Mutagenic Deimmunization of Diphtheria Toxin for Use in Biologic Drug Development

Abstract

Background: Targeted toxins require multiple treatments and therefore must be deimmunized. We report a method of protein deimmunization based on the point mutation of highly hydrophilic R, K, D, E, and Q amino acids on the molecular surface of truncated diphtheria-toxin (DT390).

Methods: Based on their surface position derived from an X-ray-crystallographic model, residues were chosen for point mutation that were located in prominent positions on the molecular surface and away from the catalytic site. Mice were immunized with a targeted toxin containing either a mutated DT390 containing seven critical point mutations or the non-mutated parental toxin form.

Results: Serum analysis revealed a significant 90% reduction in anti-toxin antibodies in mice immunized with the mutant, but not the parental drug form despite multiple immunizations. The experiment was repeated in a second strain of mice with a different MHC-haplotype to address whether point mutation removed T or B cell epitopes. Findings were identical indicating that B cell epitopes were eliminated from DT. The mutant drug form lost only minimal activity in vitro as well as in vivo.

Conclusion: These findings indicate that this method may be effective for deimmunizing of other proteins and that discovery of a deimmunized form of DT may lead to the development of more effective targeted toxin.

Keywords: biologic drug; cancer; cancer treatment; deimmunization; diphtheria; toxin.

Figure 1

Construction of the plasmid containing the dDTEGF13 gene. (A) The pET expression vector containing the dDTEGF13 target gene; (B,C) The PyMol sphere graphic was generated by downloading the Protein Data Bank [19] X-ray crystallographic structure of DT [20] into the PyMol 3D molecular modeling program [21] Shown is a frontal view of the protein and a 180° reverse view of the molecule; In (B), the amino acids associated with ADP-ribosylation (catalytic site) are bl ackened; In (C), The amino acids that were mutated for deimmunization are blackened; (D) SDS-PAGE gel analysis was performed to confirm the size and purity and stained with Coomasie blue. Photo is grayscale. Lane 1—Molecular weight standards, Lane 2—dDTEGF13 non-reduced, Lane 3—dDTEGF13 reduced. The gel was stained using Coomassie blue; (E) A HPLC trace for the purified drug is also shown illustrating mostly a single peak obtained from a TSK3000 size exclusion column. Only the single peak was collected resulting in a >95% purity.

Figure 2

Screening Chart. Chart shows our strategy of producing and screening various DTEGF13 mutants on the path to deimmunization. In phase 1, 8 triple mutants were synthesized and purified. Four of these showed less than a log of activity loss in our in vitro screening assay compared to the non-mutated parental control. In phase 2, mutations were combined to generate a quintuple mutant that still had less than a log of activity loss compared to parental. These are marked with an asterisk (*) in the figure. In phase 3, we analyzed a septuplet mutant that still had less than a log of activity loss. Glycine, alanine, and serine substitution were employed.

Figure 3

In vitro activity of dDTEGF13: (A) Bispecific DTEGF13 and its monospecific counterparts were tested and compared for their reactivity against MiaPaCa-2 cells. Proliferation assays were performed by analyzing ³H-thymidine uptake after a 72-h incubation with targeted toxins. Data are reported as percent control response. Each data point represents an average of triplicate measures ± SD; (B) Deimmunized DTEGF13 and non-mutated parental DTEGF13 were tested and compared for activity against HT-29 colon; and (C) PC-3 prostate carcinoma cells in thymidine uptake assays. Two batches are used for reproducibility; (D) A blocking assay was performed in which MiaPaca-2 pancreatic cancer cell lines were incubated with an inhibitory dose of dDTEGF13 and then blocked with increasing concentration of EGF13 ligand devoid of toxin. Thymidine uptake was then measured. The non-specific recombinant α-Ly5.2 was included as a negative blocking control.

Figure 4

In vitro activity of dDTEGF13 against negative control Raji cells. To study the selectivity of dDTEGF13, EGFR⁻IL-13⁻ Raji cells (human Burkitts’ lymphoma cell line) were studied in a proliferation assay based on ³H thymidine uptake. No inhibition of proliferation was seen with DTEGF13, dDTEGF13, DTIL13, DTEGF and DTCD3CD3. However, inhibition did occur with control DT2219ARL because DT2219 consists of DT spliced to anti-CD19 and anti-CD22scFvs. Raji expresses both CD19 and CD22. These data indicate activity is mediated through the selective ligand binding.

Figure 5

The immunogenicity of dDTEGF13. The immune response to deimmunized and non-mutated parental drug was determined by measuring anti-DT390 serum IgG on weekly samples of mice immunized with 0.25 µg of DTEGF13 (n = 5) or dDTEGF13 7 (n = 5). Measurements were made using an indirect ELISA and quantification of antibodies was determined using a standard curve generated with highly purified, high titer anti-DT antibody. Response was determined in BALB/c mice (* = P < 0.03) (A) and C57BL/6 mice (* = P < 0.01; # = P < 0.05) (B).

Figure 6

Neutralizing antibodies. Serum was collected from mice immunized with multiple injections of (A) parental DTEGF13 or (B) dDTEGF13 on day 56. Serum from individual mice was incubated with MiaPaCa-2 cells treated with a known inhibitory concentration of DTEGF13 in order to test for neutralization. Proliferation assays were performed by measuring tritiated thymidine uptake after 72 h. The serum concentration of IgG anti-toxin that was measured for each serum on day 56 is shown (μg/mL). There is a correlation between serum levels and the presence of neutralizing antibody.

Figure 7

The effect of treatment of established PC-3 flank tumors with dDTEGF13. (A) Nude mice bearing PC-3 flank tumors were treated intratumorally with dDTEGF13, control DT2219ARL, or untreated. Tumors were treated with 8 injections of dDTEGF13. Average tumor volumes are shown for each treatment group (* = P < 0.004; # = P < 0.05); (B) Individual tumor volumes are shown for both the irrelevant control BLT treated group (DT2219ARL) and the untreated mice. The growth of individual tumors is plotted over time.