Abstract
While current upfront therapy will cure over 80% of patients, B-cell precursor acute lymphoblastic leukemia ALL (BCP-ALL) remains a leading cause of cancer-related death in children, and outcomes for those patients who relapse have not been changed substantially over recent decades. Immunotherapy with a chimeric antigen receptor (CAR) targeting CD19 has resulted in impressive clinical responses, but alternative CAR targets are needed as not all patients respond and CD19 negative relapses have been observed. Thymic stromal lymphoprotein receptor (TSLPR) is coded by the CRLF2 gene. CRLF2 genetic alterations occur in 5-16% of pediatric and adult BCP-ALL and result in TSLPR over-expression that is associated with poor prognosis. Furthermore, emerging data suggests that TSLPR overexpression is functionally important for leukemic blasts. Thus, TSLPR is an ideal immunotherapeutic target. We constructed TSLPR-specific CARs and tested their therapeutic efficacy in vitro and in vivo.
Hybridomas producing TSLPR-specific antibodies were used to clone variable regions of the heavy and light chains for constructing TSLPR-specific scFv. The scFv was linked with the CD8α hinge and transmembrane domain, 4-1BB intracellular domain and CD3ζ chain, and then cloned into a 3rd generation lentiviral vector. For optimization of the CAR configuration, an IgG heavy chain constant domain (CH2CH3) spacer was fused to scFv to generate a “long” TSLPR CAR. Human T cells were transduced with CAR vectors and the transduced cells were evaluated in vitro and in vivo against TSLPR-overexpressing ALL, including a TSLPR-transduced REH cell line (REH-TSLPR) and patient-derived xenografts naturally overexpressing TSLPR.
Both long and short TSLPR CARs could be efficiently transduced (approximately 60-80%) into human T cells using a lentiviral system. T cells expressing both long and short TSLPR CARs produced high levels of cytolytic cytokines upon co-culture with of TSLPR-overexpressing ALL. However, only the short TSLPR CAR demonstrated effective killing against TSLPR-expression ALL cells in vitro. Short TSLPR-expressing T cells completely eradicated leukemia in NSG xenografts mice bearing REH-TSLPR (figure 1) as well as TSLPR-expressing patient xenografts. The long TSLPR CAR did not show any activity in vivo.
TSLPR-targeted CAR T cells is promising therapeutic approach in high risk BCP-ALL and represents the first example of an oncogene-specific cellular therapy in BCP-ALL.
Grupp:Novatis: Research Funding.
