Efficient Elimination of B-Lineage Lymphomas by Anti-CD20–Auristatin Conjugates

Abstract

The anti-CD20 antibody rituximab is useful in the treatment of certain B-cell malignancies, most notably nonHodgkin's lymphoma. Its efficacy has been increased when used in combination with chemotherapy, yet anti-CD20 monoclonal antibodies (mAbs) directly conjugated with drugs such as doxorubicin (Dox) have failed to deliver drug or to demonstrate antitumor activity. We have produced anti-CD20 antibody-drug conjugates that possess potent antitumor activity by using the anti-mitotic agent, monomethyl auristatin E (MMAE), linked via the lysosomally cleavable dipeptide, valine-citrulline (vc). Two anti-CD20 conjugates, rituximab-vcMMAE and 1F5-vcMMAE, were selectively cytotoxic against CD20؉ B-lymphoma cell lines, with IC 50 values ranging from 50 ng/mL to 1 g/mL. Unlike rituximab, which showed diffuse surface localization, rituximabvcMMAE capped and was internalized within 4 hours after binding to CD20 ؉ B cells. Internalization of rituximabvcMMAE was followed by rapid G 2 -M phase arrest and onset of apoptosis. Anti-CD20 antibody-drug conjugates prepared with Dox were internalized and localized as with rituximab-vcMMAE, yet these were not effective for drug delivery (IC 50 > 50 g/mL). Consistent with in vitro activity, rituximab-vcMMAE showed antitumor efficacy in xenograft models of CD20-positive lymphoma at doses where rituximab or rituximab-Dox conjugates were ineffective. These data indicate that anti-CD20 -based antibody-drug conjugates are effective antitumor agents when prepared with a stable, enzyme-cleavable peptide linkage to highly potent cytotoxic agents such as MMAE.

Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97

“…In contrast, conjugation of L49 to derivatives of auristatin, such as MMAE, MMAF, and AFP, endows this antibody with ability to internalize efficiently. Similar observations have previously been made for the anti-CD20 antibodies rituximab and 1F5, following conjugation to auristatins (33), and for the anti-HER2 antibody trastuzumab, on conjugation to the drug geldanamycin (34).…”

Preclinical studies of a Pro-antibody-drug conjugate designed to selectively target EGFR-overexpressing tumors with improved therapeutic efficacy

“…47 A431 cells were treated with the saturating concentration of activated PanP-DM1 conjugate at 4 C for 30 minutes. Unbound conjugate was removed by washing cells.…”

Antibody Drug Conjugates: Preclinical Considerations

“…It is also worth noting that the efficiency of internalization may vary between an unmodified mAb and an ADC (22). Experimental studies suggest that in some cases, both an ADC and an unconjugated mAb internalize at the same rate, and in other cases, the ADC may internalize with more rapid kinetics (23,24). Thus, in the context of a drug discovery program, profiling the internalization rate of both the unmodified mAb and subsequent confirmation with the ADC is paramount.…”