Computational Model of Chimeric Antigen Receptors Explains Site-Specific Phosphorylation Kinetics

Area of Science:

• Immunology
• Biochemistry
• Computational Biology

Background:

• Chimeric antigen receptors (CARs) are vital for treating hematological malignancies.
• Optimizing CAR therapies requires understanding their activation mechanisms.
• Current knowledge of CAR protein activation is limited.

Purpose of the Study:

• To quantify the in vitro kinetics of tyrosine phosphorylation on CARs.
• To elucidate the mechanistic details of CAR activation by lymphocyte-specific protein-tyrosine kinase (LCK).
• To develop a computational model for CAR signaling and T cell activation.

Main Methods:

• Phosphoproteomic mass spectrometry was employed to analyze CAR phosphorylation.
• Mechanistic computational modeling was used to study phosphorylation kinetics.
• Kinetic analysis was performed on individual tyrosine sites of CARs.

Main Results:

• Each of the 10 tyrosine sites on the CD28-CD3ζ CAR exhibits distinct phosphorylation kinetics mediated by LCK.
• Incorporating CD28 to the N-terminus of CD3ζ enhances overall CD3ζ phosphorylation rates.
• Computational modeling revealed LCK phosphorylates CD3ζ via competitive inhibition.

Conclusions:

• A quantitative modeling framework enhances understanding of CAR signaling and T cell activation.
• The study provides mechanistic insights into LCK-mediated CAR phosphorylation.
• The model predicts phosphatases interact with CD3ζ through competitive inhibition.