WP2 Production of clinical-grade EURE-CART cells
Objective of WP2
- To produce clinical-grade EURE-CART cells for distribution to all clinical participants.
The main project tasks are the following
Task 2.1 Development and validation of the EURE-CART transduction process under GMP
MLM will proceed to all necessary steps for development and GMP validation of an efficient transduction process to obtain CAR-T cells expressing the CAR CD44v6 and the suicide gene product HSV-TKmut2. The CAR CD44v6, which contains as spacer the extracellular part of the human low affinity nerve growth factor receptor (LNGFR), and the suicide gene product HSV-TKmut2, are encoded by a retroviral vector (RVV) able to efficiently transduced human T cells in defined culture conditions.
In particular, Task 2.1 includes the following activities:
i) Development of a GMP grade transduction process for the production of CAR-T cells.
The process will be developed starting from small scale to large scale conditions of cell manipulation. After this initial development phase, full-scale validation runs will be performed as transfer process to the Production Unit. The following is an example of a transduction protocol to obtain transduced T cells. Briefly, PBL are seeded in cell culture bags using an appropriate medium and are stimulated with anti-CD3/CD28-beads and GMP-grade rhIL-7 and rhIL15 for 48 hours. Activated cells are then seeded in RetroNectin-coated cell culture bags in the presence of retroviral vectors carrying the CAR CD44v6 gene and the HSV-TKmut2 suicide gene, previously manufactured and released by MLM. At the end of the transduction process, the cells are collected, washed, cultured and then selected by using microbeads conjugated to anti-LNGFR mAb (CD271 reagent by Miltenyi). The selected cells are then expanded and frozen.
The presence of process-related contaminants, critical process parameters (transduction efficiency cell expansion and cell viability) and critical quality attributes (cell differentiation, polarisation etc) are analysed at different time points of the process.
The results of these runs will be evaluated and a final protocol and a validation strategy will be designed and possibly discussed with the different National regulatory authorities (see WP3). All the results will be summarized in a development report and included in the IMPD.
ii) Development of the analytical methods to characterize the clinical-grade EURE-CART cells. Most analytical methods for the release of transduced T cells have already been developed and validated in house for similar clinical-grade protocols. Additional methods for functional evaluation of this specific process and of the final EURE-CART product will be developed by MLM. These methods will be qualified as appropriate. On the basis of results, a method transfer and validation strategy will be designed. All the results will be summarised in development/qualification reports and included in the IMPD.
iii) Validation of the final GMP-grade CAR-T cell product. The validation runs will be performed in a classified area, extensively analysed by QC and released by the Qualified Person. In the mid-term, part of these runs will be used for producing the EURE-CART cells to be investigated in the efficacy and safety study by OSR (see WP1) and to perform GLP toxicology.
Task 2.2 Preparation of the harmonised IMPD for the EURE-CART cell product.
After validating the GMP manufacturing of both the retroviral vector and the CAR-T cells, MLM will prepare a transnationally harmonised IMPD including these results, as well as those from the efficacy and safety study performed by OSR (see WP1). After verification of the compliance with the regulatory requirements identified in WP3, the IMPD will be transmitted to the National regulatory authorities of the different countries participating to the clinical study (Italy, Germany, Spain and Czech Republic) for timely approval of the EURE-CART cell product.
Task 2.3 Production and release of clinical-grade EURE-CART cells.
Following the IMPD approval by the National regulatory authorities of the different countries, clinical batches of EURE-CART cells will be produced at MLM using the transduction process validated in Task 2.1. All the manipulations will be performed in a classified area (class A located into class B dedicated room). Each batch will be analysed by the quality control unit according to a defined panel of release tests identified in WP3. After approval by the Qualified Person, EURE-CART cells will be released and shipped to all clinical participants.