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 ... -  《-》

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 ... -  《-》

Chimeric Antigen Receptors Incorporating D Domains Targeting CD123 Direct Potent Mono- and Bi-specific Antitumor Activity of T Cells.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated impressive initial response rates in hematologic malignancies. However, relapse rates are significant, and robust efficacies in other indications, such as solid tumors, will likely require novel therapeutic strategies and CAR designs. To that end, we sought to develop simple, highly selective targeting domains (D domains) that could be incorporated into complex, multifunctional therapeutics. Herein, we describe the identification and characterization of D domains specific for CD123, a therapeutic target for hematologic malignancies, including acute myelogenous leukemia (AML). CARs comprised of these D domains mediate potent T cell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We describe a strategy of engineering less immunogenic D domains through the identification and removal of putative T cell epitopes and investigate the binding kinetics and affinity requirements of the resultant D domain CARs. Finally, we extended the utility of D domains by generating functional, bi-specific CARs comprised of a CD123-specific D domain and a CD19-specific scFv. The properties of D domains suggest that this class of targeting domain may facilitate the development of multi-functional CARs where conventional, scFv-based designs may be suboptimal.

Qin H ，Edwards JP ，Zaritskaya L ，Gupta A ，Mu CJ ，Fry TJ ，Hilbert DM ，LaFleur DW ... -  《-》

Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

Thokala R ，Olivares S ，Mi T ，Maiti S ，Deniger D ，Huls H ，Torikai H ，Singh H ，Champlin RE ，Laskowski T ，McNamara G ，Cooper LJ ... -  《PLoS One》