METHOD FOR PRODUCING TUMOR-INFILTRATING T-LYMPHOCYTES (TIL) AND THEIR USE AS CELLULAR THERAPEUTICS FOR THE TREATMENT OF HUMAN TUMORS

Abstract

The invention relates to a method for isolating, activating and propagating immune cells, more particularly tumor-infiltrating autologous T-lymphocytes (TIL) from primary tumor tissue, metastatic growths, lymphatic tissue but also T cells from other tissues (e.g., blood, lymphatic fluid) in a meander perfusion bioreactor and to the production of immune cellular therapeutics therefrom for controlling tumors of the pancreas, lung, liver, prostate, breast, ovaries, stomach, colon, rectum, bones, brain, skin and other malignant tumors.

Description

Claims

1. A method for the ex-vivo extraction of TIL and other T cells and use as patient-specific cell therapeutics to combat tumors, in which a defined number of cells in a first stage of the process from comminuted tissue parts in a closed meander perfusion bioreactor vessel are introduced and distributed evenly; the cells grow into the culture medium located in a perfusion meander bioreactor, simultaneously activated and multiplied with a meandering guidance of the medium flow through a channel of the perfusion bioreactor with an overflow according to a Froud number <0.005, preferably, 0.002 and a bottom flow according to a Froud number of 0 or close to 0 and the medium flows in a directed laminar flow, which does not cause cell stress, over the sedimented TIL and, after reaching a certain density of fully grown and expanded cells is seperated from the tissue parts, in right harvested in this form, the cells separated in this way are centrifuged, resuspended in freezing medium in vials, aliquoted and cryopreserved and in a second step the cryopreserved vials are thawed, the freezing medium is washed out and the cells are resuspended in a suitable culture medium and placed in another operationally installed meander perfusion bioreactor with the same or larger settelment area in the ratio of 5 to 1 compared to the meander perfusion bioreactor of the first stage, are transferred with the culture medium being perfused in the bioreactor vessels of the first and second stages in the bioreactor vessels in a directed manner with oxygen and the culture medium is supplemented with a mixture of AB human serum, cytokines and antibodies characterized in that • the cells growing out of the pieces of tissue at the bottom of the sedimentation of the channel of the meander perfusion bioreactor,• The cells are largely dormant after sedimentation, form cell layer in which they touch, but also light ones movements occur, with TIL cell counts in the sedimented layer at 0.1 to 2 x, 106TIL/cm2, preferably 0.5 to 1 × 106TIL/cm2 for steady proliferation• the culture medium contains cytokines and antibodies, the cytokines being in the form of interleukin 12 or a mixture of interleukin 2 and interleukin 12 and the antibodies being in the form of antibody 4-1 bb and• The cells receive a hyperoxic or normoxic supply of supplemented medium and oxygen depending on the increased amount of TIL or other T cells through an automatically controlled supply.

2. The method according to claim 1, characterized in that the cells are cultivated as tumor-infiltrated lymphocytes (TIL) or tumor-infiltrated NK cells (TINK), or other cell types in the meander perfusion bioreactor in appropriately supplemented media.

3. Method according to claim 1 characterized in that • the cryopreserved vials are thawed for further expansion,• the freezing medium is washed out by centrifuging and resuspending the cells in freshly supplemented medium containing a mixture of AB human serum, cytokines and antibodies.• This cell suspension is transferred in a second stage into one or more meander perfusion bioreactors of the same design as in the first stage, but with a larger colonization area or is split• the TIL in the bioreactors expands over 12 to 20 days after the cultivation is harvested, washed in NaCl solution and centrifuged,• resuspended the cells in a solution containing 0.9% NaCl, 2% albumin and 10% contains DMSO, and this suspension is filled into defined portions in 100 ml cryobags after a conventional cooling process and stored over nitrogen, and• the TIL suspensions thus stored are retrieved, thawed and dosed appropriately to the patient.

4. Process for the production of a cell therapy according to claim 2, characterized in that the cell therapy consists of tumor-infiltrated lymphocytes (TIL), as a pharmaceutical composition for use as a cell for the treatment of tumors and infections.

5. Process for the production of a cell therapy according to, claim 2, characterized in that the cell therapy for the treatment of pancreatic, pulmonary, ovarian, breast, large intestine, bone marrow, liver, cerbral prostate, -, gastric rectum blood, glioma, melanoma, lymphoma or a combination thereof.