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. 2011 Dec;96(12):1822-30.
doi: 10.3324/haematol.2011.047159. Epub 2011 Aug 31.

The in vivo mechanism of action of CD20 monoclonal antibodies depends on local tumor burden

Peter Boross  1 , J H Marco Jansen, Simone de Haij, Frank J Beurskens, Cees E van der Poel, Lisette Bevaart, Maaike Nederend, Josée Golay, Jan G J van de Winkel, Paul W H I Parren, Jeanette H W Leusen
Affiliations

The in vivo mechanism of action of CD20 monoclonal antibodies depends on local tumor burden

Peter Boross et al. Haematologica. 2011 Dec.

Abstract

Background: CD20 monoclonal antibodies are widely used in clinical practice. Antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and direct cell death have been suggested to be important effector functions for CD20 antibodies. However, their specific contributions to the in vivo mechanism of action of CD20 immunotherapy have not been well defined.

Design and methods: Here we studied the in vivo mechanism of action of type I (rituximab and ofatumumab) and type II (HuMab-11B8) CD20 antibodies in a peritoneal, syngeneic, mouse model with EL4-CD20 cells using low and high tumor burden.

Results: Interestingly, we observed striking differences in the in vivo mechanism of action of CD20 antibodies dependent on tumor load. In conditions of low tumor burden, complement was sufficient for tumor killing both for type I and type II CD20 antibodies. In contrast, in conditions of high tumor burden, activating FcγR (specifically FcγRIII), active complement and complement receptor 3 were all essential for tumor killing. Our data suggest that complement-enhanced antibody-dependent cellular cytotoxicity may critically affect tumor killing by CD20 antibodies in vivo. The type II CD20 antibody 11B8, which is a poor inducer of complement activation, was ineffective against high tumor burden.

Conclusions: Tumor burden affects the in vivo mechanism of action of CD20 antibodies. Low tumor load can be eliminated by complement alone, whereas elimination of high tumor load requires multiple effector mechanisms.

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Figures

Figure 1.
Figure 1.
Differential requirement for FcγR for tumor killing by CD20 monoclonal antibodies in the low and high tumor burden models. EL4-CD20 intraperitoneal (i.p.) model: mice were injected i.p. with CFSE-labeled EL4-CD20 cells and 16 h later were given CD20 monoclonal antibodies (mAb) or phosphate buffered saline (PBS). After 24 h the number of tumor cells in the peritoneal wash was determined by TrueCount tubes (4–5 mice/group; ***P<0.001, **P<0.01; ANOVA). (A) Low tumor burden model: wild-type (WT) mice were injected with 5x105 EL4-CD20 cells and 10 μg ofatumumab (OFA), rituximab (RTX), 11B8 or PBS. (B) and (C) High tumor burden model: WT mice were injected with 5x106 EL4-CD20 cells and 100 μg CD20 mAb or PBS alone. FcRγ−/− (D) and NOTAM (E) mice were injected with 5x105 EL4-CD20 cells and 10 μg CD20 mAb or PBS alone. (F) FcRγ−/− mice were injected with 5x106 EL4-CD20 cells and 100 μg CD20 mAb or PBS alone.
Figure 2.
Figure 2.
Complement is essential for tumor killing by CD20 monoclonal antibody (mAb) at both low and high tumor burden. EL4-CD20 intraperitoneal (i.p.) model: mice were injected i.p. with CFSE-labeled EL4-CD20 cells and 16 h later were given CD20 mAb or phosphate buffered saline (PBS). After 24 h the number of tumor cells in a peritoneal wash was determined using TrueCount tubes. Complement was depleted with cobra venom factor (CVF) 1 day prior to the injection of tumor cells (3–5 mice/group). CVF-treated wild-type (WT) (A), FcRγ−/− (B) or NOTAM mice (C) were injected with 5x105 EL4-CD20 cells and 10 μg OFA, RTX, 11B8 or PBS alone. (D) CVF-treated WT mice were injected with 5x106 CFSE-labeled EL4-CD20 cells and 100 μg ofatumumab (OFA), rituximab (RTX), 11B8 or PBS.
Figure 3.
Figure 3.
Distribution of cell populations in the peritoneal cavity following tumor challenge and treatment. The relative numbers of tumor cells and different effector cell populations in the peritoneum at the end of experiments carried out with low and high tumor burden as determined by flow cytometry using fixed amount of beads. The effector cell populations and markers used for identification were as follows: NK cells (NK1.1), CD8+ T cells (CD8α), CD4+ T cells (CD4), B cells (CD19/B220), macrophages (CD11b).
Figure 4.
Figure 4.
Dominant role for FcγRIII in tumor killing at high tumor burden. (A) In vitro macrophage killing with wild-type (WT) and FcRγ−/− macrophages using 1 μg/mL ofatumumab (OFA). Data are representative of two independent experiments (**P<0.01; Student’s t-test). EL4-CD20 intraperitoneal model: mice were injected i.p. with CFSE-labeled EL4-CD20 cells and 16 h later were given CD20 monoclonal antibody (mAb) or phosphate buffered saline (PBS). After 24 h the number of tumor cells in the peritoneal wash was determined using TrueCount tubes (3–10 mice/group; ***P<0.001, **P<0.01; ANOVA). FcγRI−/− (B), FcγRIII−/− (C), or FcγRIIB−/− (D) mice were injected with 5x106 EL4-CD20 cells and with 100 μg OFA, rituximab (RTX), 11B8 or PBS. (E) WT and FcγRIIB−/− mice were injected with 5x106 EL4-CD20 cells and with a suboptimal amount (10 and 1 μg) of OFA.
Figure 5.
Figure 5.
Deposited complement fragments enhance macrophage-mediated tumor killing via CR3. (A) Deposition of complement fragments C3b, iC3b and C3c on the tumor cells in vivo. EL4-CD20 intraperitoneal model: mice were injected i.p. with CFSE-labeled EL4-CD20 cells and 16 h later were given CD20 monoclonal antibody (mAb) or phosphate-buffered saline (PBS). After 24 h the number of tumor cells in the peritoneal wash was determined using TrueCount tubes (3–7 mice / group, ***P<0.001, **P<0.01, *P<0.05; ANOVA). (B) CR3−/− mice were injected with 5x105 EL4-CD20 cells (low tumor burden) and 10 μg CD20 mAb. (C) CR3−/− mice were injected with 5x106 CFSE-labeled EL4-CD20 cells (high tumor burden) and 100 μg ofatumumab (OFA), rituximab (RTX), 11B8 or PBS alone. (D) In vitro macrophage killing. CFSE-labeled EL4-CD20 cells opsonized in 1 μg/mL OFA, RTX and 11B8 were incubated for 24 h with bone marrow derived macrophages (BMDM) at different E:T ratios. Prior to adding BMDM, the CD20 mAb-opsonized CFSE-labeled EL4-CD20 cells were incubated with 5% wild-type (WT) mouse serum at 37°C. The number of CFSE-labeled EL4-CD20 cells was determined by using known amount of beads. The condition without CD20 mAb was used to set the 0% level; the condition without tumor cells was used to set the 100% level and killing is expressed as percentage decrease in cell numbers compared to control. (E) As in panel (D), in vitro macrophage killing with WT macrophages at E:T = 5:1 and 1 μg/mL RTX using heat-inactivated serum. (F) As in panel (D), in vitro macrophage killing with WT and CR3−/− macrophages at E:T = 3:1 and 1 μg/mL RTX). Data are representative of at least two experiments (***P<0.001, **P<0.01; Student’s t-test).
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