Fig 2: Assessing the oligomerization state and surface organization of various chimeric antigen receptors in the unstimulated CAR T cell membrane. (a) Western blot analysis of HER2.z, HER2.CD28.z, HER2.41BB.z and HER2.CD28.41BB.z CAR T cells. The membranes were probed for anti-human CD3ζ. (b) Spot intensities were quantified using ImageJ/Fiji, and the proportion of dimer/oligomer states was determined. (c) Representative raw and segmented AiryScan images of fluorescently tagged CARs. Clusters were segregated by watershedding and quantified. The scale bar represents 2 µm. (d) Total intensity of the apical membrane slices. (e) Average number of clusters per 10 µm2 apical membrane surface. (f) Distribution of individual cluster areas. (g) Integrated intensity of individual clusters. The charts represent mean ± SEM; nHER2.z = 54, 4 donors; nHER2.CD28.z = 44, 3 donors; nHER2.41BB.z = 47, 4 donors; nHER2.CD28.41BB.z = 54, 4 donors; * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: Membrane diffusion analysis and tertiary structure modeling of HER2-specific chimeric antigen receptors. (a) Representative fluorescence fluctuation intensity traces of 10-s FCS runs. (b) Pooled autocorrelation curve for each CAR. Position of triplet lifetime (τTriplet), diffusion correlation times of the detached Alexa Fluor 647 conjugated monomer HER2 ECD (τA647HER2) and the various CAR species (τ.z; τ.CD28.z; τ.41BB.z; τ.CD28.41BB.z) are indicated by arrows. (c) Diffusion coefficients of CAR constructs. The chart represents mean ± SEM, nHER2.z = 300, 4 donors; nHER2.CD28.z = 260, 3 donors; nHER2.41BB.z = 276, 4 donors; nHER2.CD28.41BB.z = 289, 4 donors; *** p < 0.001. (d) Schematic diagram illustrating the tertiary structure of the native TCR and the CAR constructs. TCR: T Cell Receptor complex extracellular domains; CD3z TM: CD3ζ transmembrane segment; CD3z IC: CD3ζ intracellular domain; FRP5 scFv: single chain variable fragment of FRP5 antibody targeting the HER2 ECD; IgG1 SH: short hinge linker derived from IgG1; CD28 TM: transmembrane segment derived from CD28; CD28 IC: costimulatory domain from CD28; 41BB IC: costimulatory domain from 41BB. Confidence scores of the predicted models and intramolecular disulfide bonds are indicated.

Fig 4: Generation of CAR T cell products from first to third generation. (a) Representative flow cytometry histogram of non-transduced (NT) T cells, and HER2.z, HER2.CD28.z, HER2.41BB.z and HER2.CD28.41BB.z CAR T cells. (b) Expression of the HER2-CARwas quantified with indirect labeling using a HER2-Fc fusion protein and an Alexa Fluor 647-conjugated anti-human IgG (N = 3). Histograms represent mean ± SD.