Abstract A023: Highly enriched memory stem T-cell subsets (TSCM) expressing a novel CAR30 have enhanced antitumor effect in Hodgkin lymphoma
Authors: Alvarez-Fernández Carmen , Escribà-Garcia Laura , Caballero Ana Carolina , Sierra Jorge , Briones Javier 
Published: February 2019

Abstract
Background: Up to 20% of patients with advanced Hodgkin lymphoma (HL) are not cured with current chemo-immunotherapy treatments. Adoptive immunotherapy (AIT) with mature T-cells modified with a chimeric antigen receptor (CAR) holds promise for the treatment of various types of B cell lymphoma. The CD30 antigen, expressed in virtually all HL tumor cells, is absent in most healthy tissues, thus representing an ideal target for AIT of HL. In contrast to CAR19 for B cell malignancies, efficacy of CAR30 T-cells for Hodgkin and other CD30 lymphomas remains modest. Thus, novel approaches are needed to significantly improve the clinical efficacy of this approach. To this end, we used a novel antigen binding domain of a CD30 mAb, unaffected by soluble CD30 protein, as part of a chimeric antigen receptor (CAR) coupled to the 4.1BB and ζ chain endodomains, and transduced memory stem T-cells (TSCM) in an effort to ensure efficient engraftment, prolonged persistence, and enhancement of in vivo antitumor activity. To evaluate the feasibility of ex vivo expansion and antitumor efficacy of genetically-modified TSCM with a novel CD30-specific CAR that recognizes a membrane-proximal epitope of the CD30 molecule for the treatment of HL. Methods: A second-generation CD30-41BBz-EGFRt CAR was developed using a scFv that recognizes a membrane-proximal epitope of the CD30 protein. TSCM cells were generated with IL-7, IL-15 and IL-21, and transduced on day 2 of culture with a third-generation lentiviral vector encoding the CD30-CAR. The HL derived cell line L540 was used as tumor model. Tumor-specific cytotoxicity and cytokines were analysed at 24 hours. The in vivo antitumor efficacy was tested in a NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mouse model of HL xenografted with 2,5x10^6 L450 cells. Results: After 10 days, TSCM represented the majority of T-cells in culture (50.38±5.47 % for CD4+ and 70.84 ± 3.36 % for CD8+ TSCM, respectively) with a very high expression of CD30-CAR (77 ± 3.64 % in CD4+ and 83.12 ± 4.25 % in CD8+ TSCM, respectively). Expression of CD30 protein in TSCM increased over the culture period, peaking at day 8 in both CD4+ (17.5 ± 4.17 %) and CD8+ TSCM (42.37 ± 5.75 %), respectively. Despite these high levels of CD30 expression, TSCM expanded ex vivo very efficiently (CD4+ CD30-CAR: 97.24 ± 26.2 fold expansion, and CD8+ CD30-CAR: 96.79 ± 22.84 fold expansion, respectively), although lower than TSCM transduced with a CD19-CAR (CD4+ CD19-CAR: 161.96±35.9 fold expansion, and CD8+ CD19-CAR: 163.25 ± 26.51, fold expansion, respectively). Bulky CD30-CAR TSCM mediated very efficient in vitro tumor cytotoxicity (tumor cell death at 5:1 ratio: 85.25% vs. 0% with untransduced TSCM), while control CD30- targeT-cells were not killed. In addition, we evaluated the antitumor efficacy of CD30-CAR TSCM in vivo. Upon adoptive transfer of 10x10^6 bulk (CD4+ and CD8+) TSCM into NSG mice engrafted with L540 (2,5x10^6 subcutaneously), complete eradication of HL was observed. In addition, in a stress L540 model where equal numbers of CAR30 T-cells were infused at a low, suboptimal dose (i.e, 50% of effective T-cell dose to induce complete remission), highly enriched CAR30 TSCM (&gt;70% of TSCM) had significantly antitumor effect compared to the infusion of CAR30 T-cells predominantly represented by effector cells (&lt;30% TSCM) (mean survival 54 days vs 40 days, respectively; P&lt; 0.01). Conclusions: We demonstrate that TSCM can be efficiently transduced and ex vivo expanded with a novel CD30-CAR that surpasses fratricide killing of CD30 expressing T-cells and confers potent in vivo antitumor efficacy against HL. Importantly, highly enriched CAR30 TSCM products have enhanced antitumor properties compared to those comprising predominantly effector cell subsets. These proof-of-concept studies support the use of a refined CAR30 with highly enriched TSCM cell products for improving clinical efficacy of adoptive immunotherapy of patients with HL.
Citation Format: Carmen Alvarez-Fernández, Laura Escribà-Garcia, Ana Carolina Caballero, Jorge Sierra, Javier Briones. Highly enriched memory stem T-cell subsets (TSCM) expressing a novel CAR30 have enhanced antitumor effect in Hodgkin lymphoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A023.