Journal: bioRxiv
Article Title: Reprogramming BCMA-Targeted CAR-T Cells through γ-Secretase Modulation Blocks Antigen Shedding and Extends CAR-T Longevity
doi: 10.64898/2026.01.20.700575
Figure Lengend Snippet: (A) Structural modeling of murine 4C8A clone versus LLM-based humanized scFv variants showing preserved CDR orientation toward BCMA and specificity for the distinct epitope. (B) RMSD plots for the snapshots from MD simulation of the scFvs (murine and humanized) in complex with BCMA TM with three technical replicates. (C) MM/GBSA based free energy binding plots for the murine and humanized scFv-BCMA complexes. (D) Workflow of immunogenicity assay showing antibody-primed dendritic cells co-cultured with autologous PBMCs. (E-G) Cytokine analysis of IFN-γ, IL-2, and IL-4 showing lower levels for humanized CARs versus murine. Fully human IgG antibody was used as reference control. (H) Representative super-resolution imaging shows uniform membrane localization of CAR constructs (magenta). Nuclei were stained with DAPI (blue). Corresponding bar graph of the image analysis (n=6). (I1) Dot plots of flow cytometry quantification of CAR surface expression using GS-linker antibody (I2) Bar graph of the flow cytometry data showing percentage CAR-Transduction (n=5). (J1) Cell-based binding assay using flow cytometry showing affinity gain for the CDR-optimized humanized CAR (HmzCAR) (J2) Bar graph of the analysis (n=5). (K) Workflow of BLI sensorgrams and kinetic analysis. (L1, L2) Bio-layer interferometry (BLI) sensogram showing real-time binding kinetics of the indicated analytes. Colored traces represent different concentrations, with an initial association phase followed by dissociation. (M) Experimental workflow of co-culture of CAR-T cells with BCMA expressing target cells. (N) Cytotoxicity against MM.1S cells across different E:T ratios (O) Similarly, for MM.1R cells (n=5). (P) Representative flow cytometry contour plots of granzyme-B secretion (P2) Mean fluorescence intensity (MFI) of the flow cytometry contour plots. Data represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups.
Article Snippet: The cell lines MM.1R, HEK293T, HS-5, K562, HeLa, HepG2, A549, SH-SY5Y, Raji, NALM-6, and Jurkat were obtained from ATCC, while MM.1S was purchased from BPS Biosciences.
Techniques: Binding Assay, Immunopeptidomics, Cell Culture, Control, Imaging, Membrane, Construct, Staining, Flow Cytometry, Expressing, Transduction, Cell Binding Assay, Co-Culture Assay, Fluorescence