Metabolism of an Oxime-Linked Antibody Drug Conjugate, AGS62P1, and Characterization of Its Identified Metabolite

Abstract

AGS62P1 is an antibody drug conjugate (ADC) composed of a human IgG1κ monoclonal antibody against FLT3 (FMS-like tyrosine kinase 3) with a p-acetyl phenylalanine (pAF) residue inserted at position 124 of each heavy chain linked to the proprietary microtubule disrupting agent AGL-0182-30 via an alkoxyamine linker that forms an oxime upon conjugation to the antibody. AGS62P1 is currently in Phase I human clinical trials for acute myelogenous leukemia (AML). The identified primary metabolite of an oxime-linked ADC is presented for the first time. AGS62P1 metabolism was assessed in xenograft tumor-bearing mice and rats treated with the ADC using liquid chromatography and mass spectrometry-based methods described herein. In this study, we identified the metabolite of AGS62P1 as pAF-AGL-0185-30, which contains a fragment resulting from the catabolism of the antibody component of the ADC and hydrolysis of the terminal amide portion of the linker-payload. We demonstrated that the metabolite of AGS62P1 is tolerated in rats above 1.5 mg/kg and above 0.334 mg/kg in cynomolgus monkeys when given as a single dose. Furthermore, we established in vitro that pAF-AGL-0185-30 does not significantly inhibit hERG or cytochrome P450 family enzymes (CYPs).

Antibody–Drug Conjugate Payloads; Study of Auristatin Derivatives

“…First, we created a set of auristatin compounds containing heteroatoms in the central core amino acids and evaluated them to determine whether heteroatom introduction could be an effective approach to control cytotoxic activity and serve as a handle for new linker attachment at P2 or P4 instead of at P1 or P5. 18,19) 3.2.…”

“…The in vitro potency of each compound with respective P1, P2, and P4 unit modifications were summarized 19) (Fig. 12).…”

“…We revealed that introduction of hydrophilic character in the P5 subunit can control auristatin properties and generate an efficacious ADC 17) and the introduction of heteroatoms, especially azide in central core, can modulate or enhance cytotoxic activity while introducing a P2 or P4 linker attachment site (instead of the more common P1 or P5 linkage approaches) in the preparation of ADCs. 18,19) Through study of P5 modified analogues, the in vitro cytotoxicity screen showed that some weakly basic aromatic side-chain groups were tolerated with minimal loss of activity. Pyridines and anilines retained activity but the more strongly basic and aliphatic groups such as 2-and 4-imidazoles had diminished cytotoxic activity.…”

“…11,16) Thus auristatins are attractive toxic compounds on ADCs and we also have investigated the structure-activity relationship (SAR) and auristatins ADCs to create the next generation ADCs. [17][18][19][20] In this review, our recent research regarding auristatin analogues is described. The first section discusses N-terminal (P1) and C-terminal (P5) modifications and corresponding ADC data.…”

“…17) The second section discusses central peptide (P2-P4) modifications, focusing on P2 and P4 SAR. [18][19][20]…”