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

Area of Science:

• Immunology
• Oncology
• Biotechnology

Background:

• Chimeric antigen receptor (CAR) T cells are a promising cancer therapy.
• CAR T-cell efficacy and safety depend on the targeting scFv, especially when the antigen is present on healthy tissues.
• CD123 is a target antigen for certain hematologic malignancies.

Purpose of the Study:

• To evaluate the efficacy and safety of different CAR T-cell constructs targeting CD123.
• To compare on-target/off-tumor effects of various CD123-targeting scFvs.
• To identify optimal CD123 CAR T-cell candidates for clinical development.

Main Methods:

• Generated five distinct CAR T-cells by varying the scFv targeting CD123.
• Assessed cytotoxicity against leukemic cells and healthy cells (monocytes, endothelial cells, hematopoietic stem cells) in vitro.
• Utilized IncuCyte system for real-time cytotoxicity monitoring.
• Evaluated hematotoxicity using progenitor and CD34 cell assays.
• Tested efficacy in humanized mouse models of blastic plasmacytoid dendritic cell neoplasm (BPDCN).
• Performed bulk RNA sequencing on tumor samples from treated mice.

Main Results:

• Three of five CD123 CAR T-cells showed effective in vitro cytotoxicity against leukemic cells without harming monocytes or endothelial cells.
• Two CAR T-cell variants demonstrated reduced hematotoxicity.
• In vivo studies confirmed minimal elimination of CD123-negative cells.
• Two CD123 CAR T-cell constructs significantly reduced tumor burden and improved survival in BPDCN mouse models.
• RNA sequencing revealed upregulation of cytotoxicity and activation/exhaustion genes in effective CAR T-cells.

Conclusions:

• The choice of scFv significantly impacts the efficacy and safety profile of CAR T-cells.
• Screening diverse scFvs is essential for developing CAR T-cell therapies with an optimal risk-benefit ratio.
• Specific CD123 CAR T-cell variants show potential for treating BPDCN with improved safety.