# US 11,304,980 B2 · Restimulation of cryopreserved tumor infiltrating lymphocytes

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- Number: US 11,304,980 B2
- Document ID: US11304980B2
- Status: Granted
- Kind: B2
- Application number: 17/459,988
- Filed: 2021-08-27
- Published: 2021-12-30
- Granted: 2022-04-19
- Earliest claimed: 2016-10-26
- Assignees: Iovance Biotherapeutics, Inc.
- Applicants: Iovance Biotherapeutics Inc.
- Inventors: Ian Frank; Michael T. Lotze
- Web page: https://patentgrep.com/patent/US11304980B2

## CPC

- C12N 5/0636: {T lymphocytes}
- A01N 1/162: Temperature processes, e.g. following predefined temperature changes over time
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/4271: {Melanoma antigens}
- A61P 35/00: Antineoplastic agents
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 15/85: for animal cells
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- A61K 2239/57: Skin; melanoma
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2501/04: Immunosuppressors, e.g. cyclosporin, tacrolimus
- C12N 2501/2302: Interleukin-2 (IL-2)
- C12N 2502/11: blood or immune system cells
- C12N 2502/1121: Dendritic cells
- C12N 2502/99: 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- 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

The present disclosure provides methods for re-stimulating TIL populations that lead to improved phenotype and increased metabolic health of the TILs and provides methods of assaying for TIL populations to determine suitability for more efficacious infusion after re-stimulation.

## Claims

### Claim 1

A cryopreserved tumor infiltrating lymphocyte (TIL) composition comprising a therapeutic population of TILs, wherein the cryopreserved TIL composition is produced by the method comprising:
(a) performing a first expansion by (i) thawing a cryopreserved tumor digest comprising a first population of TILs from a tumor that was resected from a subject, digested after the resection, and cryopreserved after the digestion, and (ii) culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the tumor digest was prepared by incubating a sample of the tumor that was resected from the subject in an enzymatic media;
(b) 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), to produce a third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, and wherein the second expansion is performed for about 7 to 14 days in order to obtain the therapeutic population of TILs;
(c) harvesting the therapeutic population of TILs obtained from step (b);
(d) admixing the harvested therapeutic population of TILs from step (c) with a cryopreservation media to form an admixture; and
(e) cryopreserving the admixture from step (d) using a cryopreservation process to produce a cryopreserved TIL composition.

### Claim 2 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the cryopreservation media comprises a cryopreservant.

### Claim 3 (depends on 2)

The cryopreserved TIL composition according to claim 2, wherein the cryopreservant is DMSO.

### Claim 4 (depends on 3)

The cryopreserved TIL composition according to claim 3, wherein the cryopreservation media comprises 5% DMSO.

### Claim 5 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the method further comprises disrupting the tumor sample mechanically so as to dissociate the tumor sample.

### Claim 6 (depends on 5)

The cryopreserved TIL composition according to claim 5, wherein the method further comprises purifying the disassociated tumor sample using a density gradient separation.

### Claim 7 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the enzymatic media comprises DNase.

### Claim 8 (depends on 7)

The cryopreserved TIL composition according to claim 7, wherein the enzymatic media comprises 30 units/mL of DNase.

### Claim 9 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the enzymatic media comprises collagenase.

### Claim 10 (depends on 9)

The cryopreserved TIL composition according to claim 9, wherein the enzymatic media comprises 1.0 mg/mL of collagenase.

### Claim 11 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the cell culture medium is CTS Optimizer.

### Claim 12 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the APCs comprise peripheral blood mononuclear cells (PBMCs).

### Claim 13 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the first expansion is performed within from about 3 to 11 days.

### Claim 14 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein steps (a) through (e) are performed within about 24 days.

### Claim 15 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein step (a) further comprises adding the thawed tumor digest into a closed system prior to culturing the first population of TILs.

### Claim 16 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the transition from step (a) to step (b), the transition from step (b) to step (c), or the transition from step (c) to step (d) occurs without opening the system.

### Claim 17 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the transition from step (a) to step (b) occurs without opening the system.

### Claim 18 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the transition from step (b) to step (c) occurs without opening the system.

### Claim 19 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein the transition from step (c) to step (d) occurs without opening the system.

### Claim 20 (depends on 1)

The cryopreserved TIL composition according to claim 1, wherein step (a) further comprises adding the thawed tumor digest into a closed system prior to culturing the first population of TILs, wherein the transition from step (a) to step (b) occurs without opening the system, wherein the transition from step (b) to step (c) occurs without opening the system, and wherein the transition from step (c) to step (d) occurs without opening the system.