Role of Fc in antibody-mediated protection from ricin toxin

Abstract

We have studied the role of the antibody (Ab) Fc region in mediating protection from ricin toxicity. We compared the in vitro and in vivo effects of intact Ig and of Fab fragments derived from two different neutralizing Ab preparations, one monoclonal, the other polyclonal. Consistent results were obtained from each, showing little difference between Ig and Fab in terms of antigen binding and in vitro neutralization, but with relatively large differences in protection of animals. We also studied whether importing Ab into the cell by Fc receptors enhanced the intracellular neutralization of ricin toxin. We found that the imported Ab was found in the ER and Golgi, a compartment traversed by ricin, as it traffics through the cell, but intracellular Ab did not contribute to the neutralization of ricin. These results indicate that the Fc region of antibody is important for in vivo protection, although the mechanism of enhanced protection by intact Ig does not appear to operate at the single cell level. When using xenogeneic antibodies, the diminished immunogenicity of Fab/F(ab')2 preparations should be balanced against possible loss of protective efficacy.

Figure 1

Comparative binding of intact Ig and Fab fragments to ricin toxin. Two different anti-ricin Abs were titrated by ELISA for binding to ricin. Results are the mean and SEM of triplicate samples. (A) Murine mAb RAC18 (solid line) and the corresponding Fab (dashed line) and F(ab’)₂ (dotted line) fragments; (B) Polyclonal horse-anti-ricin igG (solid line) and corresponding Fab/F(ab’)₂ fragments (dashed line).

Figure 2

Neutralization of ricin toxicity by intact Ab or Fab fragments. Comparative molar concentrations of ntact IgG (1 µg/mL) or fragment (0.66 µg/mL) were mixed with the indicated concentration of ricin. H9 cells (2.5 × 10⁴ per well) were added. Two days later, cell viability was measured by MTS dye reduction. Data are mean and SEM of triplicate determinations.

Figure 3

Protective efficacy of intact IgG vs. Fab/F(ab)’₂ fragments. Mice received intact Ab (high dose: 3 mg/kg, low dose: 1 mg/kg) or Fab (mouse), or Fab/F(ab)’2 (horse) (high dose: 2 mg/kg, low: 0.66 mg/kg) four hours after a ricin challenge (20 µg/kg). Survival post-challenge is shown. Intact antibody is shown with a colored solid line, Fab a dotted line. Control animals are shown in black. Significance by Mantel-Cox log rank test (p < 0.05) is indicated with an asterisk.

Figure 4

In vivo protection from ricin toxicity by murine or chimeric RAC18. Mice received 1 mg/kg murine or chimeric mAb 4 h after ricin challenge. Survival post-challenge is shown. Statistical significance is displayed: * p = 0.05; ** p = 0.01.

Figure 5

Chimeric RAC18 is bound by human FcRI. (A) FcRγ expressing cells were incubated with Alexa-488 conjugated chRAC18 (5 µg/mL) for 1 h in PBS/BSA/Azide, washed, and then analyzed by flow cytometry; (B) FcRγI cells express CD64 but not CD16 or CD32. Cells were stained with primary Ab at 0.25–0.5 µg/mL and then FITC anti-mouse IgG secondary Ab; (C) Binding of chimeric RAC18 to FcRγI cells is only inhibited by anti-CD64, not anti-CD32. Cells were first incubated with anti-CD32 or CD64 (0.25–0.5 µg/mL) and then with Alexa-RAC18 (1 µg/mL).

Figure 6

Localization of chRAC18 internalized by FcRγI. Cells were incubated with 10 µg/mL of Alexa-labeled chRAC18 (red) for 18 h. For the final 30–60 min, cells were incubated with Bodipy-BFA (250 ng/mL), Hoechst dye (2 µg/mL), or Lysotracker Blue (LTB, 125 nM), all shown in blue. Colocalization of dye and Ab is shown in white.

Figure 7

Colocalization of internalized chRAC18 with entering ricin in FcRγI cells. Cells were incubated with 10 µg/mL of Alexa-labeled chRAC18 (red) for 18 h, washed, placed on a heated stage and Alexa-labeled ricin (3 µg/mL, green) added. Different cells were visualized at 2–3 min intervals. Representative time points are shown. Colocalization of ricin and mAb appears orange/yellow.

Figure 8

Effects of FcR on internalization of ricin and protection from ricin-mediated cytotoxicity. (A) Measurement of ricin internalization was performed by confocal microscopy over a 90 min period on a heated stage in the presence or absence of 30 µg/mL of chRAC18. The amount of internalized ricin was measured by manual partitioning and calculated from pixilated values obtained from the partitioning. Measurements performed in the presence of chRAC18 are shown in red, in the absence of mAb in black. The results are combined data from three independent experiments. Individual data points and the best fit curves are shown. Statistical significance was determined by the F test: * p < 0.05; *** p < 0.0001; (B) Neutralization by RAC18 was not affected by FcR expression on cells. MTS assay was performed in the presence (red) or absence (black) of chimeric RAC18 (20 µg/mL) at the indicated concentrations of ricin.

Figure 9

Inhibition of proteasome degradation does not affect ricin toxicity nor Ab neutralization. The drug MG132 inhibits proteasome degradation, a hallmark of TRIM21-mediated effects. An MTS assay was performed on H9 cells in the presence of 123 nM MG132 (370 nM MG132 completely suppresses MTS dye reduction) or in the absence of the drug. MG132 had no effect on toxicity or neutralization.