WP1 Efficacy and safety studies
Objective of WP1
- To evaluate the antitumor efficacy of the EURE-CART cell product.
- To evaluate the safety of the EURE-CART cell product.
CD44v6 is a tumor-associated antigen expressed by many solid and haematological malignancies and can be targeted by engineering human T cells to express a specific chimeric antigen receptor (CD44v6 CAR). The CD44v6 CAR sequence has been optimized to abrogate any potential off-target recognition by CAR-T cells, simultaneously allowing their selection/tracking. In WP1, partner OSR will validate at the preclinical level the antitumor activity and the safety of the EURE-CART cell product, namely human T cells genetically modified to express both the optimized CD44v6 CAR and an improved version of the HSV-TK (TK) suicide gene, which provides a safety switch in case of toxicity. The in vitro functionality of the CD44v6 CAR will be assayed as the capacity to kill CD44v6-expressing leukaemia or myeloma cells in co-culture assays, to proliferate and to produce effector cytokines, while the functionality of the TK suicide gene will be evaluated by measuring T-cell death induced by treatment with ganciclovir. The in vivo antitumor activity of EURE-CART cells will be evaluated in xenograft models of adoptive immunotherapy, whereas TK functionality will be evaluated in a xenograft model of graft-versus-host disease. Finally, partner OSR will perform a safety study using exactly the same EURE-CART cell product that will be used in the clinical study by monitoring the health of immunodeficient mice injected with CAR T-Cells. Partner OSR is responsible for WP1 due to its particular expertise in development and translation of innovative gene therapy concepts. Importantly, all information will be next transferred to the Coordinator thus contributing to the development of the final EURE-CART cell product.The main project tasks are the following
Task1.1 Preclinical validation of the antitumor efficacy of the EURE-CART cell product
Preclinical validation of the antitumor efficacy of the EURE-CART cell product. Workflow: The Participant OSR has already shown that T cells genetically modified with a research-grade bicistronic viral vector encoding for a CD44v6 CAR and the suicide gene TK become potent anti-tumour effectors and, at the same time, can be ablated pharmacologically (Casucci et al, Blood 2013). The CD44v6 CAR has been modified by substituting the original CH2CH3 spacer with domains from the low-affinity nerve growth factor receptor (LNGFR, WO2016042461A1). This modification completely abrogates any residual off-target recognition by CAR-T cells, simultaneously allowing their selection/tracking (Casucci et al, in preparation). In this task, Partner OSR will validate at a preclinical level the antitumor activity and the safety of human T cells genetically modified with a clinical-grade bicistronic retroviral vector (RV) encoding for both a LNGFR-spaced version of the original CD44v6 CAR and a mutated version of TK, which resists alternative splicing and reduced functionality (mut2 provided by the Coordinator MLM). TK transduction/functionality will be measured by Q-PCR and as GCVmediated killing. The overall antitumor activity of EURE-CART cells will be evaluated in xenograft models.
Task 1.2 Preclinical validation of the safety of the EURE-CART cell product
Preclinical validation of the safety of the EURE-CART cell product. Workflow: Before approving a Phase I study of cell and gene therapy, and especially in the case of first-in-human studies, National and international regulatory authorities increasingly require an IMPD describing the safety of the final ATMP in a clinically relevant model. These studies are usually performed in immunocompromised mice, in which it is possible to evaluate the risks of uncontrolled growth. After optimising the processes contributing to the EURE-CART cell product, Partner OSR will proceed to a safety study in NSG mice using exactly the same EURE-CART cell product that will be used in the clinical study and that will be produced by the Coordinator MLM (see WP2, Task 2.1 and 2.2).
Briefly, after sub-lethal irradiation, NSG mice will be infused with CAR-T cell at the same dose as in efficacy studies, see Task 1.1), NSG mice kept in IVC and given food/water ad libitum will be monitored over time for clinical signs of wellbeing (feeding, drinking, activity, weigh loss) and periodically bled for clinical chemistry studies (kidney, liver, heart function). NGFR marking of circulating T cells will crucially enable to evaluate whether transgene-expressing T cells have any competitive growth advantage over non-transgene expressing contaminants in vivo. At the time of GVHD development, defined as weigh loss >20% (usually within 30 days), NSG mice will be administered GCV over 5-7 days through a sub-cutaneous pump and monitored for survival until day 60. All animal experiments have already been approved by the IACUC and will be appropriately statistically powered to detect the expected differences between experimental groups. All these methods will be next transferred by Partner OSR to the Coordinator MLM thus contributing to the final EURE-CART cell product (see WP2, Task 2.1 and 2.2).