Figure 3. Fully human CD20 CAR exhibits potent in vitro and in vivo anti-tumor activity.

Healthy donor central memory T cells (T_CM) were stimulated with anti-CD3/CD28 beads and transduced the next day with iC9-1F5-NQ-28-BB-z, 1.5.3-NQ-28-BB-z, or 1.5.3-NQ-28-z lentiviral vectors, or a vector encoding only iC9 and tCD19 (“empty vector”), and expanded in vitro. (A) CAR-expressing cells were co-cultured in a 1:1 ratio with irradiated Granta-519 cells (left) or Raji-ffLuc cells (right), and supernatants were harvested 24 hours later and analyzed for the indicated cytokines using a Luminex assay. IL-2 and TNF-α (left axis) and IFN-γ (right axis) levels are shown. (B) Cytotoxicity was measured by using these cells as effectors in a standard 4-hour ⁵¹Cr-release assay against either Jeko (left) or Raji-ffLuc (right) cell lines. (C and D) NSG mice were injected i.v. with 5 × 10⁵ Raji-ffLuc cells followed 2 days later by 5 × 10⁶ cells transduced with the vectors described in (A). A Kaplan-Meier curve for overall survival is shown in (C) and tumor burden over time of individual mice as measured by bioluminescence is shown in (D). The survival curves for iC9_1F5-NQ-28-BB-z, 1.5.3-NQ-28-BB-z, and 1.5.3-NQ-28-z were compared using a log-rank (Mantel-Cox) test. (E) Retroorbital blood samples of mice treated with T cells transduced with 1.5.3-NQ-28-z, 1.5.3-NQ-28-BB-z, or an empty vector in the experiment described in C and D were analyzed by flow cytometry for circulating infused CAR T cells. The percentage of cells in the mCD45⁻ hCD3⁺ hCD19⁺ gate over time are shown (n = 2 mice per group). (F) NSG mice were injected i.v. with 5 × 10⁵ Granta-ffLuc cells followed 2 days later by either 5 × 10⁶ tCD19⁺ cells transduced with the 1.5.3-NQ-28-BB-z or an equal number of untransduced cells. A Kaplan-Meier curve for overall survival is shown in (F), with curves compared using a log-rank test, and tumor burden over time of individual mice as measured by bioluminescence is shown in (G).