Effective targeting of a heterogeneous tumor population in vitro and in vivo requires multi antigen targeting. (A-B) NSG mice were inoculated IV with 2 × 10⁶ U266-BCMA^(–/–) or U266-B2M^(–/–) expressing Luc2 (Addgene #72486). Tumor cells were allowed to engraft for 2 weeks before IV treatment with indicated T-cell populations (6 × 10⁶ purified eTCR-T cells or CAR T cells per mouse). Tumor signal was regularly monitored using IVIS imaging after subcutaneous injection of luciferin. For IVIS images, refer to supplemental Figure 2D-E. (C) Overnight killing of heterogeneous U266 cells by the indicated T-cell populations in vitro. (D) Survival and phenotype of heterogeneous U266 cells after 7 days of coculture with indicated T-cell populations. (E) Fluorescence-activated cell sorting plots of heterogeneous U266 showing prevalence of U266-WT, U266-BCMA^(–/–), and U266-B2M^(–/–) 7 days after coculture with indicated T cells. (C-E) Representative data showing technical duplicates from 1 of 3 independent experiments using T cells derived from independent donors. (F-G) NSG mice were inoculated IV with a 1:1:1 mix of U266 WT, U266-BCMA^(–/–), and U266-B2M^(–/–) (2 × 10⁶ cells in total) expressing Luc2. After 2 weeks, mice were IV treated with indicated T-cell populations (6 × 10⁶ purified eTCR-T cells or CAR T cells per mouse; in the combination group 3 × 10⁶ BCMA-CAR T cells were coinjected with 3 × 10⁶ BOB1-TCR T cells); n = 4 for CMV-TCR T-cell treatment group; n = 7 for other treatment T-cell groups. (H) Mice were euthanized and bone marrow was extracted for fluorescence-activated cell sorting analysis for presence of U266 cells. The percentage of U266 in the bone marrow indicates 100% × (n[Luc2-tdTom⁺])/(n[mCD45⁺ OR huCD45⁺]). Statistics depict 1-way ANOVA comparing double-treated mice to BOB1 TCR or BCMA CAR single-treated mice (Fisher's LSD test). (I) Phenotyping of U266 cells detected in the bone marrow of euthanized mice. IVIS, in vivo imaging system; ANOVA, analysis of variance; LSD, least significant difference.