Abstract
Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets. However, direct inhibition of oncogenic Ras mutants with small molecules has been extremely challenging. Here we report the development of a human IgG1 format antibody, RT11, which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of various oncogenic Ras mutants to block the interactions with effector proteins, thereby suppressing downstream signalling and exerting anti-proliferative effects in a variety of tumour cells harbouring oncogenic Ras mutants. When systemically administered, an RT11 variant with an additional tumour-associated integrin binding moiety for tumour tissue targeting significantly inhibits the in vivo growth of oncogenic Ras-mutated tumour xenografts in mice, but not wild-type Ras-harbouring tumours. Our results demonstrate the feasibility of developing therapeutic antibodies for direct targeting of cytosolic proteins that are inaccessible using current antibody technology.
Highlights
Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets
Direct inhibition of oncogenic Ras mutants has proven extremely challenging and no agents have been clinically approved to date; this is mainly due to difficulties in identifying druggable pockets for small molecule binding on the surface of Ras[13]
In the intact human IgG1 form, this can reach the cytosol of living cells after internalization through clathrin-mediated endocytosis using cell surface-expressed heparan sulfate proteoglycan (HSPG) as a receptor, and subsequent endosomal escape from early endosomes into the cytosol[22,23]
Summary
Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets. Intracellularly expressed antibody fragments (intrabodies) were developed that selectively bind to the active Ras Á GTP form to block the PPIs with effector proteins, thereby inhibiting tumorigenesis and metastasis in mouse models[20,21]. This suggests that blocking intracellular Ras Á GTP–effector PPIs using a conventional antibody regimen such as systemic administration could be an effective approach to inhibit oncogenic Ras-driven signalling. We describe the generation and therapeutic efficacy of a human IgG1 format antibody, named iMab (internalizing and PPI interfering monoclonal antibody), which directly targets the intracellularly activated GTP-bound form of oncogenic Ras mutants after internalization into the cytosol of living cells. iMab binds to the PPI interfaces between activated Ras and effector proteins to block these associations, thereby inhibiting downstream oncogenic signalling and exerting antitumour effects in mouse xenograft models when systemically administered
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