Multiple event activation of a generic prodrug trigger by antibody catalysis

Abstract

Chemotherapeutic regimes are typically limited by nonspecific toxicity. To address this problem we have developed a broadly applicable drug-masking chemistry that operates in conjunction with a unique broad-scope catalytic antibody. This masking chemistry is applicable to a wide range of drugs because it is compatible with virtually any heteroatom. We demonstrate that generic drug-masking groups may be selectively removed by sequential retro-aldol-retro-Michael reactions catalyzed by antibody 38C2. This reaction cascade is not catalyzed by any known natural enzyme. Application of this masking chemistry to the anticancer drugs doxorubicin and camptothecin produced prodrugs with substantially reduced toxicity. These prodrugs are selectively unmasked by the catalytic antibody when it is applied at therapeutically relevant concentrations. We have demonstrated the efficacy of this approach by using human colon and prostate cancer cell lines. The antibody demonstrated a long in vivo half-life after administration to mice. Based on these findings, we believe that the system described here has the potential to become a key tool in selective chemotherapeutic strategies.

Figure 1

Prodrug activation via a tandem retro-aldol–retro-Michael reaction. X stands for heteroatoms N, O, or S.

Figure 2

Synthesis of generic linkers 3 and 4, which can be used to mask functionally active amine and hydroxyl groups of drugs, respectively.

Figure 3

Doxorubicin prodrug activation via a tandem retro-aldol–retro-Michael reaction catalyzed by antibody 38C2.

Figure 4

Growth inhibition of LIM1215 human colon carcinoma cells in vitro by doxorubicin (■) and prodoxorubicin (□)(Bars indicate SD; n = 4). The data were determined by using the assay described in Fig. 5 and are summarized in Table 1. The dashed line indicates 50% decrease in the cell density as compared with the untreated control. Note the reduced capacity of prodoxorubicin for cell growth inhibition.

Figure 5

Growth inhibition of LIM1215 human colon carcinoma cells by prodoxorubicin in the presence of antibody 38C2. (A) Growth inhibition assay. Cells in quadruplicate wells in a 96-well plate were lysed 120 hr after drug addition, and the activity of the cytoplasmic enzyme lactate dehydrogenase released from the cells was detected by using a colorimetric assay. The intensity of the red color correlates with the number of cells in the well. (B) Growth inhibition in the presence of two fixed concentrations of prodoxorubicin and increasing concentrations of antibody 38C2. Controls are shown in black columns. dox, doxorubicin; prodox, prodoxorubicin; MVK, methyl vinyl ketone. Bars indicate SD; n = 4.

Figure 6

Camptothecin prodrug activation via a tandem retro-aldol–retro-Michael reaction catalyzed by antibody 38C2 followed by a spontaneous lactamization.

Figure 7

Growth inhibition of HT29 human colon carcinoma cells by procamptothecin in the presence of increasing concentrations of antibody 38C2. (▴) untreated control; (□), 1 μM procamptothecin; (■), 1 μM camptothecin. Bars indicate SD; n = 4.

Figure 8

In vivo activity of antibody 38C2. Mice were injected with 100 μl of 15 mg/ml 38C2 IgG in PBS on day 0. The concentration of 38C2 IgG in mouse sera was studied as a function of time after the injection. Activity was calculated based on the antibody 38C2-catalyzed conversion of the fluorogenic aldol sensor methodol into fluorescent 6-methoxy-2-naphtaldehyde (11). Typical data derived from one mouse are shown. The initial 38C2 IgG concentration on day 0 can be estimated to be about 6 μM based on a blood volume of 1.5 ml and 1.5 mg of injected antibody. Catalysis was not detectable in sera from mice injected with 100 μl of 15 mg/ml control antibody in PBS.