Enhanced Activity of Monomethylauristatin F through Monoclonal Antibody Delivery:  Effects of Linker Technology on Efficacy and Toxicity

Abstract

We have previously shown that antibody-drug conjugates (ADCs) consisting of cAC10 (anti-CD30) linked to the antimitotic agent monomethylauristatin E (MMAE) lead to potent in vitro and in vivo activities against antigen positive tumor models. MMAF is a new antimitotic auristatin derivative with a charged C-terminal phenylalanine residue that attenuates its cytotoxic activity compared to its uncharged counterpart, MMAE, most likely due to impaired intracellular access. In vitro cytotoxicity studies indicated that mAb-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-MMAF (mAb-L1-MMAF) conjugates were >2200-fold more potent than free MMAF on a large panel of CD30 positive hematologic cell lines. As with cAC10-L1-MMAE, the corresponding MMAF ADC induced cures and regressions of established xenograft tumors at well tolerated doses. To further optimize the ADC, several new linkers were generated in which various components within the L1 linker were either altered or deleted. One of the most promising linkers contained a noncleavable maleimidocaproyl (L4) spacer between the drug and the mAb. cAC10-L4-MMAF was approximately as potent in vitro as cAC10-L1-MMAF against a large panel of cell lines and was equally potent in vivo. Importantly, cAC10-L4-MMAF was tolerated at >3 times the MTD of cAC10-L1-MMAF. LCMS studies indicated that drug released from cAC10-L4-MMAF was the cysteine-L4-MMAF adduct, which likely arises from mAb degradation within the lysosomes of target cells. This new linker technology appears to be ideally suited for drugs that are both relatively cell-impermeable and tolerant of substitution with amino acids. Thus, alterations of the linker have pronounced impacts on toxicity and lead to new ADCs with greatly improved therapeutic indices.

Influence of the Linker on the Biodistribution and Catabolism of Actinium-225 Self-Immolative Tumor-Targeted Isotope Generators

“…In particular, a RIC with a linker incorporating the sequence Gly-Phe-Gly showed little radioactivity associated with the intact antibody and most of the activity associated with the low molecular weight fragments in a gel filtration chromatography experiment after hepatic catabolism (42). Similar to our findings, a study was recently published that describes the unexpected activity of a control immunoconjugate not designed to be able to release an active drug (43). Drug release from this conjugate was shown to take place through mAb degradation.…”

Long-term Tumor Regression Induced by an Antibody–Drug Conjugate That Targets 5T4, an Oncofetal Antigen Expressed on Tumor-Initiating Cells

“…Sulfhydryl-based bioconjugation was conducted as described (20) with modifications. Antibody was pretreated with 3 equivalents of tris(2-carboxyethyl)phosphine (TCEP) to liberate the thiol residues, and this partially reduced material was exposed to approximately 6 equivalents of maleimidocaproyl-MMAF (mcMMAF).…”

“…Optical density was measured on a spectrophotometer. To assess the released payload cys-mcMMAF (20), quantitation was achieved using an UPLC-MS/MS system (5500 Qtrap, C18 column, and cys-mcMMAD as the internal standard) with a lower limit of quantitation ranging from 0.002 to 0.1 ng/mL in plasma and tumor, respectively. Toxicokinetic analysis was done using the pharmacokinetics module within Watson LIMS v7.4 (Thermo Scientific) using a standard noncompartmental model.…”

Preclinical Antitumor Efficacy of BAY 1129980—a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody–Drug Conjugate for the Treatment of Non–Small Cell Lung Cancer

“…Here, we report the development and preclinical evaluation of BAY 1129980 (C4.4A-ADC), a C4.4A-targeting human immunoglobulin G1 (hIgG1) antibody (C4.4A-Ab) conjugated via cysteine side chains and a noncleavable alkyl hydrazide linker to a novel, highly potent microtubule-disrupting auristatin W derivative (18)(19)(20). The C4.4A-ADC is efficacious both in vitro and in vivo in a variety of cell line and patient-derived xenograft (PDX) models representing various C4.4A-expressing cancers with high efficacy in NSCLC models.…”