Dual Transgenesis of T Cells with a Novel CD44v6-Specific Chimeric Antigen Receptor and a Suicide Gene for Safe and Effective Targeting of Chemoresistance in Hematopoietic Tumors
Authors: Casucci Monica , Falcone Laura , Robilant Benedetta Nicolis di , Camisa Barbara , Genovese Pietro , Gentner Bernhard , Naldini Luigi , Savoldo Barbara , Ciceri Fabio , Bordignon Claudio , Dotti Gianpietro , Bonini Chiara , Bondanza Attilio 
Published: October 2019

Abstract
Abstract 3125

Background:
We have previously demonstrated that the genetic induction of a conditional suicidal phenotype in donor T cells allows for an operational dissociation of the GVL effect from GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Unfortunately, leukemia often escapes the immunological pressure of alloreactive donor T cells by losing “passenger” mismatched HLAs. Conversely, redirecting T cells against a non HLA-restricted antigen critically involved in the neoplastic phenotype may circumvent tumor escape due to the emergence of antigen-loss variants. The isoform variant 6 of CD44 is expressed by different epithelial and hematological cancers, and is possibly involved in tumor-cell survival and proliferation. Clinical experience with chemo-conjugated CD44v6-specific mAbs in epithelial tumors showed substantial efficacy, which was however limited by skin toxicity due to background expression of CD44v6 on keratinocytes

Aim:
By analogy with our experience in HSCT, we reasoned that CD44v6 targeting with suicide gene-modified T cells would provide a major therapeutic effect against hematological tumors, while granting a safety switch in case of toxicity. To this aim, we designed a novel CD44v6-specific chimeric antigen receptor (CAR) and developed a strategy for its co-expression with a suicide gene

Results:
CD44v6 expression by FACS was observed at high levels in 6/17 (37%) cases of acute myeloid leukemia (AML), but did not associate with enhanced leukemia initiation after infusion into NSG mice (83% vs 88%). In all cases, however, AML cells isolated from the bone marrow (BM) of engrafting mice were brightly positive for CD44v6, suggesting in vivo regulation by microenvironmental factors. In vitro, co-culturing primary AML cells with human BM-derived mesenchymal stromal cells (MSCs) caused a selective up-regulation of CD44v6 (P<0.01). The phenomenon was causally linked to MSC-induced acquisition of resistance to the chemotherapeutic agents daunorubicin and ara-c, as demonstrated by lentiviral vector (LV)-assisted short-hairpin (sh) RNA interference. At difference with AML, CD44v6 was constitutively expressed in 9/11 (81%) cases of multiple myeloma (MM). Knocking-down CD44v6 by shRNA interference significantly increased baseline sensitivity of MM cells to bortezomib. After validating CD44v6 as a common target of chemoresistant AML and MM cells, we generated a 2G CAR by cloning the scFv of a humanized CD44v6-specific mAb in a CD28/TCR zeta chain backbone and expressed it along with the Herpes simplex virus thymidine kinase (HSV-tk) suicide gene by means of a LV carrying a bi-directional promoter. After LV transduction, primary T cells concomitantly acquired CD44v6-specific in vitro cytotoxicty against autologous AML and MM cells, and a selective sensitivity to the suicide gene-activating prodrug ganciclovir. CD44v6-specific recognition associated with T-cell proliferation, IL-2 and IFN-gamma production, and complete clearance of AML cells in a BM-niche model with MSCs at very low E :T ratios (1:5–1:10). Interestingly, in the same model CD34+CD38- healthy cells were not eliminated by CD44v6-redirected T cells, consistently with stable lack of CD44v6 surface expression on healthy HSC. Once infused into NSG mice, CD44v6-redirected T cells had a major antitumor effect against previously engrafted CD44v6-positive AML and MM cell lines (THP1, P<0.001 and MM1.S, P<0.05 vs control CAR, respectively) and against autologous primary AML cells (P<0.005). Since the performance of the suicide gene is felt to be critical for controlling possible toxicities of CAR-redirected T cells, we also evaluated and inducible form of caspase 9 (icasp9) as a possible alternative to HSV-tk. Casp9 activation by its prodrug (the AP1903 dimerizer) permitted efficient CD44v6-redirected T-cell elimination even in the absence of cell proliferation and with a much faster kinetics than HSV-tk (>90% elimination: 18 hrs vs 112 hrs average, P<0.005)

Conclusions:
We demonstrated that LV-mediated dual transgenesis of primary human T cells with a novel CD44v6-specific CAR and a suicide gene is feasible, results into a powerful antitumor effect against chemoresistant AML and MM cells, and enables effective T-cell ablation in case of toxicity. The premise that suicide gene-modified CAR-redirected T cells can widen the therapeutic index of CD44v6 targeting awaits clinical confirmation

Disclosures:
Bordignon: Molmed SpA: Employment. Bonini:MolMed: Consultancy.