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.

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 × 10.sup.6TIL/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.

Description

EXAMPLE 1

[0125] Starting material for the production of TIL or other T-cells are parts of tissue, which z. B. during the surgical removal of solid organ tumors or their metastases or their immediately surrounding tissue or removed for this purpose.

[0126] A tissue volume of about 1 ml is usually sufficient as starting material.

[0127] The tissue samples are placed in aTransport vessel spent with medium.

[0128] The vessel is kept sealed at room temperature and transported from the tissue removal site to the clean room area for the production of the immune cell preparations.

[0129] The pieces of OP tissue are comminuted in a clean room under an LFB into pieces of 1 to 2 mm3 in size and, in the first stage, transferred to a meander perfusion bioreactor in accordance with DE102018000561.6.

[0130] The shredded pieces of tissue are evenly distributed in the meander perfusion bioreactor and cultivated in the perfusion mode.

[0131] The meander bioreactor is connected ready for operation in a GMP breeder and is continuously monitored by the control unit, largely automatically controlled and documented.

[0132] The meander perfusion bioreactor consists of a rectangular bioreactor vessel 1 made of preferably clear polymer material, which is sealed with a cover 2 in a sterile manner.

[0133] The bioreactor vessel 1 is divided into three chambers 3, 4, 5 arranged one above the other, with the lowest chamber 3 as an underlay chamber, the chamber 4 arranged above the underlay chamber 3 as a meander perfusion chamber and the one above the meander perfusion Chamber 4 arranged chamber 5 are designed as an overlay chamber.

[0134] The underlay chamber 3 and the meander perfusion chamber 4 are separated from one another by a perforated base plate 6 with a film 7 which is tightly fastened thereon and is permeable to oxygen.

[0135] The underlay chamber 3 has inlets and outlets 9, 10 which allow gases to flow through, in particular mixtures of air and oxygen or nitrogen and oxygen, in which the proportion of oxygen is regulated.

[0136] The oxygen diffuses preferentially to nitrogen through the gas-permeable film 7, which is tightly attached to the perforated base plate 6 of the meander perfusion chamber 4.

[0137] During the flow through the meander perfusion chamber 4, controlled amounts of oxygen enter the culture medium during the expansion of the cells both from the overlay and from the underlay chamber (through diffusion).

[0138] The meander perfusion chamber 4 is provided with inlets and outlets for culture media 11, 12, and there are also inlets and outlets 13,14 for the overlay atmosphere and an outlet connector 15 in the overlay chamber 5.

[0139] A screen fabric 16 is arranged on the outlet socket 15 inside the overlay chamber 5 in front of the outlet.At the outlet 12 of the meander perfusion chamber 4 there is also an overflow 17 for discharging the used cell broth. The overflow is adjustable in height.

[0140] The meander perfusion chamber 4 consists of the base plate 6) with strip-shaped partitions 8 arranged on the upper side 8, dividing the base plate 6 and forcing a meandering flow through the meander perfusion chamber 4 with gases and medium, with the distance A of the partitions 8 from each other as well as from the side and end walls of the meander perfusion chamber 4 is chosen such that an averaged laminar overflow with a Froude number <0.005 forms in the stream thread, in the channel formed by the partition walls 8 .

[0141] As the number of cells increases as the number of cells (TIL or other cells) multiplies, the supply of fresh medium is continuously increased, with this being automatically controlled by a corresponding algorithm. The bottom flow remains close to a Froude number of 0, which prevents the cells from being stirred up. The flow conditions described prevent cell stress and ensure a homogeneous supply of nutrients over the entire cell layer growing at the bottom of the channel.

[0142] The overlay chamber (5) has inlets and outlets 13,14 for the overflow of gases.

[0143] The overlay chamber is flown through with the same gas/gas mixture as the underlay chamber 5), whereby the cell is supplied with oxygen either hyperoxically up to 90% O2), normoxically (21% O2) or hypoxically (up to 2% O2), depending on the cell type will

[0144] In a cultivation run, cells usually grow out of the tissue pieces for 7 to 14 days, and the mature cells multiply at the same time.

[0145] In this way, TIL can be produced in larger quantities from tumor tissue.

[0146] But TINK and other cells can also be obtained from tissues in this way.

[0147] When the glucose consumption in the bioreactor reaches 100 to 300 mg per day, the TIL are separated from the tissue residues as filtrate via the outlet nozzle 15 of the bioreactor vessel 1 provided with sieve fabric 16 .

[0148] From the transfer of the tissue pieces into the bioreactor vessel 1 in the first stage, the entire process up to the filling of activated and expanded cells takes place in a completely closed vessel system.

[0149] Is cultivated in a suitable medium with a specific mixture of AB human serum, cytokines, antibodies and Irradiated human feeder cells is temporarily supplemented, with cytokines in the form of interleukin 12 or a mixture of interleukin 2 and interleukin 12 and the antibodies in the form of antibody 4-1 bb being used.

[0150] In some cases, faster propagation and activation can be achieved with supplements that are fixed to the surfaces of the bioreactor channel. In the case of TIL and other immune cells, the TIL multiplied and separated in this first stage are centrifuged, resuspended in freezing medium and aliquoted (around 50 million TIL per vial). The samples are stored in the gas phase over liquid nitrogen.

[0151] With the method according to the invention for the isolation and propagation of cells from tissue parts of tumors, metastases and other tissues, it is possible in a single closed process step to grow certain cells from small pieces of tissue, from autologous pieces of tissue, both in the culture medium located in the bioreactor and Simultaneously activate cells, multiply them and then separate them from tissue debris.

[0152] In particular, tumor-infiltrating autologous T lymphocytes (TIL) can be obtained in this way from tumor tissue, metastasis tissue, their surrounding tissue and from lymph nodes which are affected by tumor cells. Under the flow and gassing conditions in the channel of a perfusion bioreactor, the culture medium according to the invention leads to rapid growth of the cells. In the case of TIL, the cells that are particularly preferably expanded are those that have specific cytotoxicity compared to the tumor cells from which or from their environment they are obtained