Purpose: 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.

Experimental Design: 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.

Results: Although antibody-drug conjugate potency in vitro was directly dependent on drug loading (IC₅₀ 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.

Conclusions: By decreasing drug loading per antibody, the therapeutic index was increased demonstrating that drug loading is a key design parameter for antibody-drug conjugates.