We confirmed that the VCN is dependent on the multiplicity of infection and verified that the VCN of our experimental or GMP-like IL-1RAP CAR T cells met the requirement (< 5 VCN/cell) for delivery to the clinical department, similar to approved axi-cel or tisa-cel drugs. Both 28z and 28BBz ddPCR assays allow determination of the average vector copy number (VCN) per cell. By targeting the 4.1BB/CD3z (28BBz) or 28/CD3z (28z) junction area, we demonstrated that PCR assays can be applied to approved CD19 CAR T drugs.
Methodsīased on 2 experimental CAR T cell constructs, IL-1RAP and CS1, we designed 2 quantitative digital droplet (ddPCR) PCR assays. Molecular tools can meet this need to specifically monitor long-term persistent CAR T cells. Nevertheless, there is a need for additional tools, and the challenge is to develop an easy, relevant, highly sensitive, reproducible, and inexpensive detection method. Flow cytometry (FC) remains the standard for monitoring CAR T cells using a recombinant biotinylated target protein.
Four CAR T cell drugs, including tisagenlecleucel (tisa-cel) and axicabtagene-ciloleucel (axi-cel), all targeting CD19, are currently approved for treating B cell malignancies. Genetically engineered chimeric antigen receptor (CAR) T lymphocytes are promising therapeutic tools for cancer.
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