Method for the Cryopreservation of Tumour-Infiltrating Lymphocytes

• US 20180187150A1
• Filed: 06/30/2016
• Published: 07/05/2018
• Est. Priority Date: 06/30/2015
• Status: Active Application

• Current Assignee: Chu Nantes
• Original Assignee: Chu Nantes
• Application Number:
  US15/740,630
• Publication Number:
  US 20180187150A1
• Time in Patent Office: Days
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
• CPC Class Codes: A01N 1/0226 Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
• CPC Class Codes: C12N 5/0636 T lymphocytes

Abstract

The invention relates to a method for the cryopreservation of at least one sample of tumour-infiltrating lymphocytes, comprising the following steps: i) the in vitro culture of tumour-infiltrating lymphocytes using a collection of samples of in-transit skin nodules, ganglion or metastasis, from a patient with a stage III or IV melanoma, said step comprising the emergence of the tumour-infiltrating lymphocytes contained in the sample of in-transit skin nodules, ganglion or metastasis, followed by the stimulation of the tumour-infiltrating lymphocytes resulting from the emergence step, and, subsequently, the amplification of the stimulated tumour infiltrating lymphocytes; ii) the mixing of at least one sample obtained in step (i) with a composition comprising, in a physiologically acceptable medium, a) human albumin serum, b) at least one saccharide, and c) at least two ingredients selected from among DMSO, L-cysteine, coenzyme Q10 and C3-C5 alkanediols; and subsequently iii) the freezing of the mixture obtained in step (ii).

Claims

1. A process for the cryopreservation of at least one sample of tumor-infiltrating lymphocytes, comprising the following steps:

i) in vitro culture of tumor-infiltrating lymphocytes from a sample of in-transit cutaneous nodules, lymph-node or metastasis originating from a patient suffering from a stage 3 or 4 melanoma, comprising the emergence of said tumor-infiltrating lymphocytes contained in the sample of in-transit cutaneous nodules, lymph-node or metastasis, then the stimulation of the tumor-infiltrating lymphocytes resulting from the emergence step, then finally the amplification of the stimulated tumor-infiltrating lymphocytes, in order to obtain at least one sample of tumor-infiltrating lymphocytes,

ii) mixing at least one sample obtained in step i) with a composition comprising, in a physiologically acceptable medium;

a) human serum albumin,

b) at least one saccharide, and

c) at least two ingredients selected from DMSO, L-cysteine, coenzyme Q10 and C3-C5 alkanediols,

iii) freezing the mixture obtained in step ii).

2. The cryopreservation process as claimed in claim 1, wherein the saccharide is selected from monosaccharides, disaccharides and trisaccharides.

3. The cryopreservation process as claimed in claim 2, wherein the saccharide is a disaccharide selected from trehalose and sucrose.

4. The cryopreservation process as claimed in claim 1, wherein the C3-C5 alkanediol is selected from propane-1,2-diol, pentane-1,5-diol and butane-2,3-diol.

5. The cryopreservation process as claimed in claim 1, wherein the human serum albumin is present in an amount of between 2 and 10% by weight relative to the total weight of composition.

6. The cryopreservation process as claimed in claim 1, wherein the saccharide is present at a concentration of between 0.05 M and 1 M.

7. The cryopreservation process as claimed claim 1, wherein the composition used in step ii) comprises:

a) human serum albumin,

b) trehalose, and

c) DMSO and L-cysteine.

8. The cryopreservation process as claimed in claim 1, wherein the composition used in step ii) comprises:

a) human serum albumin,

b) trehalose, and

c) coenzyme Q10 and propane-1,2-diol.

9. The cryopreservation process as claimed in claim 1, wherein the freezing iii) is carried out down to a temperature of between −

100°

C. and −

180°

C.

10. The cryopreservation process as claimed claim 1, wherein the freezing iii) is carried out by placing the mixture obtained in ii) in a container submerged in a mixture of isopropyl alcohol at +4°

C., everything being brought to a temperature of between −

70°

C. and −

100°

C.

11. The cryopreservation process as claimed in claim 1, wherein the freezing iii) is carried out by means of a programmed freezer.

15. The cryopreservation process as claimed in claim 5, wherein the human serum albumin is present in an amount of between 2.5 and 6% by weight relative to the total weight of composition.

16. The cryopreservation process as claimed in claim 6, wherein the saccharide is present at a concentration of between 0.07 M and 0.5 M.

17. The cryopreservation process as claimed in claim 7, wherein the composition used in step ii) comprises:

a) the human serum albumin in an amount of between 2.5 and 6% by weight,

b) the trehalose at a concentration of between 0.07 M and 0.5 M, and

c) the DMSO and the L-cysteine respectively in an amount of between 2 and 15% by weight and at a concentration of between 0.5 mM and 2 mM.

18. The cryopreservation process as claimed in claim 8, wherein the composition used in step ii) comprises:

a) the human serum albumin in an amount of between 2.5 and 6% by weight,

b) the trehalose at a concentration of between 0.07 M and 0.5 M, and

c) the coenzyme Q10 and the propane-1,2-diol respectively in an amount of between 0.007 and 0.1% by weight and in an amount of between 3 and 10% by volume.

19. The cryopreservation process as claimed in claim 9, wherein the freezing iii) is carried out down to a temperature of between −

140°

C. and −

160°

C.

12. A method for the cryopreservation of at least one sample of tumor-infiltrating lymphocytes comprising the step of mixing a composition comprising, in a physiologically acceptable medium:

a) human serum albumin,

b) at least one saccharide, and

c) at least two ingredients selected from DMSO, L-cysteine, coenzyme Q10 and C3-C5 alkanediols,

said sample being obtained by in vitro culture of tumor-infiltrating lymphocytes from a sample of in-transit cutaneous nodules, lymph-node or metastasis originating from a patient suffering from a stage 3 or 4 melanoma, said culture comprising the emergence of said tumor-infiltrating lymphocytes contained in the sample of in-transit cutaneous nodules, lymph-node or metastasis, then the stimulation of the tumor-infiltrating lymphocytes resulting from the emergence step, then finally the amplification of the stimulated tumor-infiltrating lymphocytes.

13. The method as claimed in claim 12, wherein the tumor-infiltrating lymphocytes resulting from the emergence step are stimulated in the presence at least of non-proliferating irradiated allogeneic feeder cells.

14. The method as claimed in claim 12, wherein the tumor-infiltrating lymphocytes resulting from the emergence step are stimulated by coculture with a mixture formed of mononuclear cells originating from healthy human donor blood and of B cells transformed by the Epstein-Barr virus, all non-proliferating.