Xenograft Models and In Vivo Therapies

2 × 10⁶ tumor cells (AE17om in various mixtures with AE17o; AB12m mixed with AB12; TC-1 m mixed with TC-1; and AB-1 m mixed with AB-1) were injected into the flanks of syngeneic mice. When tumors reached ∼50–70 mm³ (4 days after tumor cell inoculation for both cell lines), 10⁷ transduced M11 CAR T cells were injected intravenously via tail vein in 2 doses, 2 days apart, and the tumors were measured by calipers every 3–4 days for up to 2 weeks. Non-treated tumor-bearing animals were used as controls. For combination treatments, mice were treated in one of four groups: (1) untreated controls, (2) M11 CAR T cells alone, (3) immune-stimulatory agent alone, or (4) a combination of M11 CAR T cell therapy and immune-stimulatory agent. The agents include anti-PD-1 antibody (BioXcell, RMPI-14) at 5 mg/kg, given via intraperitoneal (i.p.) injection biweekly; anti-CTLA-4 antibody (BioXcell, 9H10) at 200 μg per mouse, given via i.p. injection biweekly; anti-TGF-β antibody (BioXcell, ID11.16.8) at 150 μg per mouse, given via i.p. injection biweekly; agonistic CD40 (BioXcell, FGK4.5/FGK45) at 40 μg per mouse, given via i.p. injection as a single dose 1 day following CAR T cell treatment; IDO inhibitor (INCB023843) at 300 mg/kg, given via oral gavage daily for 2 weeks, starting on day 3 post-tumor inoculation; and CTX (MP Biomedicals, 150749) at 100 mg/kg, given via i.p. injection as a single dose on day 3 post-tumor inoculation, 1 day prior to CAR T cell injections.

The dosages of each agent were based on previous experiments or pilot experiments in our lab (unpublished data), where each agent was tested for its anti-tumor activity by itself. We used a standard 3- to 4-day injection of antibodies. In order to be able to detect their effects on the ability of CAR T cells to cure tumors, our goal in these studies was to choose a dose that had clear bioactivity but one that did not have anti-tumor activity so strong that it would eliminate or strongly suppress tumor growth by itself.