DE102023002417B3 - method for expanding cells - Google Patents

Abstract

The invention relates to a method for expanding cells, in particular non-adherently growing cells, such as invariant T lymphocytes or non-adherently growing T lymphocytes and NK cells, but also other immune cells, e.g. tumor-infiltrated NK cells (TINK) and mucous membrane-associated invariant T cells (MAIT) with a high proportion of CD 8+ and CD 56+ antibodies, also with a low proportion of CD3+ antibodies in a perfusion plate meander bioreactor, in which sufficient quantities of cells are made available for therapeutic treatment with cells and the high costs, in particular through the use of human sera in the expansion, are avoided, wherein in the perfusion plate meander bioreactor several superimposed cultivation carriers 7 provided with a channel 10, overflow 14 and overflow webs 33 are arranged and wherein the cultivation carriers 7 are arranged rotated by 180° to one another. The culture medium flows in the perfusion plate meander via a Luer-Lock connector 22 for the supply of culture medium, meandering from top to bottom, and is discharged from the perfusion plate meander bioreactor via a Luer-Lock connector 23, with the overlay media being fed via a feed cavity 11 and via feeds 12 above the culture medium in the channels 10 overflow in the opposite direction to the flow of the culture medium. To record and control the concentration of overlay media in the culture medium, indicator beads coated with fluorescent markers are added to the culture medium, the fluorescence brightness of which changes with the change in the concentration of overlay media.

Description

The invention describes a method for expanding cells, such as non-adherently growing cells, non-adherently growing T lymphocytes or NK cells, but also other immune cells, e.g. tumor-infiltrated NK cells (TINK) and mucosa-associated invariant T cells (MAIT) with high proportions of CD8+ and CD56+ antibodies, but also with a low proportion of CD3+ antibodies, in a perfusion plate meander bioreactor

T cells known to have a single invariant TCR are NKT cells and MAIT cells. The important role that NKT cells play in protecting an individual from bacteria and cancer, as well as their involvement in the pathology of an autoimmune disease, has been made known mainly through a study using mice. Meanwhile, MAIT cells exist in large numbers in human peripheral blood, intestine or liver, and they play an important role in mucosal immunity, but much of the details are still not clear. Although MAIT cells are abundant in humans, the production of a large amount of MAIT cells, which depends on their preparation from a human living body, has limitations because MAIT cells are difficult to propagate. MAIT cells must be obtained by preparation from a living body and purification, and there is neither a method of inducing differentiation in vitro/amplification nor a technology related to character analog cells (model cells).

The EP 38 38 288 A1 describes a composition for expanding lymphocytes comprising at least two types of cytokines selected from interleukin2 (IL-2), interleukin-15 (IL-15) and interleukin-21 (IL-21). The invention further relates to a method for producing a population of clinically relevant lymphocytes, comprising the steps of: obtaining a body sample from a mammal, in particular a tissue sample or a body fluid sample, comprising at least one lymphocyte and optionally separating the cells in the body sample, culturing the body sample in vitro to expand and/or stimulate lymphocytes in the sample, wherein the culturing comprises the use of IL-2, IL-15 and/or IL-21 and optionally determining the presence of a clinically relevant lymphocyte in the cultured sample. The present invention also relates to immunotherapy and the population of clinically relevant lymphocytes. The T cell is preferably selected from a mucosal-associated invariant T cell (MAIT). Aphaeresis was performed on a healthy donor primed with NY-ESO-1. After separation of the blood components, the product containing the leukocytes is separated from the rest. The cells are suspended in Cellgro consisting of 5% pooled human AB serum, 1000 U/ml IL-2, 10 ng/ml IL-15 and 10 ng/ml IL-21. The medium additionally contained 10 µmol NY-ESO-1 peptide as expansion antigen. Expansion of peripheral blood lymphocytes included the step of stimulation by irradiated autologous PBMCs pulsed with the antigen of interest. For this, autologous PBMCs were pulsed with 10 mM NY-ESO-1 peptide for two hours at RT and then irradiated with 55 Gy. The pulsed irradiated autologous PBMCs were added to the lymphocyte culture on day 7 and resuspended in fresh culture medium supplemented with 5% human pooled AB serum with cytokines at the concentrations defined above. On day 10, cells were expanded with Cellgro supplemented with 5% pooled human AB serum and cytokines. OKT3 antibody was added at a concentration of 30 ng/ml in the same culture medium as described above plus irradiated feeder cells (55Gy) at a 1:10 (feeder cell:T cell ratio) to the peptide and cytokines. On days 17 to 20 of culture, cells are collected for analysis or given to the patient. Pancreatic tumor tissue is obtained immediately after surgery and placed in a sterile container. Tumor tissue is cut into 1 to 2 ^mm3 pieces using a sterile scalpel. Each of the tissue pieces should be transferred to a well of a 24-well plate. 1 ml of the culture medium RPMI 1640 with 10% fetal bovine serum was added to each of the wells. The medium was changed on days 7 and 14, which means that the culture medium was removed and replaced with fresh culture medium of the same type. When the tumor cells reached a very high density, the culture was transferred to a 96-well plate.

The invention according to WO 2019 180 270 A1 provides engineered lymphocytes (e.g., γδ T cells, NK cells, NK-like T cells, engineered innate lymphoid cells, or MAIT cells) containing a heterologous targeting construct lacking an intracellular signaling domain capable of activating the lymphocyte on which the construct is expressed. For example, in some embodiments, PBMCs are pre-enriched using magnetic bead sorting, which can yield greater than 90% γδ T cells. These cells can be cultured in the presence of one or more factors (e.g., TCR agonists, co-receptor agonists, and/or cytokines, e.g., IL-4, IL-15, and/or IFN-γ) in gas-permeable bioreactor bags for up to 21 days or longer. Variations of this method and other methods for obtaining Vδ1 T cells are suitable as part of the present invention. For example, blood-derived Vδ1 T cells may alternatively be obtained by methods

In the US 2019 382 725 describe systems and methods for modulating the function of mucosa-associated invariant T cells (MAIT) in the absence of T cell receptor (TCR) signaling. The systems and methods can be used to elucidate the function of MAIT cells to evaluate potential therapeutic strategies for diseases related to inflammation. Mononuclear cells, generally including monocytes and lymphocytes, can be obtained from, for example, blood, peripheral blood, bone marrow, umbilical cord blood, mucosal tissue, gastrointestinal tract, liver, and lung. Mononuclear cells can be obtained by a known method such as centrifugation, magnetic beads, and flow cytometry. Mononuclear cells can be those derived from stem cells such as induced pluripotent stem cells, embryonic stem cells, and somatic stem cells. As particular embodiments, peripheral blood mononuclear cells (PBMC) can be used as mononuclear cells in the present disclosure. PBMCs can be isolated by density gradient centrifugation with commonly used density gradient medium such as Ficoll or Ficoll-Paque. The density gradient medium can also include sodium diatrizoate, polysaccharides, and water. The PBMCs can be cryopreserved prior to use in the methods of the present disclosure. Cryopreservation of PBMCs can include resuspension of the PBMCs in freezing medium. In particular embodiments, the freezing medium is 10-20% dimethyl sulfoxide (DMSO) and 40% fetal bovine serum (FBS) or human serum albumin (BSA) in RPMI-1640 medium. In particular, the PBMCs in freezing medium can be placed in a freezing container at -80°C overnight to allow for gradual and uniform cooling. The frozen cells can be transferred to a liquid nitrogen tank the next day for long-term storage.

Cryopreserved PBMCs may be thawed for use in the methods of the present disclosure. In particular, thawing PBMCs includes removing them from liquid nitrogen and placing them on ice, after which they are thawed in a 37°C water bath. Thawing may further include adding warm medium supplemented with 10% FBS, 1% penicillin/streptomycin, and L-glutamine, washing the cells to remove the DMSO, and resuspending the cells in medium. Thawing may also include resting the PBMCs overnight to remove apoptotic cells. After this rest period, the cells are washed and are then ready to be used in culture. Cryopreserved PBMCs were thawed and left untreated in RPMI 1640 (supplemented as described above) or stimulated with cytokines, stimulated by the T cell receptor (TCR), or stimulated by a combination of both treatments. Cytokine stimulation was performed with IL-12 (Invitrogen, Carlsbad, Calif.), IL-15 (Invitrogen, Carlsbad, Calif.), and IL-18 (MBLI, Woburn, Mass.) at 100 ng/ml. TCR stimulation was performed with Dynabead Human T Cell Activator (Invitrogen, Carlsbad, Calif.) anti-CD3/CD28 beads at a ratio of 1:1 bead/T cells. Whole PBMCs were cultured unstimulated or stimulated at a concentration of 1.25 × 106 per well using a 96-well plate for 6 and 24 hours at 37°C to allow expression of CTLA-4. MAIT cells stimulated for only 6 hours required at least 12 hours of rest in culture for sufficient induction of CTLA-4. Cytokines and TCR beads were removed by magnetic extraction and washing. Briefly, Dynabeads were removed using EasySep™ Magnet (STEMCELL Technologies Inc., Cambridge, Mass.). Cells were moved to 5 ml round polystyrene tubes and 0.5 ml of 1×PBS (phosphate buffered saline) was added to each tube. Each tube was placed in an EasySep Magnet™ for 1 min. To keep the tube in the magnet, the entire contents of the tube were poured into another tube outside the magnet. The magnetic Dynabeads remained in the first tube inside the EasySep Magnet™ while cells were moved to the new tube. Cells were washed twice by centrifuging at 2000 rpm for 3 min and resuspending in 200 µL of RPMI 1640 (supplemented as described above). After washes, cells were resuspended in 200 µL of RPMI 1640 (supplemented as described above) before transferring back to a 96-well plate to rest in culture. To block TCR signaling, cells in the cytokine-only state were treated with an anti-MR1 antibody (clone 26.5, Biolegend, San Diego, California) at 50 µg/ml. Cells were then stained with antibodies for flow cytometric detection and analysis as described above.

The WO 2014 069 072 A1 discloses a method of preparing a MAIT-like cell, consisting of introducing a reprogramming factor into a MAIT cell to obtain an induced pluripotent stem cell that retains a TCR-α chain gene rearranged into a MAIT cell-specific fashion, followed by induction of differentiation of the induced pluripotent stem cell to obtain a MAIT-like cell. The method of preparing an induced pluripotent stem cell consists of introducing a reprogramming factor into a MAIT cell to obtain an induced pluripotent stem cell that retains a TCR-α chain gene that is rearranged in a MAIT cell-specific manner. MAIT cells in the context of the present invention are T cells whose TCR-α chain gene is rearranged to a unique and uniform Vα-Jα (Vα19-Jα33 in a mouse, Vα7.2-Jα33 in humans). Most preferably, they can be defined as cells that express CD161 or IL-18Rα. Furthermore, the MAIT cell can be characterized by its TCR-α chain being restricted by the invariant MR1. Further, the MAIT cell in the present invention can be identified by expression of genes specific to the MAIT cell or by a cell biological property specific to the MAIT cell. Any culture method for obtaining MAIT-like cells by inducing differentiation in the present invention can be used as long as MAIT-like cells can be obtained, and examples include co-culture with feeder cells, float culture, hanging drop culture, spinning culture, soft agar culture, microcarrier culture. In a preferred aspect of the present invention, it is preferable to obtain MAIT-like cells by inducing differentiation of iPS cells by co-culture, and in particular, MAIT-like cells can be efficiently obtained from MAIT-iPS cells by co-culture using stromal cells such as OP9 cells as feeder cells, followed by co-culture with OP9 cells vigorously expressing a Notch ligand DLL1 (OP9/DLL1) or co-culture carried out from the beginning with the OP9/DLL1 cells. The MAIT-like cells obtained by such a method can then be subjected to cell recovery, separation and purification by known methods. The MAIT-like cells obtained by the present invention are cells having morphological, physiological and/or immunological characteristics almost equivalent to those of a MAIT cell in the living body.

In the invention WO 2021 0856 450 A1 describes the culture method for obtaining m-reMAIT-like cells by inducing differentiation, but the method is not limited to a co-culture method with feeder cells, a suspension culture method, a hanging drop culture method and a vortex culture method, soft agar culture method, microcarrier culture method and the like. In a preferred embodiment of the present invention, the co-culture is preferably suitable for inducing differentiation of iPS cells to obtain m-reMAIT cells. In particular, stromal cells such as OP9 cells are co-cultured as feeder cells and further co-cultured. By co-cultivation with OP9 cells (OP9 / dlk-1) by forced expression of the Notch ligand Delta-like 1 (dlk-1) or by co-cultivation with OP9 / dlk-1 cells from the beginning. M-re MAIT cells can be efficiently obtained from MAIT-iPS cells.

The WO 2021 122 961 A1 discloses a method for proliferation, expansion, activation and/or survival of MAIT cells. In some embodiments, the MAIT cells are incubated under stimulating conditions or in the presence of a stimulating agent. These conditions include those designed to induce proliferation, expansion, activation and/or survival of MAIT cells and/or mimic antigen exposure. The conditions may include one or more of a particular media, temperature, oxygen level, carbon dioxide level, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulating factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents to activate the MAIT cells. For example, MAIT cells may be incubated with an anti-CD3 antibody and/or an anti-CD28 antibody under conditions that stimulate proliferation of the cells. In some embodiments, the MAIT cells of the invention can be expanded in vitro by co-cultivation with tissue or cells. In some embodiments, the MAIT cells are expanded by co-cultivation with feeder cells, such as non-dividing PBMC. In some aspects, the non-dividing feeder cells can comprise irradiated PBMC feeder cells, particularly autologous or allogeneic irradiated PBMC, or other cells that express MR1. In some embodiments, MAIT cells are expanded in vitro by CD3/CD28 stimulation in the presence of autologous or allogeneic irradiated PBMCs and IL-2, IL7, IL-12, IL-18 and/or IL-15 cytokines. In further embodiments, MAIT cells are expanded and/or activated in vitro in the presence of MAIT cell activating ligands such as 5-OP-RU and/or 5-OE-RU. In some embodiments, the method comprises a step of preferably in vitro MAIT cell expansion from a cell sample from a donor, in particular from PBMCs from a donor. A preferred in vitro MAIT cell expansion can be performed by culturing PBMCs from a donor in the presence of synthetic 5-OP-RU and optionally with cytokines. In particular, PBMCs from a donor are cultured in the presence of 5-OP-RU and IL-2 (such as rhulL-2). In some embodiments, the MAIT cells are preferably ex vivo for at least about 5 days, preferably not less than about 10 days, more preferably not less than about 15 days, and most preferably not less than about 20 days prior to administration to the patient. In some embodiments, the MAIT cells have been expanded at least 100-fold, preferably at least 200-fold, and more preferably at least 400-fold, preferably at least 600-fold, more preferably at least 1000-fold, and even more preferably at least 1500-fold, compared to day 0 of expansion. prior to administration to a patient. In some embodiments, the method of manufacture includes steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering.

The WO 2020 127 513 A1 describes a mucosa-associated invariant T (MAIT) cell expressing a chimeric antigen receptor (CAR) for use in treating a cancer, immune disorder or infectious disease in a subject, wherein the MAIT cell is allogeneic to the subject. In one aspect, the CAR-MAIT cell is to be administered to an immunocompromised subject. In particular, the CAR-MAIT cell is to be administered after a conditioning treatment that renders the subject immunocompromised. The conditioning treatment may consist of chemotherapy, radiotherapy and/or lymphodeselective antibodies. Preferably, the MAIT cell expresses a CAR that specifically binds to a tumor-associated antigen (TAA). The TAA may be expressed on the surface of tumor cells. Preferably, the TAA is CD19, GD2, EGFR, CD20, CD22, CD33, CD138, CD52, CD30, ROR1, HER2, EpCAM, MUC-1, MUC5AC, BCMA, CD38, SLAMF7/CS1, CD123, IL-13Ra2, HER2, LeY, MUC16 or PSMA. More preferably, the TAA is CD19, CD20, CD22, CD33, CD138, BCMA, CD38, SLAMF7/CS1, IL-13Ra2 or HER2.The present invention provides mucosa-associated invariant T (MAIT) cells expressing a chimeric antigen receptor (CAR), referred to herein as CAR-MAIT cells, for use in the field of allogeneic adoptive immunotherapy. The CAR-MAIT cells do not exhibit alloreactive potential; They do not proliferate in vitro in response to allogeneic cells and, unlike conventional T cells, they do not participate in the induction of graft versus host disease (GVHD). According to the invention, CAR-MAIT cells can be easily produced and expanded in vitro in large quantities. CAR-MAIT cells can, for example, be stored in deep-freeze units and are "ready to use" to enable timely administration to multiple recipients. Their preparation is cost-effective and represents a universal treatment, regardless of the HLA disparity between subjects and without the risk of inducing GVHD. The cells can be obtained from blood samples (including peripheral blood and cord blood) as well as from samples resulting from one or more processing steps such as separation, centrifugation, genetic engineering, washing and/or incubation. In a particular embodiment, the donor sample comprises peripheral blood mononuclear cells (PBMCs). In a particular embodiment, MAIT cells are collected from any location where they are located in the subject, including but not limited to peripheral blood, peripheral blood mononuclear cells (PBMCs), bone marrow, umbilical cord blood. In a particular embodiment, the MAIT cells are collected by apheresis, particularly by leukapheresis. As known to one of skill in the art, various methods for isolating immune cells from a subject are available or can be adapted to the current application, for example, using the Life Technologies Dynabeads System®; STEMcell Technologies EasySep, RoboSep™, RosetteSep™, SepMate™; Miltenyi Biotec MACS Cell Separation Kits™, Cell Surface Marker Expression, and other commercially available cell separation and isolation kits (e.g., ISOCELL from Pierce, Rockford, IL). MAIT cells can be isolated by using beads or other binding agents available in such kits that are specific for MAIT cell surface markers. For example, MAIT cells can be incubated with an anti-CD3 antibody and/or an anti-CD28 antibody under conditions that stimulate proliferation of the cells. In a particular embodiment, MAIT cells are expanded and/or activated in vitro in the presence of MAIT cell activating ligands such as 5-OP-RU and/or 5-OE-RU. In another particular embodiment, the method comprises a step of preferably in vitro MAIT cell expansion from a cell sample from a donor, in particular from PBMCs from a donor. A preferred in vitro MAIT cell expansion can be performed by culturing PBMCs from a donor in the presence of synthetic 5-OP-RU and optionally with cytokines. In particular, PBMCs from a donor are cultured in the presence of 5-OP-RU and IL-2 (such as rhuIL-2). In a particular embodiment, the MAIT cells are preferably expanded ex vivo for at least about 5 days, preferably not less than about 10 days, more preferably not less than about 15 days, and most preferably not less than about 20 days prior to administration to the patient. In a further embodiment, the MAIT cells are expanded at least 100-fold, preferably at least 200-fold, and more preferably at least 400-fold, preferably at least 600-fold, more preferably at least 1000-fold, and even more preferably at least 1500-fold compared to day 0 of expansion. prior to administration to a patient.

In the EP 09 10 624 B1 A method for multiplying lymphocytes in a cell culture is described, whereby lymphocytes are cultivated and multiplied in a cell culture medium that contains a lymphocyte growth factor and additionally aurintricarboxylic acid or a substance that binds to a cyclophilin and inhibits calcineurin in this complex. The cell culture medium contains cyclosporin, ascomycin and/or tacrolimus as the cyclophilin-binding substance. The lymphocytes are leukocytes that are found in the blood, lymph, spleen, lymph nodes, tumors or in inflamed tissues and are lymphocytes, T lymphocytes, B lymphocytes or NK cells. Lymphocyte growth factors are selected from T lymphocyte, B lymphocyte or NK lymphocyte growth factors, preferably from the group IL-2, IL-10, IL-13, IL-14 and IL-15. A lymphocyte growth factor is a substance that is able to promote the cell division of lymphocytes. Lymphocyte growth factors are widely known to the person skilled in the art. Suitable T-lymphocyte growth factors are, for example, interleukin-2 (IL-2) and interleukin-15 (IL-15). A suitable NK-lymphocyte growth factor is, for example, IL-15. Suitable B-lymphocyte growth factors are, for example, interleukin-13 (IL-13), IL-14 and IL-10 ( Callard, RE, and Gearing, JH, The Cytokine Facts Book, Academic Press, London, 1994 ). Tumor-infiltrating lymphocytes are cultured as lymphocytes and after ten days of culture a mixture of T lymphocytes (CD3+, CD4+, CD8+, CD19-), NK cells (CD2+, CD3-, CD56+, CD19-) and B lymphocytes (CD19+, CD3-) is detectable.

The proliferation of tumor-infiltrating lymphocytes occurs in a medium containing interleukin-2 (IL-2) and aurintricarboxylic acid (ATA), whereby the nodule of a human colon carcinoma, which was surgically removed from the colon, is surrounded by connective tissue and
normal intestinal parts and cut into pieces of approximately 2 × 2 × 3 mm. The tumor fragments are taken up in Iscove-modified DME medium (Gibco) containing 15% FCS (BM) and distributed evenly between four culture dishes.

• • die Strömungsverhältnisse im Gerinne eines Perfusions-Bioreaktors,
• • die Ausbildung einer weitgehend ruhenden Zellschicht, in der sie sich die Zellen berühren, aber auch leichte Bewegungen auftreten können, wobei TIL-Zellzahlen in der sedimentierteSchicht bei 0,1 bis 2 × 106 TIL/cm2, vorzugsweise 0,5 bis 1× 106 TIL/cm für eine stetige Vermehrung liegen,
• • das Kulturmedium Cytokine und Antikörper enthält, wobei die Cytokine in der Form des Interleukin 12 oder aus einem Gemisch aus Interleukin 2 und Interleukin 12 und die Antikörper in der Form des Antikörper 4-1bb bestehen und
• • die hyperoxische oder auch normoxische Versorgung der Zellen mit supplementiertem Medium und Sauerstoff in Abhängigkeit von der hochgewachsenen Menge an TIL oder anderen T-Zellen

In the DE 10 2021 002 748 A1 A method is disclosed for isolating, activating and multiplying immune cells, in particular tumor-infiltrated autologous T lymphocytes (TIL) from primary tumor tissue, metastases, lymph tissue, but also T cells from other tissues (e.g. blood, lymph fluid) in a meander perfusion bioreactor and the production of immune cell therapeutics therefrom for combating tumors of the pancreas, lung, liver, prostate, breast, ovary, stomach, colon, rectum, bone, brain, skin and other malignant tumors, wherein

• • the flow conditions in the channel of a perfusion bioreactor,
• • the formation of a largely resting cell layer in which the cells touch each other, but slight movements can also occur, with TIL cell numbers in the sedimented layer being 0.1 to 2 × 106 TIL/cm2, preferably 0.5 to 1 × 106 TIL/cm for continuous 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 4-1bb and
• • the hyperoxic or normoxic supply of the cells with supplemented medium and oxygen depending on the high number of TIL or other T cells

1. (a) Gewinnung einer ersten Population von TILs aus einem Tumor, der von einem Patienten reseziert wurde
2. (b) Hinzufügen der Tumorfragmente in ein geschlossenes System;
3. (c) Durchführen einer ersten Expansion durch Kultivieren der ersten Population von TILs in einem Zellkulturmedium, das IL-2 enthält, um eine zweite Population von TILs zu erzeugen, wobei die erste Expansion in einem geschlossenen Behälter durchgeführt wird, der eine erste gasdurchlässige Oberfläche bietet, wobei die erste Expansion für etwa 3 bis 14 Tage durchgeführt wird, um die zweite Population von TILs zu erhalten, wobei die zweite Population von TILs mindestens 50-mal größer ist als die erste Population von TILs, und wobei der Übergang von Schritt (b) zu Schritt (c) erfolgt, ohne das System zu öffnen;
4. (d) Durchführen einer zweiten Expansion durch Ergänzen des Zellkulturmediums der zweiten Population von TILs mit zusätzlichen IL-2-, OKT-3- und Antigenpräsentierenden Zellen (APCs), um eine dritte Population von TILs zu erzeugen, wobei die zweite Expansion für etwa 7 bis 14 Tage durchgeführt wird, um die dritte Population von TILs zu erhalten, wobei die dritte Population von TILs eine therapeutische Population von TILs ist, wobei die zweite Expansion in einem geschlossenen Behälter durchgeführt wird, der eine zweite gasdurchlässige Oberfläche bietet, und wobei der Übergang von Schritt (c) zu Schritt (d) erfolgt, ohne das System zu öffnen;
5. (e) Entnahme der therapeutischen Population von TILs, die aus Schritt (d) gewonnen wurden, wobei der Übergang von Schritt (d) zu Schritt (e) erfolgt, ohne das System zu öffnen; und
6. (f) Übertragung der geernteten TIL-Population von Schritt e) in einen Infusionsbeutel, wobei der Übergang von Schritt (e) zu (f) erfolgt, ohne das System zu öffnen.

The EP 3 601 533 B1 describes an improved and/or shortened process for expanding TILs and producing therapeutic populations of TILs, including novel methods for expanding TIL populations in a closed system that result in improved efficacy, phenotype and increased metabolic health of the TILs in a shorter period of time while allowing for reduced microbial contamination and lower costs. Such TILs find use in therapeutic treatment regimens. The process consists of:

1. (a) Obtaining a first population of TILs from a tumor resected from a patient
2. (b) adding the tumor fragments into a closed system;
3. (c) performing a first expansion by culturing the first population of TILs in a cell culture medium containing IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas permeable surface, wherein the first expansion is performed for about 3 to 14 days to obtain the second population of TILs, wherein the second population of TILs is at least 50 times larger than the first population of TILs, and wherein the transition from step (b) to step (c) occurs without opening the system;
4. (d) 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 generate a third population of TILs, wherein the second expansion is performed for about 7 to 14 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas permeable surface, and wherein the transition from step (c) to step (d) occurs without opening the system;
5. (e) withdrawing the therapeutic population of TILs obtained from step (d), the transition from step (d) to step (e) being carried out without opening the system; and
6. (f) transferring the harvested TIL population from step e) into an infusion bag, wherein the transition from step (e) to (f) occurs without opening the system.

The methods described in the prior art for expanding cells, in particular non-adherently growing T lymphocytes or NK cells, but also other immune cells, e.g. tumor-infiltrated NK cells (TINK), are carried out in culture dishes or 24 or 96 well plates, with a high proportion of human pooled AB serum (5% to 10%) being added to the culture medium, which represents a significant cost factor in the expansion of T lymphocytes and which also means that insufficient quantities of T lymphocytes can be made available for therapeutic treatment. Furthermore, after cultivation, a mixture of T lymphocytes (CD3+, CD4+, CD8+), NK cells (CD2+, CD56+) and B lymphocytes (CD19+) can be detected, although no high proportion of T lymphocytes containing a mixture of CD8 and CD56 antibodies was expanded.

The object of the invention is to develop a method for expanding cells, in particular non-adherently growing cells, such as invariant T lymphocytes or non-adherently growing T lymphocytes and NK cells, but also other immune cells, e.g. tumor-infiltrated NK cells (TINK) and mucous membrane-associated invariant T cells (MAIT) with a high proportion of CD 8+ and CD 56+ antibodies, also with a low proportion of CD3+ antibodies, in which sufficient quantities of cells are made available for therapeutic treatment with cells and the high costs, in particular through the use of human sera in the expansion, are avoided.

The task is achieved by a method for the expansion of cells, in particular non-adherently growing cells, such as invariant T-lymphocytes or non-adherently growing T-lymphocytes and NK cells but also other immune cells, e.g. B. tumor-infiltrated NK cells (TINK) and mucous membrane-associated invariant T cells (MAIT) with a high proportion of CD 8 and CD 56 antibodies, also with a low proportion of CD3+ antibodies, in a perfusion plate meander bioreactor, wherein the culture medium guided in channels 10 is overflowed by overlay media, wherein the perfusion plate meander bioreactor consists of several channels 10 lying one above the other and rotated by 180° with respect to each other, formed by webs 13 and with overflows 14 arranged in the culture carriers and overflow webs 33 arranged in the flow direction in front of the overflows 14, and wherein the culture medium in a bioreactor vessel 1 in the channels 10 on the culture carriers 7 of the bioreactor vessel 1 horizontally and through the overflows 14 and over the overflow web 33 onto the the underlying cultivation carrier 7 is guided vertically in a meandering manner from cultivation plate 7 to cultivation plate 7 to the outflow 23 for the culture medium. For gas diffusion of the overlay media into the culture medium and for keeping the concentration of the dissolved overlay media in the culture medium constant, the overlay media are guided above the surface of the culture medium in the channels 10 from the feeding overlay cavity 12 in the direction of the overflows 14 and in a meandering manner from bottom to top to the outflow 22 for overlay media.

Chopped tissue pieces, particularly small pieces of tissue from surgical tissue or biopsies, are introduced into the culture medium and cells grow in a perfusion meander bioreactor suitable for cell growth in a first stage. After a certain density of fully grown and expanded cells has been reached, these are separated from the tissue pieces and supplemented in the culture medium, whereby the culture medium consists of conventional base media with a mixture of AB human serum, antibodies, cytokines, lymphocyte growth factors and human feeder cell glycol and is placed in a perfusion plate meander bioreactor, expanded, activated and harvested, and whereby the culture medium AB human serum is supplemented in an amount of 2% of the total amount and with membrane portions from lysed human, autologous feeder cells.

In addition, coated particles as indicator beads with a size of 10 - 100 µm, in particular made of polystyrene or ceramic hollow beads made of spray granules and coated with fluorescent markers, in particular indocyanine green, are mixed into the culture medium and illuminated by a suitable light source 18 with a wavelength of between 750 nm and 950 nm, in particular with a light source with a wavelength in the fluorescence spectrum for blood of 830 nm, whereby the fluorescence brightness of the fluorescence marker changes due to the change in the concentration of overlay media, in particular of O ₂ . The lower the concentration of overlay media, in particular of O ₂ , in the culture medium, the greater the fluorescence brightness.

The density of the culture medium is 1.077 g/cm ³ , the density of the ceramic hollow spheres with air inclusion is 1.09 to 1.1 g/cm ³ and is only slightly higher than that of the culture medium. A sinking of the spheres in the flowing medium is therefore to be expected over longer periods of time. The pulse generator 31 attached to the vibrating table 30 accelerates the culture medium in the channel 10 vertically to the direction of flow at defined time intervals, in particular 1 to 3 times per day, by jerks between 2-5 m/s ³ and a mixing of the media components and the indicator spheres is achieved.

As the number of cells increases during the cultivation process, the glucose requirement and the oxygen requirement in the overlay also increase.

If the brightness gradient of the light-emitting indicator beads is measured and evaluated during inflow and outflow, the rate of cell proliferation can be increased by changing the flow rate of the culture medium and the overlay media according to defined algorithms. This leads to time savings and cost savings in the culture medium during cell cultivation.

As described above, the glucose supply via the culture medium depends on the O ₂ concentration in the culture medium, whereby the concentration of O ₂ is determined and regulated by the fluorescence brightness of the fluorescent marker.

The width of the channels 10 is selected and the amount of culture medium supplied and discharged is adjusted so that a laminar flow with a Bodenstein number of 0.001 to 0.1 is formed in the flow threads in the channels 10 and the bottom flow remains close to a Bodenstein number of 0. The distance b of the channels from the end walls is selected so that the culture medium can flow through the overflows 14 and over the overflow webs 33 without flow losses.

The Bodenstein number is a dimensionless number that describes the ratio of moles supplied convectively to those supplied by diffusion. The Bodenstein number thus characterizes the backmixing within a system (the larger the Bodenstein number, the lower the backmixing) and enables statements to be made about whether and to what extent volume elements or substances within the channels 10 are mixed by the prevailing currents. At a Bodenstein number of 0 or close to 0, the backmixing is greatest, which means that at a Bodenstein number close to 0, the overlay media are optimally mixed with the culture medium and the cells are thus optimally supplied with the overlay media during their expansion. The Bodenstein number Bo is defined as Bo= u × L/D _ax , where u is the flow velocity, L is the length of the reactor, here the length of the channels 10, and D _ax is the dispersion coefficient. Since the length of the channels 10 is given and the Bodenstein number Bo=0 represents ideal backmixing, the dispersion coefficient and thus the distribution of the overlay media, in particular the distribution of O ₂ , depends on the flow velocity of the culture medium and the length of the reactor, which can then be recognized and regulated by the fluorescence brightness in accordance with the overlay media distribution in the culture medium in the channels 10.

The overlay media flow over the culture medium into the channel 10 arranged on the culture carrier 7 with a laminar flow which is characterized by the Froud number 0 < Fr <1.

The Froude number describes the relationship between flow velocity and the propagation velocity of a shallow water wave, whereby in a flowing flow state the wave propagation shows a parabolic pattern.

The culture medium in the perfusion plate meander bioreactor is fed via the culture carriers 7 with channels 10 and overflows 14 in a meandering manner from top to bottom and the overlay media overflow alternately in the same and opposite direction to the flow of the culture medium, whereby the cells are supplied via the overlay media during expansion normoxemically with O ₂ and also hyperoxemically with an O ₂ concentration of 23 - 25 %.

The proportion of expanded cells that are labelled with the antibodies CD 8+ and CD 56+ is 70 up to 80%, with the antibody CD3+ also docked to the cells in small amounts.

As lymphocyte growth factors, interleukin 12 alone or as a mixture of interleukin 2 and interleukin 12 or as a mixture of IL12, IL15 with additionally the antibody 4-1 BB are used.

In one embodiment of the invention, the plate plane of the cultivation carrier 7 of the perfusion plate meander bioreactor arranged on a vibrating plate 30 with a pulse generator 31 is arranged horizontally and mechanically shaken in the direction of gravity at time intervals, in particular 1 to 3 times/day, the jerk introduced being between 2-5 m/s ³ .

The plate plane of the cultivation carrier 7 can also be vibrated electromechanically.

This reduces the segregation of TIL cells due to sedimentation during flow in the channels, increases the proliferation rate and reduces the throughput time of the planned cell proliferation. This leads to an earlier start of treatment with the positive effect of higher chances of recovery, as well as lower costs.

In a further embodiment of the invention, the cells in the culture media can be subjected to shear stress during expansion, wherein a portion of the culture medium is passed through a bypass 26 outside the perfusion plate meander bioreactor through a constriction of the bypass 26 created by an electromagnetic hose clamp 27.

The invention is explained in more detail using an example, in which the 1 a flow diagram of the perfusion plate meander bioreactor, the 2 a schematic side view, the 4 a 3-D top view of the bioreactor, the 5 a representation of the cultivation vehicle 7 and the 3 a schematic side view with the bypass 26 of the perfusion plate meander bioreactor with shaking table 30 and pulse generator 31,
where

1

Bioreaktorgefäß

2

Boden

3

Deckel

4

Stirnwand

5

Stirnwand

6

Seitenwand

7

Kultivierungsträger

8

Spannbügel

9

Spannmutter

10

Gerinne

11

Zufuhrkavität

12

Zuführungen Overlay- Medien

13

Stege

14

Überlauf

15

Auffangkammer

16

Raum für Overlay- Medien

17

Fuß

18

Lichtquelle

19

Lichtfenster

20

Luer- Lock- Verbinder für die Zuführung Overlay- Medien

21

Luer- Lock- Verbinder für die Zuführung Kulturmedium

22

Luer- Lock- Verbinder für die Abführung Overlay- Medien

23

Luer- Lock- Verbinder für die Abführung Kulturmedium

24

Overlay- Kavität

25

Kulturmedien

26

Bypass

27

elektromagnetische Schlauchklemme

28

Dreiwegekükenhahn

29

Luer- Lock- Verbinder

30

Rütteltisch

31

Impulsgeber

32

Kamera

33

Überlaufsteg

bedeuten.

1

bioreactor vessel

2

Floor

3

Lid

4

bulkhead

5

bulkhead

6

side wall

7

cultivation carrier

8

tensioning bracket

9

clamping nut

10

channel

11

supply cavity

12

overlay media feeders

13

footbridges

14

overflow

15

collection chamber

16

space for overlay media

17

Foot

18

light source

19

light window

20

Luer-Lock connector for supplying overlay media

21

Luer-Lock connector for supplying culture medium

22

Luer-Lock connector for the removal of overlay media

23

Luer-Lock connector for the removal of culture medium

24

overlay cavity

25

cultural media

26

bypass

27

electromagnetic hose clamp

28

three-way cock

29

Luer-Lock connector

30

vibrating table

31

initiator

32

camera

33

overflow bridge

mean.

A culture medium consists of a cell growth medium that is temporarily supplemented with a specific mixture of AB human serum, cytokines, antibodies and irradiated human feeder cells. The autologous tumor tissue pieces taken from the patient during an operation and chopped up in a size of 1 to 2 mm ³ as well as polystyrene particles coated with indocyanine green in a size of 10 -100 µm are added to this culture medium and fed with the culture medium to the bioreactor vessel 1 via the Luer-Lock connector 21 for the supply of culture medium. The culture medium is only supplemented with AB human serum in an amount of 2% of the total amount and with membrane parts from lysed human, autologous feeder cells. mented, with the membrane portions of the lysed feeder cells being added 4 - 5 days after the start of the expansion. The chopped tissue pieces are evenly distributed in the bioreactor and cultivated in perfusion mode, with the culture medium being supplied with the appropriate overlay atmosphere in a controlled manner by the overlay media fed into the overlay spaces 16 via the feed cavity 11 and the feeds 12 for overlay media. In a cultivation run over 7 to 14 days, cells (TIL or other cells) grow in sufficient quantities. As the number of cells increases, the supply of fresh medium is continuously increased as the number of cells (TIL or other cells) increases, with the increased supply of fresh medium being automatically controlled by an appropriate algorithm.

The perfusion plate meander bioreactor consists of a bioreactor vessel 1 made of polystyrene, which is made up of a base 2, a lid 3, end walls 4, 5 and side walls 6. Several, in particular eight, cultivation supports 7 are fastened in the bioreactor vessel 1 via the clamping nuts 9 and a clamping bracket 8 at a distance of 11.5 mm. On the cultivation supports 7, channels 10 are formed by webs 13 through which the culture medium and the gaseous overlay media are guided and the culture medium is discharged via an overflow web 33 of 6 mm height and through one or more overflows 14 onto the cultivation support 7 below. This underlying cultivation carrier 7 is mounted with its overflow 14 and its overflow web 33 rotated by 180° relative to the cultivation carrier 7 above, so that the culture medium flows in a meandering manner from top to bottom into the collecting chamber 15 for the culture medium and is discharged from the bioreactor vessel 1 via the Luer Lock connector 23. The distance a between the webs 13 is selected and the amount of culture medium supplied and discharged is set so that a laminar flow with a Bodenstein number of 0.001 to 0.1 is formed in the flow thread, in the channel 10 formed by the webs 13, for the optimal supply of the expanding cells, and the bottom flow remains close to a Bodenstein number of 0. This is mainly achieved by the overflow web 33 with a height of up to 6 mm, because the flow velocity (u) in the laminar boundary layer at the bottom of the channel 10 is close to 0 and thus the Bodenstein number Bo is approximately 0) (u is proportional to Bo).

A supply cavity 11 for overlay media is incorporated into the front wall 4, which is connected via feeds 12 for overlay media to overlay spaces 16, which are located above the cultivation supports 7 with the flowing culture medium. Via the Luer-Lock connector 20, which is connected to the supply cavity 11 for overlay media, the overlay media is guided in countercurrent over the cultivation medium into the collection chamber 24 for overlay media and is discharged from the bioreactor vessel 1 via the Luer-Lock connector 22 for the discharge of the overlay media.

The bottom 2 of the bioreactor vessel 1 has a light window 19, under which a light source 20 with a wavelength in the fluorescence spectrum for blood of 830 nm with a video camera 32 is mounted, with the aid of which the backmixing of the overlay media in the culture media is monitored and controlled, wherein the culture medium is provided with indocyanine green coated polystyrene particles to detect the backmixing, the color change of which indicates the change in the concentration of overlay media, in particular the concentration of O _2. The lower the concentration of overlay media, in particular of O ₂ in the culture medium, the greater the fluorescence brightness.

Instead of the Luer-Lock connector 20, a three-way cock 28 can be arranged in the front wall 4 and a Luer-Lock connector 29 connected to the space for the culture medium 25 of this culture carrier 7 can be arranged in the front wall 5 above the centrally arranged cultivation carrier 7, wherein the three-way cock 28 and the Luer-Lock connector 29 are connected to one another via a bypass 26 and wherein an electromagnetic hose clamp 27 is arranged in the bypass 26.

This means that by switching the three-way cock 28 and the Luer lock connector 29, part of the culture medium can be passed through the bypass 26 and by narrowing the cross-section of the bypass 26, a definable shear stress can be exerted on the expanding cells in the culture media. The activation of cells, particularly T cells, is both a mechanical and a biochemical process. With the electromagnetic hose clamp 27, a reproducible, defined shear stress is exerted on the cells, particularly T cells, and thus the activation is mechanically stimulated.

The bioreactor vessel 1 is arranged on a vibrating table 30 with a pulse generator 31, wherein a light source 18 with a wavelength in the fluorescence spectrum for blood of 830 nm, with a camera 32 is attached to the bottom 2 of the bioreactor vessel 1 under a polished light window 19.

The control of glucose supply via the culture medium is dependent on the O ₂ concentration concentration in the culture media, whereby the concentration of O ₂ is determined from the fluorescence brightness of the fluorescent marker and the glucose supply is thus regulated.

The culture medium, which flows in 10 parallel, hydraulically separated channels 10 on the cultivation support 7 through the channels 10 and at the end of the cultivation plate 7 flows through the overflows 14 onto the cultivation plate 7 below and then flows in the opposite direction through the channels 10, has a very low flow velocity of approx. 1 cm/min and thus meanders vertically. In relation to the entire channel area, this results in a Reynolds number between 10<Re<50 <<2320 =Rekrit., which, at the low flow velocity, leads to a guaranteed laminar flow with a Bodenstein number Bo close to zero.

The Bodenstein number Bo is defined with Bo= u × L/Dax, where u is the flow velocity, L is the length of the reactor, here the length of the channels 10, and D _ax is the dispersion coefficient. Since the length of the channels 10 is given and the Bodenstein number Bo=0 represents ideal backmixing, the dispersion coefficient and thus the distribution of the overlay media, in particular the distribution of O ₂ , depends on the flow velocity of the culture medium, which can then be recognized and regulated by the fluorescence brightness in the channels 10 according to the overlay media distribution in the culture medium.

The overlay media flow over the culture medium into the channel 10 arranged on the culture carrier with a laminar flow characterized by the Froud number close to 0.

The culture medium in the perfusion plate meander bioreactor is guided via the culture carriers 7 with the channels 10 and overflows 14 in a meandering manner from top to bottom and the overlay media overflow from bottom to top in the opposite direction to the flow of the culture medium, whereby the cells are supplied with O ₂ via the overlay media during expansion normoxemically or hyperoxemically with an O ₂ concentration of 23 - 25%.

When expanded using this method, the proportion of expanded cells that are provided with the antibodies CD 8+ and CD 56+ is 70 to 80%, with the antibody CD3+ also docked to the cells in small amounts.

As lymphocyte growth factors, interleukin 12 alone or as a mixture of interleukin 2 and interleukin 12 or as a mixture of IL12, IL15 with additionally the antibody 4-1 BB are used.

The plate plane of the cultivation carrier 7 of the perfusion plate meander bioreactor arranged on a vibrating plate 30 with a pulse generator 31 is arranged horizontally and is mechanically jerked in the direction of gravity at time intervals, in particular 1 to 3 times / day, whereby the jerk introduced is between 2-5 m/s ³ .

The plate plane of the cultivation carrier 7 can also be vibrated electromechanically.

The cells in the culture medium will be subjected to shear stress during expansion, with a portion of the culture medium being passed through a bypass 26 outside the perfusion plate meander bioreactor through a constriction of the bypass 26 created by an electromagnetic hose clamp 27.

Claims (16)

Method for the expansion of cells, such as non-adherently growing cells, invariant T lymphocytes, non-adherently growing T lymphocytes, NK cells, tumor-infiltrated NK cells (TINK), mucous membrane-associated invariant T cells (MAIT) or other immune cells with a high proportion of CD8+ and CD56+ antibodies and with a low proportion of CD3+ antibodies in a perfusion plate meander bioreactor with culture medium and overlay media streams that are fed in countercurrent to one another, whereby cells grow out of chopped tissue pieces from surgical tissue parts or from biopsies in a closed perfusion meander process in a first stage and, after reaching a certain density of fully grown and expanded cells, are separated from the tissue parts, supplemented in suitable culture medium and placed in a perfusion plate meander consisting of several culture carriers (7) arranged vertically one above the other and rotated by 180°. Bioreactor, are introduced, expanded, activated and harvested, whereby channels (10), overflows (14) and overflow webs (33) are arranged on the cultivation carrier (7) and the culture medium from usual base medium with a mixture of AB human serum, antibodies, cytokines, lymphocyte growth factors and human feeder cells with supplemented glycol and in the bioreactor vessel (1) in the channels (10) on the cultivation carriers (7) of the bioreactor vessel (1) flows horizontally and through the overflows (14) into the cultivation carrier (7) and over the overflow webs (33) in a meandering vertical manner from cultivation plate (7) to cultivation plate (7) to the outlet (23) for the culture medium and the Overlay media overflow in the opposite direction to the flow of the culture medium in the channels (10) for gas diffusion of the overlay media into the culture medium and for keeping the concentration of the dissolved overlay media in the culture medium constant above the surface of the culture medium in the channels (10), characterized in that • the culture medium is supplemented with AB human serum in an amount of 2% of the total amount and with membrane portions from lysed human, autologous feeder cells and • indicator beads in a size of 10 -100 µm, which are coated with fluorescent markers, are mixed into the culture medium and illuminated by a light source with a wavelength of between 750 nm and 950 nm, whereby the fluorescence brightness of the fluorescent marker changes due to the change in the O ₂ concentration in the overlay media procedure according to claim 1 , characterized in that the cells are supplied via the overlay media during expansion normoxemically with O ₂ or hyperoxemically with an O ₂ concentration of 23 - 25 %. procedure according to claim 1 , characterized in that the indicator beads consist of polystyrene or hollow ceramic beads made from spray granules. procedure according to claim 3 , characterized in that the coated indicator beads, which consist of hollow ceramic beads made from spray granulate, consist of mixtures of ZrO ₂ and Al2O ₃ . procedure according to claim 1 and 2 that the culture medium, which is guided in parallel, hydraulically separated channels (10) on the cultivation carrier (7) through the channels (10) and flows at the end of the cultivation plate (7) through the overflows (14) onto the underlying cultivation plate (7) and then in the opposite direction through the channels (10), has a flow velocity of 1 cm/min and meanders vertically, whereby, based on the entire channel area, a Reynolds number between 10<Re<50 <<2320 =Re _crit . and a laminar flow with a Bodenstein number Bo close to zero is established. procedure according to claim 1 - 4 , characterized in that the fluorescent marker consists of indocyanine green. procedure according to claim 1 , characterized in that the culture medium with the admixed particles is irradiated with a light source with a wavelength in the fluorescence spectrum for blood of 830 nm. procedure according to claim 1 - 7 , characterized in that the proportion of cells with the CD 8+ and CD 56+ antibodies is 70 to 80%. procedure according to claim 1 - 8 , characterized in that the lymphocyte growth factors consist of interleukin 12 alone or of a mixture of interleukin 2 and interleukin 12 or of a mixture of IL12, IL15 and all mixtures additionally contain the antibody 4-1 BB. procedure according to claim 1 - 9 , characterized in that the membrane components from lysed human, autologous feeder cells are added to the culture medium 4 - 5 days after the start of the expansion of the cells. procedure according to claim 1 - 10 , characterized in that the control of the glucose supply via the culture medium takes place as a function of the O ₂ concentration in the culture medium, wherein the concentration of O ₂ is determined and regulated by the fluorescence brightness of the fluorescent marker. Method according to claims 1 - 11, characterized in that in the culture medium during the meandering flow through the perfusion meander plate bioreactor, jerky liquid turbulences are generated by one or more jolts from the outside on the perfusion plate meander bioreactor in the culture medium flowing through the channels (10) of the cultivation carriers (7). procedure according to claim 1 - 12 , characterized in that the plate plane of the cultivation carriers (7) lie horizontally on a perfusion plate meander bioreactor arranged on a shaking device (not shown) and is mechanically shaken in the direction of gravity at time intervals, the jerk impulse introduced being between 2-5 m/s ³ . procedure according to claim 1 - 13 , characterized in that the shock pulse is introduced into the culture medium at a time interval of 1 to 3 times / day. procedure according to claim 1 - 14 , characterized in that the plate plane of the cultivation carrier (7) is moved electromechanically. procedure according to claim 1 - 15 , characterized in that the culture medium is a Luer-Lock connector (29) and a three-way cock (28) via a bypass (26) is subjected to a defined shear stress by an electromagnetic hose clamp (27).

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