CD19 CAR antigen engagement mechanisms and affinity tuning

Area of Science:

• Immunology
• Biochemistry
• Structural Biology

Background:

• Chimeric antigen receptor (CAR) T cell therapy is a promising cancer treatment that utilizes engineered T cells to target cancer cells.
• The efficacy of CAR T cell therapy is significantly influenced by the affinity of the single-chain variable fragment (scFv) binder, which recognizes specific cell surface antigens.
• CAR T cells targeting CD19 have shown remarkable success in treating B cell malignancies and are approved by the FDA.

Purpose of the Study:

• To elucidate the structural basis of CAR T cell function by determining the cryo-electron microscopy (cryo-EM) structures of CD19 in complex with key binders.
• To leverage structural insights and molecular dynamics simulations to engineer CAR T cell binders with modulated affinities.
• To evaluate how variations in binder affinity impact CAR T cell performance, including tumor recognition sensitivity, cytolysis induction, and trogocytosis.

Main Methods:

• Obtained cryo-EM structures of the CD19 antigen complexed with the FMC63 and SJ25C1 binders.
• Performed molecular dynamics simulations based on the obtained structures.
• Engineered novel binders with altered affinities (lower and higher) based on simulation data.
• Assessed the functional performance of CAR T cells engineered with these modified binders in vitro.

Main Results:

• Determined high-resolution cryo-EM structures of CD19 bound to FMC63 and SJ25C1.
• Successfully designed and created CAR T cell binders with precisely tuned affinities.
• Demonstrated that CAR T cells with altered binder affinities exhibit distinct sensitivities to target antigen density.
• Observed differential induction of cytolysis and trogocytosis based on CAR T cell binder affinity and target cell antigen expression.

Conclusions:

• Structural information of CAR binders and their target antigens is crucial for understanding and optimizing CAR T cell function.
• Binder affinity can be rationally modulated using structural and computational approaches to fine-tune CAR T cell therapeutic potential.
• This study provides a framework for engineering CAR T cells with tailored specificities for diverse cancer antigen expression levels.