CAR-T In Vitro Assay

• • The evaluation standard of a therapeutic antibody is first its biological function and then affinity etc..
  • So functional assays are conducted right after the primary screening
  • The screening scale is larger to increase the chance of getting leads with good function.
• • ScFv, but not mAb, is expressed in CAR lead generation.
  • Because the role of an antibody in a CAR-T development is mainly a targeting tool, affinity is the top consideration when selecting scFv or sdAb candidates.
  • Specificity test can be performed according to customer requirements.
  • The function of CAR-T cells not the scFv or sdAb itself should be validated in a CAR-T development project.

CAR Expression

Figure 1. Representative of flow cytometry analysis of anti-CD19 CAR-T cells and non-transduced control cells 10 days after transduction. Cells were stained with anti-CD3 antibody and anti-FLAG (CD19-FLAG) antibody.

Functional Validation

• A

  

• B

  

Figure 2. Functional validations of anti-CD19 CAR-T cells. (A) Cytotoxicity assay was performed to validate anti-CD19 CAR-T cytotoxic function at a ratio of 20:1 (CAR-T: Raji) for 24 hours. Raji (CD19 positive) cells were engineered to express luciferase and the cytotoxicity effects can be inferred by the reduction of bioluminescence signal. (B) Cytokine production by activated CD19 CAR-T cells in response to its target - Raji cells. The supernatant was analyzed by ELISA for IFN-γ secretion from anti-CD19 CAR-T cells.