41BB-based and CD28-based CD123-redirected T-cells ablate human normal hematopoiesis in vivo

Abstract

BackgroundAcute myeloid leukemia (AML) is a hematopoietic malignancy which is biologically, phenotypically and genetically very heterogeneous. Outcome of patients with AML remains dismal, highlighting the need for improved, less toxic therapies. Chimeric antigen receptor T-cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are challenging because of the absence of a universal pan-AML target antigen and the shared expression of target antigens with normal hematopoietic stem/progenitor cells (HSPCs), which may lead to life-threating on-target/off-tumor cytotoxicity. CD33-redirected and CD123-redirected CARTs for AML are in advanced preclinical and clinical development, and they exhibit robust antileukemic activity. However, preclinical and clinical controversy exists on whether such CARTs are myeloablative.MethodsWe set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-based and CD28-based CARCD123. We analyzed 97 diagnostic and relapse AML primary samples to investigate whether CD123 is a suitable immunotherapeutic target, and we used several xenograft models and in vitro assays to assess the myeloablative potential of our second-generation CD123 CARTs.ResultsHere, we show that CD123 represents a bona fide target for AML and show that both 41BB-based and CD28-based CD123 CARTs are very efficient in eliminating both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-based and CD28-based CD123 CARTs ablate normal human hematopoiesis and prevent the establishment of de novo hematopoietic reconstitution by targeting both immature and myeloid HSPCs.ConclusionsThis study calls for caution when clinically implementing CD123 CARTs, encouraging its preferential use as a bridge to allo-HSCT in patients with R/R AML.

Bone marrow MSC from pediatric patients with B-ALL highly immunosuppress T-cell responses but do not compromise CD19-CAR T-cell activity

“…We first evaluated in vitro the impact of HD and B-ALL BM-MSC on CD19-CAR T-cell performance. We generated CD19-CAR T-cells following a standard protocol with anti-CD3/anti-CD28 plus hIL-7 and hIL-15, 29 and studied the effects of BM-MSC on CD19-CAR T-cell cytotoxicity against NALM6 and SEM cells cultured at a 1:1 E:T ratio for 2 and 6 days ( figure 4A ). We found that the number of CD19-CAR-resistant NALM6 or SEM cells cultured in the presence of either HD or B-ALL BM-MSC was very similar to that of target cells exposed to CD19-CAR T-cells alone, a non-protective role of BM-MSC on NALM6 ( figure 4B ) and SEM ( figure 4C ) cell survival in CD19-CAR T-cell therapy, at least in vitro.…”

“…T-cells were activated by plate-coating with anti-CD3 and anti-CD28 mAbs in complete RPMI medium for 2 days and were then transduced with a CAR-expressing lentivirus at a multiplicity of infection of 10 in the presence of hIL-7 and hIL-15 (10 ng/mL). 29 T-cells were expanded in complete RPMI medium plus hIL-7 and hIL-15 for up to 6 days. CAR transduction efficiency in T-cells was analyzed by FACS.…”

Hematopoeitic Cell Transplantation and CAR T-Cell Therapy: Complements or Competitors?

“…Preclinical work suggests that it is a viable CAR target for AML, although it could lead to myeloablation requiring allo-HCT rescue. It is also expressed on vascular endothelium, heightening the risk for toxicity such as capillary leak syndrome (133)(134)(135). One risk mitigation strategy has been the production of transient CAR T cells infused serially, a concept that was safe and feasible in a pilot study, but which was discontinued during phase I (136).…”

Novel TCR-like CAR-T cells targeting an HLA∗0201-restricted SSX2 epitope display strong activity against acute myeloid leukemia

“… 23 , 24 While CAR T cell therapy has experienced exciting successes for the treatment of patients with ALL, the lack of well-characterized and tumor-specific surface antigens in AML has necessitated consideration of CAR T cell strategies that may also affect normal tissues. 25 , 26 One such example is the well-known surface antigen CD123, which is expressed on the majority of AML blasts, but is also expressed on many normal hematopoietic progenitor cells (HPCs). 26 , 27 Re-expression of CTAs, as well as tumor suppressor genes by inhibiting DNA methyltransferase, has been used with success for the treatment of hematologic malignancies including AML.…”

“… 25 , 26 One such example is the well-known surface antigen CD123, which is expressed on the majority of AML blasts, but is also expressed on many normal hematopoietic progenitor cells (HPCs). 26 , 27 Re-expression of CTAs, as well as tumor suppressor genes by inhibiting DNA methyltransferase, has been used with success for the treatment of hematologic malignancies including AML. 28 , 29 Because SSX2 expression in tumor cells has been reported to occur either as naturally expressed or having its expression induced by demethylation agents in tumor cells, our group sought to evaluate a novel scFv specific for SSX2 and develop a CAR with specific activity against AML tumors.…”