Simple automated manufacturing of gene engineered T cells from lymphoma and melanoma blood samples ISCT North America 216 Regional Meeting Nadine Mockel-Tenbrinck Miltenyi Biotec GmbH
Macrophage Tumour cell Typical workflow FOCUS for ON gene-engineering t UmOUR ImmUNOl of T Og cells y & ImmUNOt for adoptive REVI h ERa T EWS cell py MDSC therapy research NK cell a Tumour sample from patient TCR T cell Tumour- CAR TCR Fragmentation of tumour mass Tumour Expand cell populations in culture T Reg cell T cell Patient Chemotherapy Irradiation Blood sample T cells isolated from blood tumour Avidity Activation testing Viral vector encoding CAR Human T cell engineered to express tumour- CAR harvest modified from Restifo et al. Nat rev immunol. 212 b T cell growth factors (such as IL-2) Infusion of tumourinto patient Typical workflow: Blood c Genetically engineered activating motif Mouse transgenic for human MHC class I molecules Clone chimeric Figure 1 antigen receptor into retroviral vector Adoptive cell transfer (ACT). The administration of tumour-specific lymphocytes (obtained from the patient (autologous) or from a donor (allogeneic)) following a lymphodepleting preparative regimen. Lymphodepletion The use of total-body irradiation or cytotoxic drugs to deplete the lymphoid Quality control Activation and of T cells Human T cell expressing chimeric antigen a tumour antigen T cell populations Nature Reviews Immunology expanding T cell populations. The TILs are dissociated tumour cells and normal tissues that are dispensable? from immunosuppressive cell populations such as Is there a window such that T cells can recognize myeloid-derived suppressor cells (MDSCs) and possibly tumour cells but not normal cells? What proportion of exposed to lower levels of immunosuppressive cytokines malignant cells in a tumour stably express the candidate during this early period in culture. The ex vivo antigen, and at what level? Is it strictly necessary to target Isolate mouse T cell Clone mouse Human T cell Storage Application compartment in a patient. 2. October 216; ISCT NA Miltenyi of TIL populations Biotec to more GmbH favourable numbers followed all cancer cells, or will bystander killing result in tumour Page 2
The CliniMACS Prodigy T Transduction (TCT) platform enables the automated gene-engineering of T cells Blood Storage Application + Integrated cell processing from starting material to final cellular : Sample preparation washing & density gradient separation MACS cell separation Genetic modification culture Final Enabling complex processes Automated & controlled system Closed single-use tubing set 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 3
Proof of concept: Automated T cell Transduction and Expansion Process Whole blood or apheresis (fresh or frozen) CliniMACS CD4 Reagent + CliniMACS CD8 Reagent MACS GMP T TransAct or MACS GMP TransAct Kit Lentiviral vector (encoding CD2 CAR) TexMACS GMP Medium + MACS GMP rec. hum.il7/15 Re and harvest of cellular Closed single-use CliniMACS Prodigy TS 52 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 4
The TCT Software Program enables Simple and Automated separation, and gene-modification of T cells Guided User interface (GUI) for culture: Activity matrix All parameters, so called activities, have to be defined before starting the culture: Culture set-up (integrity test, priming of TS, cell isolation, stimulation) Transduction Shaking conditions Feeding (Feed or Media exchange) culture wash Culture process is running until final harvest User Interactions required: connection of fresh virus (day 1) New medium bag (2L for 5-7 days) Connection of solution for harvest (day 1-14) In-between samples for in-process control (+ pictures) can be taken (at any time-point of culture) 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 6
cell count [x1 8 ] Viability The CliniMACS Prodigy TCT application Robustness and Reproducibility of culture 5 4 3 2 1 Robust non-transduced lentiviral transduced 2 4 6 8 1 12 14 time [days] 1 8 6 4 2 2 4 6 8 1 12 14 time [days] count (day 1-14) T cells black: non-trans. grey: CD2 CAR CD2 CAR CAR + T cells non-transduced 3.11E9 (±1.3) - transduced 3.13E9 (±.9) 1.3E9 (±.3) 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 7
CD8 The CliniMACS Prodigy TCT application Results: Automated T cell Selection Process 1 5 Melanoma Melanoma patient T cells: 4.2% 71.1% WB enriched 1 5 Lymphoma Lymphoma patient T cells: 19.4% 53.9% LP enriched CD4 CD8 NKT cells NK cells Monocytes B cells Granulocytes CD4 Preenrichment postenrichment 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 8
The CliniMACS Prodigy TCT application Results: Automated T cell Activation indirect analysis by detection of T cell clustering using the integrated microscope camera 24h 72h post Normal material Lymphoma material 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 9
frequency of GFP+ efficiency cells (in %) SSC efficiency The CliniMACS Prodigy TCT application Results: Automated lentiviral CD2 CAR T cell Transduction optimal time-point for lentiviral after TransAct stimulation 1 8 5 6 4 2 TransAct automated lentiviral CD2 CAR modification of T cells (day 1) mock CD2 CAR 38% normal material (N) 4% 5 healthy untransduced day patient day 1 day 2 day 3 CD2 CAR Lymphoma material (L) N healthy L patient 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 1
The CliniMACS Prodigy TCT application Results: Automated T cell cultivation Monitoring of T cell growth after polyclonal T cell stimulation followed by lentiviral gene-modification Melanoma Lymphoma normal material * frozen lymphoma material 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 11
The CliniMACS Prodigy TCT application Results: Automated T cell Expansion material type T cell count for culture final T cell count T cell Final CAR + T cell count Melanoma (M35-WB).21E8 2.35E9 112 fold 1.38E9 Lymphoma (L42-LP) 1.E8 3.68E9 37 fold 1.54E9 Lymphoma (L43-LP) 1.E8 3.31E9 33 fold - Lymphoma (L55-LP)*.55E8 4.9E9 89 fold 1.9E9 Lymphoma (L56-LP)*.55E8 3.2E9 58 fold.72e9 * frozen start material SUM.66E8 (±.33) 2.89E9 (±.7) 66 fold (±34) 1.2E9 (±.4) 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 12
T B M NK NKT G T B M NK NKT G melanoma or lymphoma T B M NK NKT G T B M NK NKT G normal The CliniMACS Prodigy TCT application Results: Quality control ular composition enriched population: HEALTHY 1 enriched population final : HEALTHY 1 final cell 5 5 1 1 5 5 T: T cell B: B cell M: Monocytes NK: NK cell NKT: NKT cell G: Granulocytes 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 13
CD4 CD8 CD4 CD8 melanoma or lymphoma (%) melanoma or lymphoma CD4 CD8 CD4 CD8 Normal material Normal material The CliniMACS Prodigy TCT application Results: Quality control T cell Phenotype 1 enriched population 1 final cell 1 enriched population 1 final cell 5 5 5 5 1 T N T CM T EM 1 T N T CM T EM 1 1 5 5 5 5 T N T CM T EM T N T CM T EM 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 14
GM-CSF IFN-g IL-2 IL-9 IL-1 IL-17A TNF-a GM-CSF IFN-g IL-2 IL-9 IL-1 IL-17A TNF-a pg/ml pg/ml % killing of CD2 + targets normalized to mock control The CliniMACS Prodigy TCT application Results: Quality control Functionality of CD2 CAR T cells cytokine secretion after co-culture with target cells (24 hours) 5 1 4 4 1 4 normal 5 1 4 4 1 4 lymphoma In vitro CD2 target cell killing 1 healthy normal Lymphoma patient 3 1 4 2 1 4 3 1 4 2 1 4 5 1 1 4 1 1 4 1:1.2:1 1:1.2:1 E:T Ratio 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 15
The CliniMACS Prodigy TCT application for simple automated manufacturing of gene-modified T cells The process can be started from several types of blood CD4/CD8 enables reduction of unwanted cell populations Efficient stimulation of selected T cells from different types of blood Automated lentiviral modification enables simple manufacturing of functional CAR specific T cells Good of T cells during automated process yielding sufficient number of CAR T cells Automated manufactured CAR T cells from different material are poly-functional 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 16
Acknowledgement Miltenyi Biotec Andrew Kaiser Daniela Mauer Carola Barth Katharina Drechsel Dominik Lock Jörg Mittelstät Ian Johnston Thomas Schaser Constanze Lehmann Juliane Stuth Kristina Reck Rene Hamannt Michael Apel Manal Hadenfeld Stefan Miltenyi Mario Assenmacher Anne Richter Volker Huppert Miriam Haak Jürgen Schmitz Ulf Bethke Martin Meyer Bernd Schröder Christophe Klumb Burgund Kauling Georg Rauser Katharina Winnwmöller Liane Preußner Yingzi Ge Marion Jurk LTI Rimas Orentas Dina Schneider Boro Dropulic UKK Prof. Dr. Hinrich Abken Prof Dr. Hallek Natali Pflug Udo Holtick Peter Borchman Max Schlaak Philipp Gödel Philipp Köhler Michael Von Bergwelt Michael Hallek Thanks for your attention 2. October 216; ISCT NA Miltenyi Biotec GmbH Page 16