Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment

Abstract

The chimeric anti-CD30 IgG1, cAC10, conjugated to eight equivalents of monomethyl auristatin E (MMAE) was previously shown to have potent antitumor activity against CD30-expressing tumors xenografts in mice. Moreover, the therapeutic index was increased by lowering the stoichiometry from 8 drugs/antibody down to 2 or 4. Limitations of such 'partially-loaded' conjugates are low yield (10-30%) as they are purified from mixtures with variable stoichiometry (0-8 drugs/antibody), and heterogeneity as the 2 or 4 drugs are distributed over eight possible cysteine conjugation sites. Here, the solvent-accessible cysteines that form the interchain disulfide bonds in cAC10 were replaced with serine, to reduce the eight potential conjugation sites down to 4 or 2. These Cys-->Ser antibody variants were conjugated to MMAE in near quantitative yield (89-96%) with defined stoichiometries (2 or 4 drugs/antibody) and sites of drug attachment. The engineered antibody-drug conjugates have comparable antigen-binding affinities and in vitro cytotoxic activities with corresponding purified parental antibody-drug conjugates. Additionally, the engineered and parental antibody-drug conjugates have similar in vivo properties including antitumor activity, pharmacokinetics and maximum tolerated dose. Our strategy for generating antibody-drug conjugates with defined sites and stoichiometries of drug loading is potentially broadly applicable to other antibodies as it involves engineering of constant domains.

Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

“…The ADCs contained only low levels of aggregates (V2.5%) as assessed by analytical size-exclusion chromatography (Table 1). Competition binding experiments with 786-O cells and fluorescently labeled anti-CD70 IgG1 revealed that drug conjugation did not impair binding by any of the IgG variants (Table 1), as reported for other ADCs (13). This presumably reflects that the solvent accessible cysteine residues used for drug conjugation are distant from the antigen-binding residues in the variable domains.…”

“…The anti-CD70 IgG variants were purified by protein A chromatography using an Ä KTAexplorer FPLC (GE Healthcare) as described (13) with modifications provided in Supplementary Materials. 1 Purified antibodies were analyzed by SDS-PAGE and TSK-Gel G3000SW highperformance liquid chromatography size exclusion chromatography (Tosoh Bioscience).…”

“…Conjugates were diluted 10-fold with 20 mmol/L sodium acetate (pH 5.0) and bound to the membrane, washed with additional sodium acetate buffer, and eluted with 3Â PBS. Drug loading was determined by reversephase high-performance liquid chromatography under reducing conditions (13,14) and by hydrophobic interaction chromatography (15).…”

Brentuximab Vedotin (SGN-35) for Relapsed CD30-Positive Lymphomas

“…In vitro, the drug was found to be potent and selective against CD30-positive tumor-cell lines, and activity was observed in models of Hodgkin's lymphoma and ALCL in mice with severe combined immunodeficiency. [18][19][20] To assess the safety and clinical activity of brentuximab vedotin, we treated patients with relapsed or refractory CD30-positive hematologic cancers in a phase 1, open-label, dose-escalation trial. Furthermore, because serum levels of TARC have been shown to correlate with disease activity in patients with Hodgkin's lymphoma, 21, 22 we evaluated serum levels of TARC and various cytokines in patients in the expansion phase of the study.…”

Protein Engineering with Non-Natural Amino Acids

“…The therapeutic index will be highest for a molecule with the ideal number of drugs attached, for example, the maximum tolerated dose and clearance rate improved by 4-5 fold when comparing ADCs containing exactly 2 auristatin analogs versus 8 (Hamblett et al, 2004). One approach to limit product heterogeneity is to replace some of the natural cysteines with serines (McDonagh et al, 2006). Engineering in new Cys residues has also allowed more control over the degree of conjugation, but the reaction product still carries a distribution of species (Junutula et al, 2008).…”