvesting the therapeutic population of TILs obtained from step (b), wherein the transition from step (c) to step (d) occurs without opening the system;
(e) administering a therapeutically effective dosage of the harvested therapeutic population of TILs from step (d) to the subject.

### Claim 22 (depends on 21)

The method of claim 21, wherein after step (d) and before step (e) the method further comprises the step of transferring the harvested therapeutic population of TILs from step (d) to an infusion bag, wherein the transfer occurs without opening the system, and wherein step (e) is performed by administering a therapeutically effective dosage of the harvested therapeutic population of TTLs from the infusion bag to the subject.

### Claim 23 (depends on 21)

The method of claim 21, wherein after the step of transferring the harvested TIL population from step (d) to an infusion bag the method further comprises the step of cryopreserving the infusion bag comprising the harvested therapeutic population of TTLs using a cryopreservation process.

### Claim 24 (depends on 21)

The method of claim 21, further comprising administering to the subject a therapeutically effective dosage of aldesleukin or a biosimilar thereof concomitantly with or after the administration of the therapeutically effective dosage of the harvested therapeutic population of TILs.

### Claim 25 (depends on 21)

The method of claim 21, further comprising administering to the subject a therapeutically effective dosage of a PD-1/PD-L1 inhibitor concomitantly with, before or after the administration of the therapeutically effective dosage of the harvested therapeutic population of TILs.

### Claim 26 (depends on 25)

The method of claim 25, wherein the PD-1/PD-L1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, avelumab, durvalumab, atezolizumab, and biosimilars thereof.

### Claim 27 (depends on 21)

The method of claim 21, wherein the cancer is selected from the group consisting of melanoma, cervical cancer, head and neck squamous cell cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, pancreatic cancer, and sarcoma.

### Claim 28 (depends on 21)

The method of claim 21, wherein the culture medium of step (c) further comprises one or more AKT pathway inhibitor.

### Claim 29 (depends on 28)

The method of claim 28, wherein the AKT pathway inhibitor is selected from the group consisting of afuresertib, uprosertib, ipatasertib, AT7867, AT13148, AZD8055, MK-2206, LY294002, and pharmaceutically acceptable salts, solvates, hydrates, cocrystals, or prodrugs thereof.

### Claim 30

A method for expanding tumor infiltrating lymphocytes (TTLs) into a therapeutic population of TTLs comprising:
(a) obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into multiple tumor fragments;
(b) performing a priming first expansion by culturing the first population of TTLs in a cell culture medium comprising IL-2, OKT-3, one or more AKT pathway inhibitor (AKTi), and antigen presenting cells (APCs) to produce a second population of TTLs, wherein the priming first expansion is performed in a container comprising a first gas-permeable surface area, wherein the priming first expansion is performed for first period of about 1 to 7 days to obtain the second population of TTLs, wherein the second population of TILs is greater in number than the first population of TTLs;
(c) performing a rapid second expansion by supplementing the cell culture medium of the second population of TTLs with additional IL-2, OKT-3, and APCs, to produce a third population of TTLs, wherein the number of APCs added in the rapid second expansion is at least twice the number of APCs added in step (b), wherein the rapid second expansion is performed for a second period of about 1 to 11 days to obtain the third population of TTLs, wherein the third population of TTLs is a therapeutic population of TTLs, wherein the rapid second expansion is performed in a container comprising a second gas-permeable surface area;
(d) harvesting the therapeutic population of TILs obtained from step (c); and
(e) transferring the harvested TIL population from step (d) to an infusion bag.

### Claim 31 (depends on 30)

The method of claim 30, wherein the culture medium of step (c) is further supplemented with one or more AKT pathway inhibitor (AKTi).