Precision Co-Targeting of the Thymic Stromal Lymphopoietin Receptor in Childhood CRLF2-Rearranged Acute Lymphoblastic Leukemia
Authors: Bagashev Asen , Loftus Joseph Patrick , Ross Savannah , Niswander Lisa M , Qin Haiying , Fry Terry J. , Tasian Sarah K 
Published: November 2021

Abstract
Introduction : Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with high rates of chemoresistance and relapse. CRLF2 (cytokine receptor-like factor 2) rearrangements occur in 50% of Ph-like and 60% of Down Syndrome (DS)-associated ALL and induce constitutive JAK/STAT and other kinase signaling. Current clinical trials are studying chemotherapy with the JAK inhibitor ruxolitinib in patients with CRLF2-rearranged Ph-like ALL, but results are not yet known. While chimeric antigen receptor T-cell (CART) immunotherapies have induced remarkable remissions in children with relapsed/refractory B-ALL, approximately 50% of CD19CART-treated patients will relapse again, many with CD19 antigen loss. New therapies are needed to prevent relapse and overcome immunotherapeutic resistance.
Methods : We previously developed CAR T cells targeting the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) and demonstrated potent preclinical activity in Ph-like ALL models (Qin Blood 2015), which has led to a soon-to-open phase 1 clinical trial for patients with relapsed/refractory CRLF2-overexpressing ALL. In the current preclinical studies, we hypothesized that combinatorial targeting with bispecific TSLPRxCD19CART or TSLPRxCD22CART (Ross Cancer Res 2020) or with TSLPRCART + ruxolitinib will have superior activity against CRLF2-rearranged Ph-like and DS-ALL.
Results : TSLPRCART treatment of CRLF2-rearranged ALL cell line (n=1) and patient-derived xenograft (PDX) models potently inhibited leukemia proliferation in vitro and in vivo and induced long-term 'cure' of xenograft mice. However, co-administration of TSLPRCART + ruxolitinib markedly diminished in vivo T cell numbers, blunted cytokine production, and facilitated leukemia relapse, which could be abrogated by delaying ruxolitinib. Importantly, ruxolitinib co-treatment prevented severe TSLPRCART-induced cytokine release syndrome (CRS) and animal death. Interestingly, ruxolitinib withdrawal led to return of T-cell functionality with re-detection of TSLPRCART in peripheral blood, induction of IFN-γ production, and leukemia clearance upon CRLF2+ ALL rechallenge (Figure 1).
Conclusions: In these preclinical studies, we report potent activity of TSLPRCART in cell line (n=1) and PDX models of childhood CRLF2-rearranged Ph-like ALL (n=2) and DS-ALL (n=2) and, interestingly, deleterious effects of concomitant JAK inhibition upon CAR T cell functionality. We demonstrated that ruxolitinib co-administration impaired in vivo TSLPRCART-induced ALL cell killing but was also beneficial in protection against life-threatening cytokine release syndrome in co-treated animals. Importantly, TSLPRCART was not eliminated, only suppressed, by JAKi co-treatment with restoration of T cell functionality upon ruxolitinib removal and/or leukemia relapse/rechallenge studies. Ongoing studies are defining optimal TSLPRCART + ruxolitinib sequence(s) to maximize both anti-leukemia efficacy and potential CRS mitigation, as well as assessing in vivo efficacy of bispecific TSLPRCARTs in CRLF2-R Ph-like ALL and DS-ALL PDX models for future translation and clinical evaluation in next-generation trials.
Figure 1 Figure 1.

Disclosures
Fry: ElevateBio: Research Funding; Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tasian: Aleta Biotherapeutics: Consultancy; Kura Oncology: Consultancy; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.