Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies

Abstract

Fc γ receptor (FcγR) coengagement can facilitate antibody-mediated receptor activation in target cells. In particular, agonistic antibodies that target tumor necrosis factor receptor (TNFR) family members have shown dependence on expression of the inhibitory FcγR, FcγRIIB. It remains unclear if engagement of FcγRIIB also extends to the activities of antibodies targeting immunoregulatory TNFRs expressed by T cells. We have explored the requirement for activating and inhibitory FcγRs for the antitumor effects of antibodies targeting the TNFR glucocorticoid-induced TNFR-related protein (GITR; TNFRSF18; CD357) expressed on activated and regulatory T cells (T reg cells). We found that although FcγRIIB was dispensable for the in vivo efficacy of anti-GITR antibodies, in contrast, activating FcγRs were essential. Surprisingly, the dependence on activating FcγRs extended to an antibody targeting the non-TNFR receptor CTLA-4 (CD152) that acts as a negative regulator of T cell immunity. We define a common mechanism that correlated with tumor efficacy, whereby antibodies that coengaged activating FcγRs expressed by tumor-associated leukocytes facilitated the selective elimination of intratumoral T cell populations, particularly T reg cells. These findings may have broad implications for antibody engineering efforts aimed at enhancing the therapeutic activity of immunomodulatory antibodies.

Figure 1.

Activating, rather than inhibitory, FcγRs are necessary for the antitumor activity of an agonistic antibody to GITR. Efficacy study of anti-GITR antibody (DTA-1 rIgG2b; 5 mg/kg i.p.) in wild type (A), FcγRIIB^−/− (B), and Fc common γ chain^−/− (C) BALB/c mice bearing Colon26 tumors (n = 6–10 mice per treatment group). Day 0 refers to treatment day, 6–8 d after tumor inoculation. Data is a representative of two or more independent experiments.

Figure 2.

Engagement of FcγRs by DTA-1 is required for antitumor activity. (A and B) GITR-binding assay. Primary splenocytes stimulated with CD3- and CD28-specific antibodies served as targets. DTA-1 variants were detected using rat IgG2b-specific (A) or murine IgG2a-specific (B) PE-conjugated antibodies. (C and D) In vitro activity of GITR-specific antibodies at various concentrations tested on GITR-expressing NF-kB 293 reporter cell line (C) and splenocytes incubated with suboptimal doses of anti-CD3 and anti-CD28 antibodies (D). The in vitro data are derived from triplicates and are a representative of two or more independent experiments. (E) Efficacy study of 5 mg/kg DTA-1 mIgG2a in wild-type, FcγRIIB^−/−, and Fc common γ chain^−/− mice bearing Colon26 tumors (n = 7–10 mice per treatment group). Mean and standard errors are based on triplicates, and the data is a representative of two or more independent experiments. (F) In vivo efficacy study after treatment with the DTA-1 variant antibodies (n = 7). The efficacy data are a representative of two or more independent experiments. (G) Saturation of GITR on T reg cells in the tumor and draining lymph node by the three versions of DTA-1.

Figure 3.

Engagement of FcγRs by DTA-1 induces loss of intratumoral T reg cells early after treatment. (A) Cell surface expression of GITR on day 0 (n = 3). (B and C) Dot plots of CD11b⁺ myeloid and DX5⁺ NK cells, gated on live CD45⁺ leukocytes (B) and expression of FcγRII/III and IV by the two cell populations in the tumor (C). (D) Dot plots of T cells 3 d after treatment with the DTA-1 variant antibodies. Percentages of live CD45⁺ CD3⁺ T cells are indicated. The data are a representative of two or more independent experiments. (E and F) Density of T reg cells (E) and FoxP3-CD4⁺ T cells and CD8⁺ T cells (F) in the tumor after treatment with the 5 mg/kg DTA-1 variants. (G) Ratio between CD8⁺ T cells and T reg cells in the tumor. (H) Percentage of intratumoral CD45⁺ leukocytes. (I) T cell number in the draining lymph nodes after treatment with 5 mg/kg of the DTA-1 variants. Mean and standard errors are based on triplicates from two independent experiments. P-values were calculated using an unpaired Student’s t test (**, P < 0.001).

Figure 4.

Activating, but not inhibitory, FcγRs are required for intratumoral T reg cell depletion by antibodies targeting GITR. Intratumoral T cell density and CD8⁺ T cells to T reg cells ratios 5 d after treatment with 5 mg/kg DTA-1-mIgG2a using wild-type (A), FcγRIIB^−/− (B), or Fc common γ chain^−/− (C) mice bearing Colon26 tumors. (D–F) Cell surface expression of GITR on T cells. Mean and standard errors are based on triplicates from two independent experiments. P-values were calculated using an unpaired Student’s t test (**, P < 0.001).

Figure 5.

Engagement of activating FcγRs is also required for the antitumor activity of an antibody targeting the non-TNFR CTLA-4. (A) Efficacy study using 15 mg/kg CTLA-4–specific antibody (9D9) in wild-type (top) or Fc common γ chain^−/− (middle) mice bearing Colon26 tumors (n = 8–10 mice per treatment group). (B) Intratumoral T cell density and CD8⁺ to T reg cells ratios 5 d after treatment. (C) Cell surface expression of CTLA-4 on T cells. The efficacy data are a representative of two or more independent experiments. Mean and standard errors are based on triplicates from two independent experiments. P-values were calculated using an unpaired Student’s t test (*, P < 0.01).