Fig. 2

Construction and identification of effector cells, target cells, and bifunctional molecules. a Characterization of CD4⁺ T cells or CD8⁺ T cells enriched with the magnetic microbeads by flow cytometry. With anti-CD4-PE and anti-CD8-APC antibodies, the sorting efficiency of CD4⁺ T cells and CD8⁺ T cells was determined to be 92.4 and 90.7%, respectively (shown on the upper). At the single-cell level, our flow cytometry data indicated the size and morphology of cells attached to antibodies (shown on the lower). (Ch02 bright channel, Ch03 PE fluorescent channel, Ch11 APC fluorescent channel) b sdCAR-engineered T cells were successfully constructed by electroporation. We evaluated the transduction efficiency by flow cytometry with endogenous BFP expression to quantify fractions of sdCAR-CD4⁺ T cells and sdCAR-CD8⁺ T cells. c To assess sdCAR-T cell activity, K562 cells were lentivirally transduced to stably express human tumor antigens MSLN or CEA, respectively. Western blotting results confirmed the expression of cognate antigen (MSLN) on MSLN⁺ K562 cells (shown on the upper), non-cognate antigen (CEA) on CEA⁺ K562 cells (shown on the middle), and cognate antigen (MSLN) on MSLN⁺ HT29 cells (shown on the lower). We used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. d Western blot results showed that the integrin αvβ3 was highly expressed on the surface of K562 cells (shown on the left). In contrast, HT29 cells did not express integrin αvβ3 (shown on the right). GAPDH was used as an endogenous control. e Analysis of site-specific FHBM conjugates after isolation and purification. Chromatography of obtained FHBM demonstrated that the final product was of high purity (> 98%) and can be used to regulate CAR-T activity (shown on the left). Characteristic ion peaks from mass spectrometry, such as [M+4H]⁴⁺, [M+3H]³⁺, and [M+2H]²⁺, showed that the molecular weight of FHBM was approximately 2565 and the ratio of FITC to HM-3 in FHBM conjugate was about 1.97 (shown on the right)