Abstract 1570: PermaLink®, a stable and scalable bioconjugation platform as an alternative to maleimide-based conjugation

Abstract IKSUDA Therapeutics’ proprietary PermaLink® platform comprises simple, stable, and adaptable conjugation chemistry that overcomes the instabilities associated with the retro-Michael exchange of traditional maleimide-based chemistry. This platform enables ADC generation with a range of drug-to-antibody ratios (DAR) and can be applied to multiple payloads and linker chemistries to improve conjugate properties and maximize the therapeutic index of ADC therapeutics. ADCs are generated by reducing the hinge region disulfides and/or decapping engineered cysteines with TCEP, conjugation with PL-payload followed by purification. DAR was measured by HPLC. The stability of PL-based ADCs was compared to traditional maleimide-based ADCs in murine and human plasma. In vitro activity and bystander effect were assessed using mono or co-culture experiments by CellTiter-Glo assay. In vivo efficacy studies were conducted in SCID mice with human cancer xenografts. Toxicology and PK studies were conducted in cynomolgus monkeys. PL-based conjugation followed a simple process of reducing the antibody disulfides with TCEP, conjugation with PL-payload and removal of excess linker toxin by size exclusion chromatography, desalting or filtration. Control of process parameters such as TCEP and linker-payload excesses yielded conjugates with DARs ranging from 4-8 for native wildtype antibodies. Additionally, DAR 10 was achieved for an antibody with two engineered cysteines. The resulting ADCs demonstrated favorable biophysical properties with minimal aggregation by SEC and DAR assessment was consistent by PLRP-HPLC and MS. Typical yields at the research scale ranged from 70-90%. For preclinical proof-of-concept, MMAE conjugates of Isumab01, targeting FRA, were prepared with a protease-cleavable valine-citrulline (vc) and enzymatically cleavable β-glucuronide (glu) linker. Both PL-based conjugates showed excellent stability in murine and human plasma, whereas FDA-approved maleimide-based ADCs brentuximab vedotin and trastuzumab deruxtecan displayed a significant decrease in DAR. PL-MMAE conjugates were highly potent against JEG-3 cancer cells in vitro, with IC50 values in the pM range. Isumab01-PL-glu-MMAE resulted in complete tumor regression in a JEG-3 xenograft model following a single dose of 2.5 mg/kg and was very well tolerated in cynomolgus monkeys at 12 mg/kg single dose and 10 mg/kg Q3W x2 (highest doses tested), showing improved tolerability over MMAE-based ADCs with maleimide-cathepsin B cleavable linkers. The Isumab01-PL-glu-MMAE conjugate demonstrates a favorable PK profile in monkeys with high stability in circulation consistent with improved bioconjugation. PermaLink offers a simple, stable alternative to maleimide-based ADC bioconjugation moieties and confers a favorable preclinical anti-tumor activity and safety profile Citation Format: Adam Lodge, Justyna Mysliwy, Will A. Stanley, James Stephenson, Sarah Robinson, Alexander Sanders, Robert Murawski, Bryony Moss, Patrick Higgs, Jutta Deckert, Jenny Thirlway, Robert J. Lutz. PermaLink®, a stable and scalable bioconjugation platform as an alternative to maleimide-based conjugation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1570.