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. 2011 Aug 19;333(6045):1030-4.
doi: 10.1126/science.1206954.

Inhibitory Fcγ receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies

Fubin Li  1 , Jeffrey V Ravetch
Affiliations

Inhibitory Fcγ receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies

Fubin Li et al. Science. .

Abstract

CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, is expressed on antigen-presenting cells (APCs) and is essential for immune activation. Although agonistic CD40 antibodies have been developed for immunotherapy, their clinical efficacy has been limited. We have found that coengagement of the Fc domain of agonistic CD40 monoclonal antibodies (mAbs) with the inhibitory Fcγ receptor FcγRIIB is required for immune activation. Direct comparison of mAbs to CD40 enhanced for activating FcγR binding, hence capable of cytotoxicity, or for inhibitory FcγRIIB binding, revealed that enhancing FcγRIIB binding conferred immunostimulatory activity and considerably greater anti-tumor responses. This unexpected requirement for FcγRIIB in enhancing CD40-mediated immune activation has direct implications for the design of agonistic antibodies to TNFR as therapeutics.

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Figures

Fig. 1
Fig. 1
FcγRIIB-engagement is required for the adjuvant activity of agonistic CD40 antibodies. (A) WT and FcγR−/− (Fcgr2b−/−Fcer1g−/−, deficient in all FcγRs) mice were injected i.p. with DEC-OVA(hIgG1) in the presence or absence of 1C10. Spleen cells were harvested 7 days later and analyzed by flow cytometry for OVA-specific CD8+ T cells, detected by OVA tetramer staining or IFN-γ expression upon in vitro OVA peptide stimulation as described in “Materials of methods” (5). (B–D) WT, Fcer1g−/− (FcRγ-deficient), and Fcgr2b−/− (FcγRIIB-deficient) mice were immunized with DEC-OVA(hIgG1)N297A (the N297A variant of DEC-OVA(hIgG1), null for FcγR binding) in the presence or absence of 1C10, or deglycosylated 1C10, or 1C10 F(ab’)2 fragment, and analyzed as in (A). Shown are representative contour plots gated on CD4−CD8α+ cells, with gate and percentage (mean ± S.D) of tet-OVA+ or IFN-γ+ cells, of two or more independent experiments with 4–5 mice per group with similar results.
Fig. 2
Fig. 2
The adjuvant effect of agonistic CD40 antibodies can be modulated by manipulating their binding affinities to FcγRIIB. (A–C) WT, Fcgr2b−/−, FcγR−/−, and FcγR−/− mice with human FCGR2A and/or FCGR2B transgenes (FcγR−/−hFCGR2A+, FcγR−/−hFCGR2B+, or FcγR−/−hFCGR2A+hFCGR2B+) were immunized with DEC-OVA(hIgG1)N297A in the presence or absence of CD40 antibodies with the indicated Fc’s, and analyzed for OVA-specific CD8+ T cells in peripheral blood (A) or spleen (B–C) as in Fig. 1. Shown are representative contour plots gated on CD4−CD8α+ cells, with gate and percentage (mean ± S.D) of tet-OVA+ or IFN-γ+ cells, of two or more independent experiments with 4~5 mice per group with similar results. (D) Increased in vivo DC maturation induced by anti-CD40 with enhanced hFcγRIIB-binding affinity. FcγR−/−hFCGR2B+ mice (3 per group) were either untreated or injected i.p. with 100µg of the indicated CD40 antibodies. Three days later, CD80 and CD86 expression were analyzed on splenic dendritic cells by flow cytometry. Presented is a bar-graph showing mean fluorescence intensity (MFI) values with S.D. * p < 0.05; ** p < 0.01; *** p < 0.001. One-way ANOVA with Dunnett post hoc test was used to compare: all groups to the untreated control group; and the αCD40:hIgG1 and αCD40:hIgG1(S267E) treated groups.
Fig. 3
Fig. 3
Anti-tumor activities of CD40 antibodies correlate with their adjuvant effects in CD40− tumor models. (A–B) WT and FcγR−/−hFCGR2B+ mice were inoculated with MO4 tumor cells subcutaneously, and treated with DEC-OVA(hIgG1)N297A plus low doses (30µg) of control IgG or the indicated CD40 antibodies with either mouse (A) or human (B) IgG Fc’s after tumor establishment. Tumor growth curves (3–5 mice per group) are shown. (C) WT mice were inoculated intravenously with B6BL tumor cells and treated with high doses of control IgG or the indicated CD40 antibodies (d3: 200µg; d4; 200µg). (D) WT and T cell deficient mice (Tcrb−/− Tcrd−/−) were inoculated with B6BL as in (C) and treated with control IgG or the indicated CD40 antibodies (d3: 200µg) with or without αCD4 or αCD8 depleting antibodies; (E) Fcgr2b−/−(Tg−) and Fcgr2b −/−hFCGR2B+ (Tg+) mice were inoculated with B6BL as in (C) and treated with high doses (d3: 200µg; d4: 200µg) or lower doses (d3: 40µg; d4: 40µg) of the indicated CD40 antibodies or control IgG. Long-term survivors were re-challenged at 10 weeks with B6BL tumor cells. (C–E) Survival curves of 4–5 mice are shown. Error bars are S.D. *p<0.05, **p<0.01, ***p<0.001. One-way ANOVA with Dunnett post hoc test (A–B) or Logrank test (C–E) was used to compare: all groups to the control groups (“mIgG” in (A), “hIgG” in (B), “mIgG” in (C), “WT:mIgG” in (D), or Tg+:hIgG(400µg) in (E), respectively); or “αCD40:hIgG1” to “αCD40:hIgG1(S267E)” in (B); or between Tg+ mice treated with same doses of αCD40:hIgG1 or αCD40:hIgG1(S267E) in (E).
Fig. 4
Fig. 4
Anti-CD40 enhanced for FcγRIIB-binding has greater anti-tumor activities than anti-CD40 enhanced for activating FcγR-binding in CD40+ tumor models. (A) BALB/c mice were treated with the indicated CD40 antibodies or control IgG (200µg) 1h before the subcutaneous inoculation of A20 tumor cells. Tumor growth curves of 5 mice per group are shown. WT (B) and Fcer1g−/− (D) mice were inoculated with B6BL-CD40 tumor cells and treated with the indicated CD40 antibodies or control IgG (d3: 200µg; d6: 200µg) with or without αCD8 depleting antibodies. Survival curves of 4–6 mice per group are shown in (B, D). (C) WT mice (3 per group) were treated with 200µg of the indicated CD40 antibodies or control IgG. Six days later, the percentage of CD40+ cells and the ratio of CD8+ to CD4+ T cells were analyzed in peripheral blood and presented in the bar-graph. Error bars are S.D. **p<0.01, ***p<0.001. One-way ANOVA with Dunnett post hoc test (A, C) or Logrank test (B, D) was used to compare: all groups to the “mIgG” control groups; “αCD40:mIgG1” to “αCD40:mIgG2a” in (C).
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