(M1330-04-24) Understanding Mechanisms behind Low Conjugation Efficiencies of Thiol-Maleimide Reactions to Synthesize Antibody-Drug Conjugates

Purpose: Previously, our lab developed an anti-perlecan IgG antibody AM6 that was shown to be effective in recognizing triple-negative breast cancer (TNBC) cells.1 Encouraged by this observation, we investigated whether AM6 could be utilized as an antibody platform for creating an ADC. We determined the intracellular internalization dynamics of the AM6-based ADC and the effect of linker chemistry on intracellular drug release. Our initial studies with a clinically relevant cytotoxin MMAE did not yield ADCs with the expected drug-to-antibody ratio (DAR) under the reaction conditions that were optimized using cetuximab. We, therefore, investigated the thiolation reaction with AM6 antibody in greater detail and determined optimal conjugation conditions.

Methods: AM6 antibody and commercial cetuximab were conjugated to different payloads including doxorubicin, MMAE and AF647 via thiol maleimide conjugation following hinge disulfide reduction with tris(2-carboxyethyl) phosphine (TCEP) at different molar excess (1.15x, 2.3x, 3.45x, and 15x molar equivalent of antibody). The reduced antibody was then reacted with the payload via different linkers. The reaction mixture was purified with Zeba Spin columns. Each reaction sample was then analyzed with hydrophobic interaction chromatography utilizing the Tosoh Butyl NPR column. Internalization of doxorubicin-AM6 conjugate and the cytotoxicity of MMAE-AM6 conjugate was investigated in a model TNBC cell line MDA-MB-231-LM2.

Results: Interestingly, AM6 was characterized by quick cellular internalization compared to that with the benchmark anti-EGFR antibody cetuximab (Fig. 1a). Our initial studies utilizing doxorubicin and MMAE as model payloads suggested that AM6 ADC delivered the payload inside the cells and using lysosomal cleavable linkers facilitated the release of the payload (Fig. 1b and 1c). At low reductive concentrations (1.15, 2.3, 3.45 molar equivalent of TCEP), AM6 showed lower DAR (DAR=0.56, 1.96, 3.16) than benchmark cetuximab with the more hydrophobic vcMMAE (respective DARs for cetuximab=1.16, 2.55, 4.84) (Fig. 2). A similar result was also observed with hydrophilic mc-AF647 (Fig.3). However. with a high TCEP concentration (15 molar equivalents), both antibodies achieved DAR 8, suggesting all the interchain disulfides generated have reactivity towards maleimide.

Conclusion: These results suggest that AM6-based ADCs were effective in delivering the payload into the cells. While the thiols produced by interchain disulfide reduction of AM6 reacted with maleimide, at least some of the interchain disulfides of the AM6 may be difficult to access or reduce. We are currently utilizing mass spectrometry analysis to confirm the above results to further understand the potential issues, such as protein oxidation, that may contribute to the lower-than-expected DAR.

References: 1. Kalscheuer, Stephen, et al. "Discovery of HSPG2 (Perlecan) as a therapeutic target in triple negative breast cancer." Scientific reports 9.1 (2019): 12492.

Fig.1 AM6 shows quicker internalization and facilitates payload intracellular delivery and release. A) Antibody internalization imaging by live cell confocal microscopy, 3 different endocytic inhibitors are used to determine the internalization pathway. B) Doxorubicin ADC internalization and drug release imaging. Doxorubicin nucleus colocalization and amount by pixel volume is reported in the histogram. C) Cell viability test with AM6 and Ctx MMAE conjugates on MDA-MB-231-LM2 cells

Fig. 2 HIC analysis of vcMMAE ADC with AM6 and cetuximab using different TCEP concentrations.

Fig. 3 HIC analysis of Antibody-AF647 conjugate with the step-up conjugation.