Figure 2: CD28^iKO hBCMAmBBmζ CAR T cells are functionally impaired in vivo.

(A) Schematic depicting the process of manufacturing mouse CD28 knockout (CD28^iKO) hBCMAmBBmζ CAR T cell.

(B) Surface CD28 protein expression on CD28^fl/fl versus CD28^iKO mouse T cells during CAR T cell manufacture.

(C) Median fluorescent intensity (MFI) of CD28 measured by flow cytometry at the conclusion of CD28^fl/fl versus CD28^iKO mouse CAR T manufacture. Data shown as mean ± SD from 10+ independent experiments. *p<0.05, ****p<0.0001 by one-way ANOVA.

(D) CD28^fl/fl versus CD28^iKO hBCMAmBBmζ CAR T cell cytotoxic activity during 24 hr. co-culture with luciferase-tagged 5TGM1^hBCMA mouse myeloma cells. Cell viability was assessed by luciferase assay. Data are shown as mean ± SD and representative of at least 3 independent experiments. *p<0.05, **p<0.01 by two-way ANOVA with Tukey’s multiple comparison test.

(E) Heatmap representation of culture supernatant mouse cytokine concentrations measured by multiplexed Luminex assays at the conclusion of a 24-hr. co-culture of hBCMAmBBmζ CD28^fl/fl or CD28^iKO CAR T cells with 5TGM1^hBCMA myeloma cells. Log₂ transformed cytokine concentrations represent the mean of 4 independent experiments.

(F) Diagram of experimental setup used to evaluate CD28^fl/fl versus CD28^iKO hBCMAmBBmζ CAR T cell therapy in a mouse MM xenograft model. RAG2^−/− mice were inoculated intravenously with 2 × 10⁶ 5TGM1^hBCMA-luc cells on day −14 and treated with CD28^fl/fl or CD28^iKO CAR T cells on day 0. Tumor burden was monitored by IVIS bioluminescent imaging (BLI) 2x/week through endpoint.

(G) Tumor burden expressed as relative photon flux measured by BLI from 5TGM1^hBCMA-luc bearing mice treated with CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T or control T cells. Each line represents an individual mouse (n = 6 mice per CAR T cell treated group).

(H) Kaplan – Meier analysis of survival of CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cell or control T cell treated 5TGM1^hBCMA-luc bearing mice (n = 6 mice per CAR T cell treated group). Median survival of CD28^fl/fl hBCMAmBBmζ CAR T treated mice was 38 days post-CAR T cell infusion vs. 24 days for CD28^iKO hBCMAmBBmζ CAR T treated mice. *p<0.05, **p<0.01, ***p<0.001 by log-rank Mantel-Cox test.

(I) Heatmap representation of cytokine levels in the MM BME 7 days following infusion of CD28^fl/fl or CD28^iKO hBCMAmBBmζ CAR T cells into 5TGM1^hBCMA-luc bearing mice. Bilateral hind limbs were harvested, and BM was flushed into 15 μL PBS for multiplexed cytokine analysis. Log₂ transformed cytokine concentrations represent the mean of 3 mice per group.