Co-Expression of a Suicide Gene in CAR-Redirected T Cells Enables the Safe Targeting of CD44v6 for Leukemia and Myeloma Eradication
Authors: Casucci Monica , di Robilant Benedetta Nicolis , Falcone Laura , 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 949

Background:
Acute myeloid leukemia (AML) and multiple myeloma (MM) share common features, such as preferential homing to the bone marrow (BM) and the tendency to relapse after chemotherapy. These phenomena are possibly linked, as signals provided within the BM niche induce chemoresistance. Relapsing AML and MM can however be cured by allogeneic hematopoietic stem cell transplantation (HSCT), suggesting that allospecific T may specifically sterilize tumor cells that survive chemotherapy. Unfortunaley, the universal application of HSCT is severely limited by the occurrence of life-threatening GVHD and by the fact that leukemia often escapes the alloimmune pressure by losing specific HLAs, as we have recently demonstrated (Vago et al., NEJM 2009). The isoform variant 6 of the adhesion receptor CD44 (CD44v6) is widely expressed in AML and MM, and is crucially involved in BM homing. In phase I/II trials, targeting CD44v6 with mAb conjugated to chemotherapeutic agents showed substantial efficacy, but also toxicity due to background expression of CD44v6 on the skin. Thanks to the chimeric antigen receptor (CAR) technology it is now possible to efficiently redirect T cells against virtually any antigen for which there is availability of a specific mAb. Our experience with suicide gene therapy in the context of HSCT (Ciceri et al., Lancet Oncol 2009) clearly indicates that genetic switches are efficient tools for controlling the off-tumor toxicities of T cells.

Aim:
To develop a strategy for the safe targeting of CD44v6 through the co-expression of a suicide gene in CAR-modified T cells for AML and MM treatment

Results:
CD44v6 surface expression was observed in 11/14 (79%) AML cases. Co-culturing primary leukemic cells with BM-derived mesenchymal stromal cells (MSCs) ex vivo caused the significant up-regulation of CD44v6 expression (P<0.05). At difference with AML, CD44v6 expression was constitutively high in the majority of MM cases (13/15, 87%). Upon MSC co-culturing, both primary leukemic cells and malignant plasma cells (mPCs) became resistant to cell death induced by daunorubicin (P<0.01) and bortezomib (P<0.05), respectively. Pre-incubating tumor cells with a CD44v6-blocking mAb reverted the chemoresistant phenotype. Importantly, knocking-down CD44v6 expression by (LV)-assisted short-hairpin (sh) RNA interference completely abated tumor-cell homing to the BM and tumorigenesis in NSG mice (P<0.001). We therefore constructed a CAR by cloning the scFv of a humanized CD44v6 mAb in a CD28/TCR zeta chain backbone and expressed it with the Herpes simplex virus thymidine kinase (HSV-tk) suicide gene by means of a LV carrying a PGK bi-directional promoter. After LV transduction, primary T cells concomitantly acquired potent and CD44v6-specific cytotoxicity against autologous leukemic cells and mPCs, and a selective sensitivity to the prodrug ganciclovir. Importantly, adding MSCs did not interfere with effector functions. Once infused into NSG mice, CD44v6-redirected T cells persisted long-term and completely eradicated previously engrafted autologous leukemic cells (P<0.005). Antitumor efficacy was dependent on transduction with CD3/CD28-beads and culture with IL-7/IL-15, according to a protocol that preserves the expression of the self-renewal marker IL-7 receptor and of the BM addressin CXCR4 (Bondanza, Blood 2011). Interestingly, CD44v6-redirected T cells were not cytotoxic against CD34+CD38- hematopoietic stem cells, but recognized mature monocytes. To overcome the problem of HSV-tk immunogenicity, we alternatively evaluated the novel, humanized suicide gene inducible caspase 9 (icasp9). Differently form HSV-tk, icasp9 activation by the specific chemical inducer of dimerization allowed the ablation of CD44v6-redirected T cells even in the absence of proliferation and with a much faster kinetics (<90% in 4 hrs vs 84 hrs, P<0.05)

Conclusions:
CD44v6 CAR-redirected suicidal T cells have the potential to eradicate AML and MM and can be ablated at will. We are currently designing a phase I/II clinical trial where CD44v6 CAR-redirected suicidal T cells will be applied soon after autologous HSCT for sterilizing residual tumor, and routinely ablated thereafter not to interfere will full hematological reconstitution. The co-expression of a suicide gene will also allow to control any potential toxicity deriving from background expression of CD44v6 on healthy tissues.

Disclosures:
Bordignon: Molmed SpA: Employment.