Abstract 3234: Multi-antigen targeting of CD19, CD22 and TSLPR to prevent Ph-like ALL resistance
Authors: Ross Savannah , Bagashev Asen , Schneider Dina , Hu Peirong , Tasian Sarah , Fry Terry 
Published: September 2020

Abstract
Background: Despite high remission induction rates after CD19 chimeric antigen receptor T cell (CART) therapy in patients with B-acute lymphoblastic leukemia (ALL), many patients eventually relapse with loss of CD19 antigen. Strategies to overcome antigen-loss relapse include co-targeting of multiple antigens and selection of alternative functionally important surface targets. The thymic stromal lymphopoietin receptor (TSLPR), encoded by CRLF2 (cytokine receptor-like factor 2), is overexpressed in &gt;50% of patients with Ph-like ALL and is associated with constitutively active JAK/STAT signaling targetable by ruxolitinib. Our groups previously demonstrated potent activity of TSLPRCART against TSLPR+ ALL (Qin Blood 2015). Our current study aims to develop (1) multi-specific CAR T cells targeting TSLPR and CD19 or CD22 and (2) TSLPRCART and ruxolitinib combinatorial approaches to decrease antigen-negative relapse potential after CART therapy.
Methods: Lentiviral transduction of activated healthy donor T cells was used to introduce monovalent TSLPR or CD19 CARs and bivalent CD19xTSLPR or CD22xTSLPR CARs. CAR expression was quantified via flow cytometry. CRISPR/Cas9-modified Nalm-6 (human B-ALL) cells were transduced with a TSLPR expression vector to generate cell lines expressing combinations of CD19, CD22, and TSLPR antigens. In vitro functionality was assessed using enzyme linked immunosorbent assays to detect IL-2 and IFN-γ production following CART co-culture with Nalm-6. In vivo evaluation was performed against luciferase-transduced Nalm-6 murine xenograft models treated with 5e6 CART and monitored by bioluminescent imaging. TSLPRCART was also tested against MUTZ5 (TSLPR-overexpressing Ph-like ALL) cells in vitro and in vivo in combination with ruxolitinib to determine effects on CAR T cells.
Results: TSLPRCART and CD19xTSLPRCART induced in vitro cytokine production when co-cultured with CD19+/TSLPR+ Nalm-6 cells with higher levels than those produced by CD19CART. Greater cytokine production was observed with co-incubation of TSLPR+ Nalm-6 cells with CD22xTSLPRCART vs TSLPRCART. In vivo studies demonstrated equivalent leukemia eradication by TSLPRCART and CD19CART compared to CD19xTSLPRCART in CD19+/TSLPR+ Nalm-6 xenograft models. Additional in vivo studies in MUTZ5 xenograft models co-treated with TSLPRCART and ruxolitinib demonstrated impaired T cell expansion and cytokine production, which could be overcome via higher CART cell dose or delayed ruxolitinib administration.
Conclusions: TSLPRCART is a robust immunotherapeutic strategy for CRLF2-rearranged Ph-like ALL. We demonstrate potent in vitro and in vivo functionality of novel bivalent CD19xTSLPRCART and CD22xTSLPRCART. Ongoing studies are also evaluating bicistronic CD19xTSLPR CAR constructs. We further demonstrate that combining TSLPRCART with ruxolitinib moderately blunts T cell expansion and cytokine production, which could be an attractive strategy for management of CAR T cell-associated toxicities such as cytokine release syndrome.
Citation Format: Savannah Ross, Asen Bagashev, Dina Schneider, Peirong Hu, Sarah Tasian, Terry Fry. Multi-antigen targeting of CD19, CD22 and TSLPR to prevent Ph-like ALL resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3234.