Abstract
Antibody drug conjugates (ADCs) are no longer an unknown entity in the field of cancer therapy with the success of marketed ADCs like ADCETRIS and KADCYLA and numerous others advancing through clinical trials. The pursuit of novel cytotoxic payloads beyond the mictotubule inhibitors and DNA damaging agents has led us to the recent discovery of an mRNA splicing inhibitor, thailanstatin, as a potent ADC payload. In our previous work, we observed that the potency of this payload was uniquely tied to the method of conjugation, with lysine conjugates showing much superior potency as compared to cysteine conjugates. However, the ADC field is rapidly shifting towards site-specific ADCs due to their advantages in manufacturability, characterization and safety. In this work we report the identification of a highly efficacious site-specific thailanstatin ADC. The site of conjugation played a critical role on both the in vitro and in vivo potency of these ADCs. During the course of this study, we developed a novel methodology of loading a single site with multiple payloads using an in situ generated multi-drug carrying peptidic linker that allowed us to rapidly screen for optimal conjugation sites. Using this methodology, we were able to identify a double-cysteine mutant ADC delivering four-loaded thailanstatin that was very efficacious in a gastric cancer xenograft model at 3mg/kg and was also shown to be efficacious against T-DM1 resistant and MDR1 overexpressing tumor cell lines.
Highlights
Antibody-drug conjugates have proven to be a promising new area of cancer therapy with over 50 Antibody drug conjugates (ADCs) in clinical trials and numerous more in the pre-clinical pipeline [1, 2]
In this study we describe the identification of a highly efficacious, site-specific thailanstatin trastuzumab ADC with a unique cytotoxic mechanism of action
Screening for an effective site of conjugation on the antibody was challenging as it defied our prior experience with tubulysin and auristatin payloads, where site of conjugation did not significantly impact the in vitro cytotoxicity of the ADC
Summary
Preparation of hinge-cysteine ADCCommercially available trastuzumab (Genentech Inc) was dialyzed into Dulbecco’s Phosphate Buffered Saline (DPBS, Lonza). Conjugation was performed in 50 mM borate buffer, pH 8.5 by addition of 11 equiv of iodoacetamide thailanstatin payload (1–3) [20] that was dissolved as a 10 mM solution in dimethylacetamide (DMA). The conjugation was performed at 10 mg/mL for 16 h at room temperature. The reaction mixture was buffer exchanged into DPBS Crude material was purified by size exclusion chromatography (SEC) using a GE AKTA Explorer system with a GE Superdex 200 column and DPBS The resulting ADCs were filtered through sterile 0.22 μm Ultrafree-MC Centrifugal Filter Units (Millipore, Billerica, MA) and concentration was measured spectrophotometrically. The ADCs were further characterized via size exclusion chromatography (SEC) for purity and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS) to calculate drug antibody ratio (loading) and confirm mass shift
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