Apoptotic and antitumor activity of death receptor antibodies require inhibitory Fcγ receptor engagement

Abstract

By virtue of their ability to induce apoptosis and regulate growth, differentiation, and cytokine responses, the tumor necrosis factor receptor (TNFR) superfamily members have emerged as attractive targets for anticancer therapeutics. Agonistic antibodies to apoptosis-inducing TNFRs, such as death receptor 5 (DR5), although displaying impressive activities against a variety of tumors in preclinical models, appear to be less active in clinical trials. We report that the in vivo apoptotic and antitumor activities of these antibodies have an absolute requirement for the coengagement of an inhibitory Fcγ receptor, FcγRIIB. Anti-DR5 antibodies of the type currently in clinical trials have weak FcγRIIB binding and thus are compromised in their proapoptotic and antitumor activities in both colon and breast carcinoma models. Enhancing FcγRIIB engagement increases apoptotic and antitumor potency. Our results demonstrate that Fc domain interactions are critical to the therapeutic activity of anti-DR5 antibodies and, together with previous reports on agonistic anti-CD40 antibodies, establish a common requirement for FcγRIIB coengagement for optimal biological effects of agonistic anti-TNFR antibodies.

Fig. 1.

MD5-1 uniquely depends on FcγRIIB for its tumoricidal activity in the MC38 model. MC38 cells were inoculated s.c. into WT (A), Fcgr2b^−/− (B), and Fcer1g^−/− (C) mice on the B6 background, then treated with MD5-1 antibodies at the indicated doses at the time points indicated by arrows. Representative tumor growth curves of four to six mice are shown. The cross represents mortality. Error bars represent SD. *P < 0.05; **P < 0.01.

Fig. 2.

MD5-1 uniquely depends on FcγRIIB for its hepatotoxic effect. (A–C) WT, Fcgr2b^−/−, Fcer1g^−/−, and Fcer1g^−/−Fcgr2b^−/− mice on the B6 background (five mice per group) were treated with high-dose MD5-1 or hamster control IgG (300 μg/mouse repeated at 3-d intervals for a total of 1.2 mg/mouse), and analyzed for serum AST level (A), jaundice (B), and survival (C). (D) Serum AST levels of WT and Fcer1g^−/− mice on the BALB/c background (four mice per group) treated with 300 μg of MD5-1 7 d earlier. ***P < 0.001. Error bars represent SD.

Fig. 3.

FcγRIIB is necessary and sufficient for the in vitro proapoptotic activity of MD5-1. (A) Percentage of MC38 cells with active caspase-3. MC38 cells were treated with hamster control IgG, MD5-1, or a combination of MD5-1 and the FcγRIIB/III blocking antibody 2.4G2 in the absence or presence of splenocytes isolated from the indicated mice, and then analyzed for caspase-3 activation. ***P < 0.001. Error bars represent SD. Data shown are representative of three experiments. (B) SPR analysis of the binding of MD5-1 to mouse FcγRs. Real-time sensorgrams with affinity constants (K_D = mean ± SD) are shown.

Fig. 4.

Tumoricidal and hepatotoxic effects of agonistic anti-DR5 antibodies can be enhanced by FcγRIIB-targeted Fc engineering. (A and B) Fcgr2b^−/−huFCGR2B⁺ mice on the B6 background (five to nine mice per group) were implanted with MC38 cells s.c. and treated with 100 μg (A) or 33 μg or 11 μg/mouse (B) of the indicated control or anti-DR5 antibodies at the indicated times. Tumor growth curves are presented. (C) Fcgr2b^−/− and Fcgr2b^−/−huFCGR2B⁺ mice on the BALB/c background (four to six mice per group) were inoculated with 4T1.2 cells i.v. and treated with 33 μg/mouse of the indicated control or anti-DR5 antibodies at the indicated times. Survival curves are shown. (D) Fcgr2b^−/−huFCGR2B⁺ on the B6 background (three mice per group) were treated with 300 μg of the indicated control or anti-DR5 antibodies and analyzed for serum AST level 7 d later. *P < 0.05; **P < 0.01; ***P < 0.001. The cross represents mortality. Error bars represent SD.

Fig. 5.

Proapoptotic activities of agonistic anti-DR5 antibodies can be enhanced by FcγRIIB-targeted Fc engineering. MC38 or 4T1.2 cells were treated with the indicated control IgG or anti-DR5 antibodies in the absence or presence of splenocytes isolated from the indicated mice and then analyzed for caspase-3 activation. The percentages of MC38 (A) and 4T1.2 (B) cells with active caspase-3 are shown. Note the different scales for each tumor line. Error bars represent SD. Results are representative of two experiments.

Fig. 6.

αDR5:hIgG1(S267E) exert its antitumor activity through apoptosis. Fcer1g^−/−Fcgr2b^−/−huFCGR2B⁺ mice on the B6 background (three mice per group) were inoculated s.c. with MC38 cells on the left flank and MC38-cFLIP cells on the right flank, and then treated with 33 μg/mouse of the indicated anti-DR5 antibodies at the indicated time. Shown are a photo of representative mice treated with the indicated anti-DR5 antibodies (A) and tumor growth curves of MC38 and MC38-cFLIP in the αDR5:hIgG1(S267E)-treated mice (B). *P < 0.05. Error bars represent SD.