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Differential Fc-Receptor Engagement Drives an Anti-tumor Vaccinal Effect - PubMed

  • ️Thu Jan 01 2015

Differential Fc-Receptor Engagement Drives an Anti-tumor Vaccinal Effect

David J DiLillo et al. Cell. 2015.

Abstract

Passively administered anti-tumor monoclonal antibodies (mAbs) rapidly kill tumor targets via FcγR-mediated cytotoxicity (ADCC), a short-term process. However, anti-tumor mAb treatment can also induce a vaccinal effect, in which mAb-mediated tumor death induces a long-term anti-tumor cellular immune response. To determine how such responses are generated, we utilized a murine model of an anti-tumor vaccinal effect against a model neoantigen. We demonstrate that FcγR expression by CD11c(+) antigen-presenting cells is required to generate anti-tumor T cell responses upon ADCC-mediated tumor clearance. Using FcγR-humanized mice, we demonstrate that anti-tumor human (h)IgG1 must engage hFcγRIIIA on macrophages to mediate ADCC, but also engage hFcγRIIA, the sole hFcγR expressed by human dendritic cells (DCs), to generate a potent vaccinal effect. Thus, while next-generation anti-tumor antibodies with enhanced binding to only hFcγRIIIA are now in clinical use, ideal anti-tumor antibodies must be optimized for both cytotoxic effects as well as hFcγRIIA engagement on DCs to stimulate long-term anti-tumor cellular immunity.

Copyright © 2015 Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1

Fc-FcγR interactions are required for the clearance of lymphoma by mAb and the initiation of an anti-tumor vaccinal effect. A, Experimental protocol: Mice were injected i.v. with 5×105 EL4-hCD20 lymphoma cells on day 0 (red arrow), and received 100μg of mIgG2a isotype anti-hCD20 mAb (clone CAT13.6E12) on days 1, 4, 7, 10, and 13 (blue arrows). On day 90, surviving mice were re-challenged i.v. with 5×106 EL4-hCD20 lymphoma cells (green arrow) or EL4-WT cells, a 10-fold greater dose of tumor compared to the primary lymphoma challenge, and survival was monitored daily. Alternatively, surviving mice were re-challenged with 5×104 B6BL-hCD20 or B6BL-mCD20 cells i.v. B, Wildtype (red circles) or FcRα-null mice (blue squares) were injected with EL4-hCD20 cells and treated with IgG2a isotype anti-hCD20 mAb, with survival assessed daily (n=9-11 mice per group). C, After 90 days, surviving (primed) mice treated with mIgG2a anti-hCD20 mAb from (b) were re-challenged with EL4-hCD20 cells (green circles) or EL4-WT cells (blue squares) with survival assessed daily. For comparison, naïve mice were also challenged with EL4-hCD20 cells (gray diamonds) or EL4-WT cells (filled triangles). n=10-13 mice per group. D, Mice were primed with EL4-hCD20 lymphoma cells and mAb as in (A-B) before re-challenge on day 90 with B6BL tumor cells expressing either hCD20 (green circles) or msCD20 (blue squares). n=10 mice per group.

Figure 2
Figure 2

Expression of mFcγRIV on CD11c+ cells is required for the generation of an anti-tumor vaccinal effect. A, mFcγRIV expression levels on spleen innate cell subsets. Spleen lymphocytes were harvested from Fcgr4fl/fl (red line), Fcgr4flfl;cd11c-cre (blue line), or Fcgr4−/− (shaded line) mice and mFcγRIV expression levels on CD11c+ dendritic cells and CD11b+Gr-1lowSSClow resident monocytes was assessed. Representative flow cytometry histograms from three independent experiments are shown. B, Fcgr4fl/fl (red circles; n=15) or Fcgr4flfl;cd1c-cre (blue squares; n=14) mice were given EL4-hCD20 cells and treated with mIgG2a anti-hCD20 mAb, with survival monitored daily. C, After 90 days, surviving Fcgr4fl/fl (green circles) or Fcgr4flfl;cd1c-cre (blue squares) mice treated with mIgG2a isotype anti-hCD20 mAb from (B) were re-challenged with EL4-hCD20 cells, with survival assessed daily (n=14-16 mice per group). Significant differences between groups are indicated: **, p=0.0065; n.s., not significant

Figure 3
Figure 3

CD11c+ cell-specific expression of mFcγRIV is required for the generation of anti-tumor cellular immunity. A, Experimental protocol: Mice were injected i.v. with EL4-hCD20 lymphoma cells on day 0 (red arrow), and received mIgG2a anti-hCD20 mAb (blue arrows). On day 30, spleens were harvested and total splenocytes were isolated or CD3+ cells were purified. Then, 50×106 total splenocytes or 15×106 CD3+ cells were adoptively transferred into naïve mice one day before i.v. challenge with EL4-hCD20 lymphoma cells (green arrow). B, Survival was measured in naïve mice receiving CD3+ cells (green filled circles) or total splenocytes (green open circles) from tumor and mAb-primed wild-type mice, or CD3+ cells (blue filled squares) or total splenocytes (blue open squares) from naïve mice. Another group of naïve mice received no adoptive transfer (black triangles). n=4-6 mice per group. C, Survival in naïve mice receiving total splenocytes from tumor and mAb-primed Fcgr4fl/fl (green circles; n=25) or Fcgr4flfl;cd1c-cre (blue squares, n=34) mice before EL4-hCD20 cell challenge. Other groups of naïve mice received splenocytes from naïve mice (black triangles; n=7) or no adoptive transfer (gray diamonds; n=15). Significant differences between groups are indicated: **, p=0.0041.

Figure 4
Figure 4

Human FcγR expression on innate cells in FcγR-humanized mice. A, Representative flow cytometry dot plots show hFcγRIIA vs. hFcγRIIIA/B expression on spleen cells from FcγR-humanized or FcRα-null mice. Numbers represent the frequency of cells in the indicated gate. B, DCs and monocytes from FcγR-humanized mouse spleens (red lines) were stained for hFcγRIIA, hFcγRIIIA/B, or hFcγRIIB. Background staining by hFcγR− cells is shown (gray lines). C, Frequencies of hFcγR+ cells among spleen DCs and monocytes (n=3 per group), with frequencies generated by background staining subtracted.

Figure 5
Figure 5

Differential hFcγR engagement mediates tumor cytotoxicity. A, Anti-hCD20 hIgG1 mAb Fc mutants for selectively-enhanced engagement of hFcγRs. Relative binding capabilities to the indicated hFcγRs are shown, based on binding affinities from biacore experiments (Supplementary Table 2). B, hFcγRIIIA engagement mediates cytotoxic clearance of tumor cells by mAb. FcγR-humanized mice were given EL4-hCD20 cells and treated with hIgG1 mutant versions of anti-hCD20 mAb: GASDALIE mutant (enhanced engagement of hFcγRIIA and hFcγRIIIA; red circles; n=20), GA mutant (preferential hFcγRIIA engagement; blue squares; n=6), ALIE mutant (preferential hFcγRIIIA engagement; gray diamonds; n=12), or PBS (black triangles; n=17), with survival monitored daily. C. hFcγRIIIA is necessary and sufficient to mediate the immediate cytotoxic clearance of EL4-hCD20 lymphoma cells. EL4-hCD20 cells were injected into hFCGR2A-Tg mice that were given GASDALIE mutant anti-hCD20 mAb (filled red circles; n=11), hFCGR3A/B-Tg mice given GASDALIE mutant anti-hCD20 mAb (filled blue squares; n=11), or wild-type mice given PBS (filled triangles; n=10) with survival monitored daily.

Figure 6
Figure 6

Selective engagement of hFcγRIIA mediates an anti-tumor vaccinal effect. A, Experimental protocol: FcγR-humanized mice were injected i.v. with 5×105 EL4-hCD20 lymphoma cells on day 0 (red arrow), and received 250 μg of hIgG1 mutant anti-hCD20 mAb on days 1 and 2 (blue arrows). On day 60, surviving mice were re-challenged i.v. with 5×106 EL4-hCD20 lymphoma cells (green arrow), and survival was monitored daily. B, Surviving tumor-primed mice that received GASDALIE hIgG1 anti-hCD20 mAb (green circles; n=28) or ALIE hIgG1 anti-hCD20 mAb (blue squares; n=10) from Fig. 5B, or naïve mice (black triangles; n=15) were re-challenged with EL4-hCD20 cells. C, Surviving tumor-primed FcγR-humanized (green circles; n=28) or hFcγRIIIA/B-Tg mice (blue squares, n=11) that received GASDALIE hIgG1 anti-hCD20 mAb from Fig. 5C, or naïve mice (black triangles; n=10) were re-challenged with EL4-hCD20 cells. Significant differences between groups are indicated: **, p<0.01.

Figure 7
Figure 7

Model for the generation of an anti-tumor vaccinal effect. Anti-tumor mAb opsonizes tumor cells and targets them for killing by FcγR-mediated ADCC, a process that generates antibody:tumor antigen immune complexes. These immune complexes engage activating FcγRs expressed by mouse or human CD11c+ cells, which results in stimulation of DC maturation and presentation of tumor antigens to T cells, thereby leading to long-term anti-tumor cellular memory formation

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