Improving antibody internalization with proteolysis targeting chimeras

Advances in antibody engineering have already impacted clinical practice with regulatory approvals of antibody-drug conjugates, bispecific antibodies and bispecific T cell engaging antibodies. Many novel antibody formats, antibody targets and payloads are in various stages of development and may also impact clinical care. Antibody drug conjugates are perhaps the most common antibody format currently entering clinical trials in oncology [1].
In prior work led by Fiona Simpson, it was shown that prochlorperazine, an antiemetic, could reduce endocytosis of membrane proteins [2]. Prochlorperazine was then shown to improve natural killer cell-mediated antibody-dependent cellular cytotoxicity by delaying the endocytosis of membrane proteins that were targeted with IgG1 antibodies.
We were very intrigued by a corollary of these findings in light of the significant development of antibody-drug conjugates. As antibody-drug conjugates primarily rely on internalization to release their cytotoxic payloads in the acidic environment of the lysosome, we wondered whether the activity of antibody-drug conjugates could be improved by increasing internalization. Instead of delaying internalization to improve natural killer cell-mediated antibody-dependent cellular cytotoxicity, we sought to enhance it to improve release of the cytotoxic conjugates.
In an ongoing project with my collaborator Amarnath Natarajan, we developed a library of proteolysis targeting chimeras (PROTACs) based on the small molecule capmatinib to degrade the cell surface oncogene MET [3]. As we had seen that these PROTACs could degrade the membrane protein MET, in a mechanism dependent on the proteasome, we thought that PROTACs that target the same oncogenic cell surface proteins as antibodies could potentially internalize these antibodies. We decided to investigate EGFR, HER2 and MET as we already had cell lines with known expression of these oncogenes, antibodies have been approved by the FDA to target these proteins, and PROTACs had been developed by others and ourselves against these proteins.
In short, we found that PROTACs improve internalization of antibodies and their derivative antibody-drug conjugates when both agents target the same oncogenic cell surface proteins. As PROTACs are likely to enter clinical practice, it is conceivable that they could be combined with antibody-drug conjugates with the appropriate supporting preclinical data.
Although not the focus of our publication, we learned that the binding of an antibody to its target does not guarantee its internalization, and the rates of internalization vary across cell lines. Based on what membrane protein is being targeted, and in which disease setting, an understanding of the rates of target internalization, and the subsequent impact on antibody-dependent cellular cytotoxicity or payload release could rationally guide antibody design efforts.
References:
1. Tsuchikama, K., Anami, Y., Ha, S.Y.Y. et al. Exploring the next generation of antibody–drug conjugates. Nat Rev Clin Oncol 21, 203–223 (2024).
2. Chew, H., De Lima, J., Gonzalez Cruz, J., et al. Endocytosis Inhibition in Humans to Improve Responses to ADCC-Mediating Antibodies. Cell Volume 180, Issue 5, 895-914 (2020).
3. Mallareddy JR, Yang L, Lin WH et al. Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders. bioRxiv [Preprint]. 2024 Nov 25:2024.11.22.624922.
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