Initial characterization of an immunotoxin constructed from domains II and III of cholera exotoxin

Abstract

Immunotoxins are antibody-toxin fusion proteins under development as cancer therapeutics. In early clinical trials, immunotoxins constructed with domains II and III of Pseudomonas exotoxin (termed PE38), have produced a high rate of complete remissions in Hairy Cell Leukemia and objective responses in other malignancies. Cholera exotoxin (also known as cholix toxin) has a very similar three-dimensional structure to Pseudomonas exotoxin (PE) and when domains II and III of each are compared at the primary sequence level, they are 36% identical and 50% similar. Here we report on the construction and activity of an immunotoxin made with domains II and III of cholera exotoxin (here termed CET40). In cell viability assays, the CET40 immunotoxin was equipotent to tenfold less active compared to a PE-based immunotoxin made with the same single-chain Fv. A major limitation of toxin-based immunotoxins is the development of neutralizing antibodies to the toxin portion of the immunotoxin. Because of structure and sequence similarities, we evaluated a CET40 immunotoxin for the presence of PE-related epitopes. In western blots, three-of-three anti-PE antibody preparations failed to react with the CET40 immunotoxin. More importantly, in neutralization studies neither these antibodies nor those from patients with neutralizing titers to PE38, neutralized the CET40-immunotoxin. We propose that the use of modular components such as antibody Fvs and toxin domains will allow a greater flexibility in how these agents are designed and deployed including the sequential administration of a second immunotoxin after patients have developed neutralizing antibodies to the first.

Fig. 1

CET40 immunotoxin. a Shown is the DNA and protein sequence of the immunotoxin HB21-CET40. The initiating methionine is followed by the variable portion of the heavy chain, a glycine-serine linker, the variable portion of the light chain, a short connector sequence (including the HindIII site), and amino acids 270–634 of cholera exotoxin. b Sequence alignment via ‘ClustalX’ (2.09) analysis. Shown in descending order are amino acids 270–634 of cholix toxin [30], amino acids 270–634 of CET (corresponding to domains II and III from the exotoxin gene of Vibrio cholerae strain 1587), and finally domains II and III of exotoxin A, PE40. Key common features include the location of a furin cleavage site, an NAD binding glutamic acid, and a KDEL-like motif at the C-terminus. Coloring was per the default settings for ClustalX as defined by ‘colprot.xml’ (colprot.par). For alignment: “*” = fully conserved; “:” = conserved within a ‘strong’ group; and “.” = conserved within a ‘weak’ group

Fig. 2

Cytotoxicity of HB21-CET40 compared with HB21-PE40. Immunotoxin concentrations from 0.1 to 100 ng/ml were added to each of six cell lines for 48 h. DLD-1 (colon), A549 (lung), KB 3-1 (epidermoid), 293TT (kidney), Raji (B-cell), or HUT102 (T-cell) cells were used as representative cell lines of various common cancers. Cell viability was then determined using the WST-1 reagent. Error bars represent one standard deviation (SD) of five replicate wells per data point

Fig. 3

Immunotoxin specificity. a Excess HB21 antibody competes for killing activity on DLD1 cells. Cells were pretreated or not with the HB21 antibody (10 µg/ml) for 1 h at 37°C and then incubated with HB21-CET40 at 10 and 1 ng/ml. b Immunotoxin activity on a mouse cell line. HB21-CET40 or HB21-PE40 was added to L929 cells at concentrations from 0.1 to 100 ng/ml. Cell viability was assessed after 48 h using the WST-1 reagent. Error bars represent one SD of five replicate wells per data point

Fig. 4

Toxin reactivity via western blot analysis. a Western blot analysis of HB21-PE40 and HB21-CET40. Immunotoxins ~30 ng per lane were separated on a reducing 8–16% Gel and transferred to a PVDF membrane. Immunotoxins and a lane with molecular weight (MW) markers were each probed with one of three anti-PE antibodies (from left to right: M40-1, rabbit anti-PE from Sigma–Aldrich and rabbit anti-PE raised at NCI. b Western blot analysis of CET and PE probed with anti-CET40 antibodies. CET or PE at 30 and 3 ng per lane was probed with a rabbit anti-HB21-CET40 antibody preparation

Fig. 5

a, b Neutralization activity of anti-PE antibody preparations. Antibody preparations were mixed with twice the desired final immunotoxin concentration for 1 h at room temp. At the end of the incubation the mixture was added to each well of DLD-1 cells in a 96-well format. After a 48-h incubation, cell viability was assessed using the WST-1 reagent. Each bar represents a replicate of five with the error bar indicating one SD. Comparisons of immunotoxin activity with and without anti-PE antibodies are indicated with thin black lines. Each experiment was conducted independently at least twice per antibody preparation

Fig. 6

Neutralizing activity of pre and post-treatment sera from four individual patients treated with a PE38-immunotoxin. Patient sera (at 1:100) were mixed with either 5 or 1 ng/ml of immunotoxin for 1 h at room temp. a Reports activity with HB21-PE40 and b with HB21-CET40. At the end of the incubation the mixture was added to DLD-1 cells in a 96-well format. After a 48-h incubation, cell viability was assessed using the WST-1 reagent. Each bar represents a replicate of five with the error bar indicating one SD. Direct comparisons of pre- and post-treatment serum samples are indicated with thin black lines. Each experiment was conducted independently twice per patient sample