Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia.

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5'-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells.

El Khawanky N ，Hughes A ，Yu W ，Myburgh R ，Matschulla T ，Taromi S ，Aumann K ，Clarson J ，Vinnakota JM ，Shoumariyeh K ，Miething C ，Lopez AF ，Brown MP ，Duyster J ，Hein L ，Manz MG ，Hughes TP ，White DL ，Yong ASM ，Zeiser R ... -  《Nature Communications》

T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.

Mardiros A ，Dos Santos C ，McDonald T ，Brown CE ，Wang X ，Budde LE ，Hoffman L ，Aguilar B ，Chang WC ，Bretzlaff W ，Chang B ，Jonnalagadda M ，Starr R ，Ostberg JR ，Jensen MC ，Bhatia R ，Forman SJ ... -  《-》

Improved Activity against Acute Myeloid Leukemia with Chimeric Antigen Receptor (CAR)-NK-92 Cells Designed to Target CD123.

Morgan MA ，Kloos A ，Lenz D ，Kattre N ，Nowak J ，Bentele M ，Keisker M ，Dahlke J ，Zimmermann K ，Sauer M ，Heuser M ，Schambach A ... -  《Viruses-Basel》

Impact of scFv on Functionality and Safety of Third-Generation CD123 CAR T Cells.

Chimeric antigen receptor (CAR) T cells express an extracellular domain consisting of a single-chain fragment variable (scFv) targeting a surface tumor-associated antigen. scFv selection should involve safety profiling with evaluation of the efficacy/toxicity balance, especially when the target antigen also is expressed on healthy cells. Here, to assess differences in terms of efficacy and on-target/off-tumor effects, we generated five different CARs targeting CD123 by substituting only the scFv. In in vitro models, T cells engineered to express three of these five CD123 CARs were effectively cytotoxic on leukemic cells without increasing lysis of monocytes or endothelial cells. Using the IncuCyte system, we confirmed the low cytotoxicity of CD123 CAR T cells on endothelial cells. Hematotoxicity evaluation using progenitor culture and CD34 cell lysis showed that two of the five CD123 CAR T cells were less cytotoxic on hematopoietic stem cells. Using a humanized mouse model, we confirmed that CD123- cells were not eliminated by the CD123 CAR T cells. Two CD123 CAR T cells reduced tumor infiltration and increased the overall survival of mice in three in vivo models of blastic plasmacytoid dendritic cell neoplasm. In an aggressive version of this model, bulk RNA sequencing analysis showed that these CD123 CAR T cells upregulated genes associated with cytotoxicity and activation/exhaustion a few days after the injection. Together, these results emphasize the importance of screening different scFvs for the development of CAR constructs to support selection of cells with the optimal risk-benefit ratio for clinical development.

Fredon M ，Poussard M ，Biichlé S ，Bonnefoy F ，Mantion CF ，Seffar E ，Renosi F ，Bôle-Richard E ，Boidot R ，Chevrier S ，Anna F ，Loustau M ，Caumartin J ，Gonçalves-Venturelli M ，Robinet E ，Saas P ，Deconinck E ，Daguidau E ，Roussel X ，Godet Y ，Adotévi O ，Angelot-Delettre F ，Galaine J ，Garnache-Ottou F ... -  《-》

Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia.

We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA-electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti-CD123-4-1BB-CD3ζ T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML.

Tasian SK ，Kenderian SS ，Shen F ，Ruella M ，Shestova O ，Kozlowski M ，Li Y ，Schrank-Hacker A ，Morrissette JJD ，Carroll M ，June CH ，Grupp SA ，Gill S ... -  《-》