Evaluation of Fab and F(ab')2 fragments and isotype variants of a recombinant human monoclonal antibody against Shiga toxin 2

Abstract

5C12 HuMAb is a human monoclonal antibody against the A subunit of Shiga toxin 2 (Stx2). We have previously shown that 5C12 HuMAb effectively neutralizes the cytotoxic effects of this toxin by redirecting its transport within the cell and also by neutralizing the toxin's ability to inhibit protein synthesis. The 5C12 HuMAb and its recombinant IgG1 version protect mice at a dose of 0.6 microg against a lethal challenge of Stx2. The contribution of the Fc region to this observed neutralization activity of the 5C12 antibody against Stx2 was investigated in this study. Using recombinant DNA technology, 5C12 isotype variants (IgG1, IgG2, IgG3, and IgG4) and antibody fragments [Fab, F(ab')(2)] were expressed in Chinese hamster ovary cells and evaluated in vitro and in vivo. All four 5C12 isotype variants showed protection in vitro, with the IgG3 and IgG4 variants showing the highest protection in vivo. The Fab and F(ab')(2) fragments also showed protection in vitro but no protection in the mouse toxicity model. Similar results were obtained for a second HuMAb (5H8) against the B subunit of Stx2. The data suggest the importance of the Fc region for neutralization activity, but it is not clear if this is related to the stability of the full-length antibody or if the Fc region is required for effective elimination of the toxin from the body.

FIG. 1.

(A) Neutralization of Stx2 by r5C12 isotype variants in a HeLa cell cytotoxicity assay. The percent survival of HeLa cells in the presence of 5 ng of Stx2 and various quantities of r5C12 IgG1, IgG2, IgG3 or IgG4 (0.3 to 10 ng) was measured. The percent survival values are from at least two independent assays (triplicate wells) and represent the mean of these values. (B) Neutralization of Stx2 by r5H8 antibody or Fab. The neutralization activities of the 5H8 Fab fragments were compared to those of their parent IgG1 antibodies. Although neither Fab fragment showed good neutralization activity, the parent IgG1 antibodies showed good protection (>40%) only at doses of 25 and 50 ng.

FIG. 2.

Comparison of the neutralization activities of the 5C12 antibody fragments to those of their parent IgG1 antibodies in a HeLa cell cytotoxicity assay. The percentage of increased survival of HeLa cells in the presence of 5 ng of Stx2 and various amounts of antibody or antibody fragments (0.8 to 25 ng) was determined. The percent survival values are from at least two independent assays (triplicate wells) and represent the mean of these values. Overall, the majority of the observed differences in neutralization activities were not statistically significant when analyzed by one-way ANOVA.

FIG. 3.

Survival rates of antibody-treated groups against the lethal effects of Stx2 in the mouse toxicity model. Mice (10 per group) were treated with either four (5C12) or five (5H8) doses of IgG antibodies or one or two doses of Fab antibodies to evaluate their neutralization activities against Stx2. For the multiple-dose groups, the concentration of antibody was doubled. To simplify the survival data, all dose groups were combined for each antibody (left panels). The mean survival data for all groups are shown in the right panels. Detailed survival data are included in Tables 1 to 3. (A) r5C12 isotype variant groups and two r5C12 Fab groups. For the isotype variant groups, doubling the concentration from 0.08 to 1.2 μg/mouse (five doses) was tested. The Fab antibodies were tested at a single dose only (1.2 μg/mouse). PBS control group survival is indicated by a dotted line (3 days). (B) 5C12 IgG1 and 5C12 Fab groups (r5C12 and HuMAb). The IgG1 and Fab antibodies were tested at four doses, 0.15 to 1.2 μg/mouse and 0.6 to 5 μg/mouse, respectively. PBS control group survival is indicated by a dotted line (2 days). (C) 5H8 IgG1 and 5H8 Fab groups (r5H8 and HuMAb). The two IgGl antibodies were tested at five doses (0.6 to 10 μg/mouse) and the two Fab antibodies at two doses (5 and 10 μg/mouse). PBS control group survival is indicated by a dotted line (2.25 days).