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

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

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

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)

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

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