Effects of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate

TLDR

An antibody‑drug conjugate of MMAE linked to the anti‑CD30 mAb cAC10, carrying eight drug molecules per antibody, has been shown to kill CD30⁺ malignant cells potently. The study aimed to evaluate how varying drug loading on cAC10 affects its therapeutic potential in vitro and in vivo. The authors generated ADCs with two, four, and eight MMAE molecules per antibody, purified them by hydrophobic interaction chromatography, and assessed their potency against CD30⁺ cell lines and in xenograft mouse models while determining maximum‑tolerated doses and pharmacokinetics. In vitro potency rose with drug loading (IC₅₀ E8 < E4 < E2), yet in vivo E4 achieved antitumor activity comparable to E8 at the same antibody dose while containing half the MMAE; E2 was active only at higher doses, and higher drug loading produced lower maximum‑tolerated doses and faster clearance, so reducing drug loading per antibody increased the therapeutic index.

Abstract

An antibody-drug conjugate consisting of monomethyl auristatin E (MMAE) conjugated to the anti-CD30 monoclonal antibody (mAb) cAC10, with eight drug moieties per mAb, was previously shown to have potent cytotoxic activity against CD30(+) malignant cells. To determine the effect of drug loading on antibody-drug conjugate therapeutic potential, we assessed cAC10 antibody-drug conjugates containing different drug-mAb ratios in vitro and in vivo.Coupling MMAE to the cysteines that comprise the interchain disulfides of cAC10 created an antibody-drug conjugate population, which was purified using hydrophobic interaction chromatography to yield antibody-drug conjugates with two, four, and eight drugs per antibody (E2, E4, and E8, respectively). Antibody-drug conjugate potency was tested in vitro against CD30(+) lines followed by in vivo xenograft models. The maximum-tolerated dose and pharmacokinetic profiles of the antibody-drug conjugates were investigated in mice.Although antibody-drug conjugate potency in vitro was directly dependent on drug loading (IC(50) values E8<E4<E2), the in vivo antitumor activity of E4 was comparable with E8 at equal mAb doses, although the E4 contained half the amount of MMAE per mAb. E2 was also an active antitumor agent but required higher doses. The maximum-tolerated dose of E2 in mice was at least double that of E4, which in turn was twice that of E8. MMAE loading affected plasma clearance, as E8 cleared 3-fold faster than E4 and 5-fold faster than E2.By decreasing drug loading per antibody, the therapeutic index was increased demonstrating that drug loading is a key design parameter for antibody-drug conjugates.