D3.403 - Protein Profiling of CAR-Engineered NK92-Derived Exosomes Using Western Blot

Exosomes derived from Chimeric Antigen Receptor (CAR)-engineered Natural Killer (NK) cells are emerging as promising cell-free therapeutic agents, as they may retain key functional proteins from their parental cells, including cytotoxic molecules and potentially the CAR construct itself. Demonstrating the presence of CAR within exosomes is essential to support their role in antigen-specific targeting. This study aimed to perform protein-level characterization of exosomes derived from NK92 wild-type (WT) and anti-HER2 CAR-engineered NK92 cells by Western Blot (WB) to confirm selective CAR incorporation.

Exosomes from NK92 WT and CAR-NK92 were previously isolated, quantified, and their protein content was measured. Exosome lysates were denatured, separated on a 10% polyacrylamide gel, and transferred to a nitrocellulose membrane. The membranes were incubated overnight at 4 °C with primary antibodies targeting exosome-specific markers (CD63 and Alix), β-actin, granzyme B, and CAR-associated components (CD3ζ and the G4S linker), followed by incubation with HRP-conjugated secondary antibodies. The protein bands were visualized using enhanced chemiluminescence. Whole cell lysates were used for comparison purposes.

Both NK92 WT and CAR-NK92 cell lysates expressed granzyme B (monomers and multimers) and CD3ζ, while CAR-NK92 cells showed two CD3ζ bands corresponding to endogenous CD3ζ and the CAR-associated fusion protein. Exosomal analysis confirmed the presence of CD63 and Alix in both groups, supporting proper exosome biogenesis, while β-actin was absent, indicating minimal cellular contamination. Importantly, CD3ζ and granzyme B were also identified in exosomes from both populations, and the CAR-specific G4S linker was detected exclusively in CAR-NK92-derived exosomes, confirming selective incorporation of the engineered-CAR molecule into exosomes.

WB analysis is a reliable method for characterizing exosomal protein content, confirming the presence of canonical exosomal markers and the absence of cellular contamination. Importantly, it demonstrated that CAR-NK92-derived exosomes selectively incorporate the CAR molecule, while preserving cytotoxic components such as granzyme B. These findings provide a foundation for future research exploring CAR-engineered exosomes as a potential cell-free immunotherapeutic approach.