Custom Antibody Services Case Study: Unique Solutions for Custom Chimeric Antigen Receptor (CAR) Design
A customer was seeking custom CARs against multiple targets. Their request was for multiple CAR configurations to identify the optimal construct for targeting T cell expansion and persistence in their model.
CARs are critical components of adaptive cell therapies and cancer treatments. They are engineered chimeric receptors that give T cells the ability to target a specific protein. In CAR T cell therapy, CARs are transduced into T cells and infused to a patient with the goal of targeted tumor killing and persistence. CARs are composed of an extracellular antibody binding domain fused to the intracellular T cell signaling domain, thus merging antigen-binding and T-cell activating functions into a single receptor. The two domains are joined by a flexible hinge, which modulates the target binding and activation. Our brick system allows us to rapidly generate diverse configurations of CARs with hinge signaling domains and reporters tailored to our customers specifications (Figure 1). Antibodies can be custom engineered or selected from a library of CAR validated single chains.
Figure 1. Our brick system allows for the rapid generation of CAR constructs that can be tailored for specific applications.
The customers chose existing single chains from our validated CAR library and requested predefined signaling domains with EGFP reporters. Through modular cloning, the custom CARs were assembled with a panel of diverse hinges to optimize target binding and activation. The constructs were transfected into HEK293 cells, and membrane orientation and target binding were validated by fluorescent microscopy (Figure 2). The data showed that the Hinge 2 construct was best at positioning the single chain at the cell surface.
Figure 2. Immunofluorescence was used to validate target binding by CAR constructs.
The Hinge 2 construct was further validated by flow cytometry using a fluorescent-conjugated target protein (Figure 3). The targets were ultimately passed to the customer for testing on their platform.
Figure 3. Flow cytometry was used to validate target binding by the Hinge 2 CAR construct.