Anti-CTLA-4 Antibodies of IgG2a Isotype Enhance Antitumor Activity through Reduction of Intratumoral Regulatory T Cells (1)
Antibody blockade of CTLA-4 was shown to exacerbate autoimmunity. By extension to tumor immunity, the ability of anti-CTLA-4 to cause regression of established tumors provided a dramatic example of the therapeutic potential of CTLA-4 blockade. Anti-CTLA-4 9D9-IgG2b has been tested in a variety of mouse subcutaneous tumor models, such as Sa1N fibrosarcoma, MC38 and CT26 colon adenocarcinomas, and B16 melanoma.
C57BL/6 or BALB/c mice were subcutaneously injected with 2 million MC38 or 1 million CT26 tumor cells, respectively. After 7 days, tumor volumes were determined and mice were randomized into treatment groups so as to have comparable mean tumor volumes (45–50 mm3/2). Antibodies formulated in PBS were administered intraperitoneally (i.p.) on days 7, 10, and 14 at 200 μg per dose in a volume of 200 μL for MC38 and on days 7, 10, 14, and 17 for CT26. Tumor volumes were recorded 3 times weekly. 9 female C57BL/6 mice were injected intraperitoneally with 10 mg/kg of each isotype of anti-CTLA-4 (IgG1, IgG1-D265A, IgG2a, or IgG2b).
Antitumor activity in therapeutic MC38 and CT26 colon adenocarcinoma tumor models was tested with treatment initiated 7 days after implantation. Anti-CTLA-4 9D9-IgG2a treatment resulted in nearly complete tumor rejection, whereas 9D9-IgG2b showed moderate tumor growth inhibition in both models. Unexpectedly, anti-CTLA-4 containing IgG1 or IgG1-D265A showed little activity; the growth of tumors in mice treated with these antibodies was comparable with those in the control IgG1 treatment group.
Anti-CTLA-4–IgG2a treatment resulted in the most pronounced increases in intratumoral levels of both T-helper (TH)1 and TH2 cytokines, with significant enhancement of IFN-γ, TNF-α, IL-13, and IL-10 compared with each of the other isotype variants. This upregulation was unique to treatment with the IgG2a isotype and not simply associated with tumor destruction and regression as this increase in IL-1α was not observed with MC38 tumor-bearing mice treated with the combination of anti-PD-1 and anti-CTLA-4–IgG2b antibodies.
Interestingly, the antitumor activity of anti-CTLA-4 follows the hierarchy defined by the A/I ratio: IgG2a > IgG2b > IgG1, and suggests that maximal antitumor activity of anti-CTLA-4 requires binding to activating FcγR. Anti-CTLA-4 IgG1 and IgG1-D265A were equally inactive in antitumor activity, suggesting that FcγRIIB and FcγRIII do not appreciably contribute to the antitumor effect of anti-CTLA-4-IgG2a and IgG2b isotypes. Anti-CTLA-4-IgG2a, and to a lesser extent anti-CTLA-4-IgG2b, results in the elimination or depletion of Tregs from the tumor site consistent with their ability to bind to activating FcγRs.