# US 2021/0274776 A1 · Expansion of TILs from Cryopreserved Tumor Samples

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- Number: US 2021/0274776 A1
- Document ID: US20210274776A1
- Status: Published application
- Kind: A1
- Application number: 17/172,840
- Filed: 2021-02-10
- Published: 2021-09-09
- Earliest claimed: 2018-09-20
- Assignees: Iovance Biotherapeutics, Inc.
- Applicants: Iovance Biotherapeutics, Inc.
- Inventors: Anand Veerapathran; Kenneth Onimus
- Web page: https://patentgrep.com/patent/US20210274776A1

## CPC

- A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
- 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 40/42: Cancer antigens
- C12N 5/0636: {T lymphocytes}
- C12N 2501/2302: Interleukin-2 (IL-2)
- C12N 2501/2315: Interleukin-15 (IL-15)
- C12N 2501/2321: Interleukin-21 (IL-21)
- C12N 2509/10: Mechanical dissociation
- C12N 2523/00: Culture process characterised by temperature
- 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)
- 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 from cryopreserved tumor tissue and methods of using the expanded tumor infiltrating lymphocytes in the treatment of human diseases, including cancers, are disclosed In some embodiments, compositions of cryopreserved tumor tissues are disclosed.

## Claims

### Claim at position 1

1-75. (canceled)

### Claim 76

A method of cryopreserving tumor tissue for the manufacture of tumor infiltrating lymphocytes (TILs), the method comprising:
(i) fragmenting the tumor tissue;
(ii) incubating the fragments in a cryopreservation medium; and,
(iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen.

### Claim 77 (depends on 76)

The method of claim 76, wherein the tumor tissue is fragmented into approximately spherical fragments having a diameter of about 1.5 mm to 6 mm.

### Claim 78 (depends on 76)

The method of claim 76, wherein the tumor tissue is fragmented into generally rectangular fragments having a shortest edge length of at least 1.5 mm and a longest edge length of about 6 mm.

### Claim 79 (depends on 76)

The method of claim 76, wherein the tumor tissue is fragmented into generally cubical fragments having edge lengths of between about 1.5 mm and about 6 mm.

### Claim 80 (depends on 76)

The method of claim 76, wherein the tumor tissue is from a dissected tumor.

### Claim 81 (depends on 76)

The method of claim 76, wherein the cryopreservation medium comprises 2% v/v to 15% v/v dimethyl sulfoxide (DMSO).

### Claim 82 (depends on 76)

The method of claim 76, wherein the cryopreservation medium comprises at least one antimicrobial agent.

### Claim 83 (depends on 82)

The method of claim 82, wherein the at least one antimicrobial agent is gentamicin at a concentration of at least 50 μg/mL.

### Claim 84 (depends on 76)

The method of claim 76, wherein fragmenting the tumor comprises digestion.

### Claim 85 (depends on 84)

The method of claim 84, wherein the digestion comprises incubation in enzyme media followed by mechanical dissociation.

### Claim 86 (depends on 85)

The method of claim 85, wherein the enzyme media comprises DNase and collagenase.

### Claim 87 (depends on 76)

The method of claim 76, wherein the tumor fragments are incubated in the cryopreservation medium at a temperature of about 2° to 8° C.

### Claim 88 (depends on 76)

The method of claim 76, wherein in step (ii) the tumor fragments are incubated in the cryopreservation medium for at least about 30 minutes.

### Claim 89 (depends on 76)

The method of claim 76, wherein the tumor tissue is washed in a physiologically buffered isotonic saline solution prior to incubation.

### Claim 90 (depends on 89)

The method of claim 89, wherein the washing comprises three serial washes of at least three minutes each, with the physiologically buffered isotonic saline solution replaced after each serial wash.

### Claim 91 (depends on 76)

The method of claim 76, wherein the freezing takes place at a temperature in the range from of −125° C. to about −196° C.

### Claim 92 (depends on 76)

The method of claim 76, wherein the freezing takes place at a temperature of about −145° C.

### Claim 93 (depends on 76)

The method of claim 76, further comprising the step of (iv) storing the frozen fragments below at least −130° C.

### Claim 94 (depends on 93)

The method of claim 93, wherein the frozen fragments are stored in the vapor phase of liquid nitrogen.

### Claim 95 (depends on 93)

The method of claim 93, wherein the frozen fragments are stored submerged in liquid nitrogen.

### Claim 96

A cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by a process comprising the steps of:
(i) fragmenting a dissected tumor or tumor biopsy sample;
(ii) incubating the fragments in a cryopreservation medium; and,
(iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen.

### Claim 97 (depends on 96)

The cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by the process of claim 96, wherein step (ii) further comprises incubating the fragments at about 2° C. to about 8° C. in the cryopreservation medium, the cryopreservation medium comprising 2% v/v to 15% v/v DMSO.

### Claim 98 (depends on 96)

The cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by the process of claim 96, wherein the incubating is performed for about 30 minutes to about 60 minutes.

### Claim 99 (depends on 96)

The cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by the process of claim 96, wherein fragmenting comprises digestion.

### Claim 100 (depends on 99)

The cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by the process of claim 99, wherein the digestion comprises incubation in enzyme media followed by mechanical dissociation.

### Claim 101 (depends on 100)

The cryopreserved tumor fragment for the manufacture of tumor infiltrating lymphocytes (TILs), prepared by the process of claim 100, wherein the enzyme media comprises DNase and collagenase.

### Claim 102

A method for preparing tumor infiltrating lymphocytes (TILs) for adoptive T-cell therapy, the method comprising:
(a) obtaining a cryopreserved tumor fragment by:
(i) fragmenting the tumor tissue by enzymatic digestion,
(ii) incubating the fragments in a cryopreservation medium, and
(iii) freezing the fragments wherein the freezing is flash freezing using the vapor phase of liquid nitrogen;
(b) treating the cryopreserved tumor fragment in a gas permeable container with a first cell culture medium comprising interleukin 2 (IL-2) and optionally OKT-3 antibody to provide TILs;
(c) expanding the TILs with a second cell culture medium comprising cell culture media, antigen presenting cells (APCs), OKT-3 antibody, and IL-2 in a gas permeable container to provide an expanded number of TILs; and,
(d) optionally cryopreserving the expanded number of TILs.

### Claim 103 (depends on 102)

The method of claim 102, wherein enzymatic digestion comprises incubation in enzyme media followed by mechanical dissociation, the enzyme media comprising DNase and collagenase.

### Claim 104 (depends on 102)

The method of claim 102, wherein the tumor fragments are incubated in the cryopreservation medium for at least about 30 minutes.

### Claim 105 (depends on 102)

The method of claim 102, wherein tumor fragments are incubated in the cryopreservation medium at a temperature of about 2° to 8° C.