Fig 2: T7011 introduces CD19 and BCMA on tumor cells, which direct activation and cytotoxicity of CAR-TCD19 and CAR-TBCMA cells. (A-F) HEp-2, A375, and PC-3 cells were infected with T7011 at an indicated MOI of 0.01, 0.03, 0.1, 0.3, or 1 for 24 hours (black line) and then cocultured with untransduced T cells, CD19 specific CAR T cells [CAR-TCD19, (A–C)], or BCMA specific CAR T cells [CAR-TBCMA, (D-F)] at an effector: target (E: T) ratio of 4:1 for 24 hours. Cell viability values for tumor cells cocultured with untransduced T cells (green square) or CAR-TCD19 cells (blue triangle) are indicated by a single data point. The live cells were determined with CellTiter-Glo®. Results are expressed as the mean of the cell viability ± SEM compared to the mock treatment control (as 100%). (G–L) HEp-2, A375, and PC-3 cells were cocultured with CD19-specific CAR T cells [CAR-TCD19, (G–I)], or BCMA-specific CAR T cells [CAR-TBCMA, (J–L)] at an effector: target (E: T) ratio of 4:1 in the presence or absence of infection of T7011 or T3011 at 1 PFU per cell. The live cells were determined with CellTiter-Glo®. Results are expressed as the mean of the cell viability ± SEM compared to the mock treatment control (as 100%). Statistically significant differences are indicated by an asterisk (**p < 0.01).

Fig 3: Morphological characterization of BCMA+ cells in PB samples: (A) Representative candidate rare cells with comparative BCMA and CD138 expression across different cell sizes. (B) UMAP projection of all candidate cells using all 761 single-cell morphology and marker intensity features, colored by CD138 expression. (C) UMAP projection, colored by BCMA expression. (D) UMAP projection, colored by CD45 expression. (E) Density plots showing channel intensities for each disease state.

Fig 4: Oncolytic virus T7011 effectively expresses payload genes and delivers truncated CD19 and BCMA to solid tumor surface in vitro. (A) Schematic representation of the expression cassette of oHSV T7011. Under the control of HSV-1 immediate early (IE) promoter, two truncated non-signaling variant tumor associate antigens CD19 and BCMA, and chemokine CCL5 were inserted between UL37 and UL38 genes in the T7011 genome, tailed with SV40 polyA. (B) Total viral yields were assayed at the indicated time points post infection with T7011. HEp-2 cells were seeded into a 12-well plate at a density of 5×105 cells per well. After overnight incubation, the cells were mock infected or infected with T7011 or HSV-1 (F) at an MOI of 1. After 2-hour incubation, the inoculum was replaced with fresh culture medium. The cell pellets were harvested at 2, 24, 48, and 72-hour post infection (h p.i.) and lysed with three freeze-thaw cycles for titration to detect the virus titer. Statistically significant differences between HSV-1 (F) and T7011 infection groups are indicated by asterisks (**P < 0.01). (C) Accumulation of human IL-12, anti-PD-1 antibody, and CCL5 in T7011 infected Vero cells. Vero cells were seeded into a T150 flask at a density of 6×106 cells per flask. After overnight incubation, the cells were infected with 0.01 PFU of T7011 per cell. The supernatant was collected at 48 h p.i. for ELISA assay. Data are shown as means ± standard error of mean (SEM). (D) The time-course of CCL5 protein expression in T7011 infected HEp-2 cell. HEp-2 cells were seeded into the T25 flask at a density of 1×106 cells per flask. After overnight incubation, the cells were mock infected or infected at 1 PFU of T7011 per cell. The cell supernatant was harvested at 0, 4, 8, 12, 24, 48, 72, and 96 h p.i. for ELISA assay. Data are shown as means ± SEM. (E) The time-course of truncated CD19 and BCMA cell surface localization in T7011 infected HEp-2 cell. HEp-2 cells cultured in coverslip were mock infected or infected with T7011 at 5 PFU per cell. The cells were fixed at indicated hour (h) post infection and then stained with antibodies against CD19 (green) and BCMA (red) as described in Methods and Materials. The images including the differential interference contrast (DIC) images were captured and processed using a Nikon confocal laser-scanning microscope. Scale bars = 10 μm.

Fig 5: Assay rationale and Immunofluorescence targeting: (A) B-cell differentiation and BCMA expression during the B-cell maturation spectrum from GC B to mature plasma cells [7,8]. (B) Clinical studies involving BCMA as a therapeutic target of interest. Data from ClinicalTrials.gov (accessed on 1 May 2022) with “BCMA” as the search key term, as of April 2022. “Not Applicable” = study phase explicitly marked as “not applicable”. “Unknown” = no data entered for study phase. The low count in the year 2020 reflects the effect of the COVID-19 pandemic on clinical enrollment. (C) Immunofluorescence targeting with different antibodies for the detection of BCMA+ and CD138+ cells in the enrichment-free HDSCA workflow with histological features of plasma cells and B cells. Large CD138+ cells with eccentric nuclei represent the plasma cell compartment, while small CD45+ and BCMA+CD138- cells represent the B-cell fraction. Gt = goat, Ms = mouse, Rb = rabbit, Ig = immunoglobulin, A555 = Alexa Fluor 555 dye, A488 = Alexa Fluor PLUS 488 dye, A647 = Alexa Fluor 647 dye.