Oncogenic RAS mutant (RAS^MUT) proteins have been considered undruggable via conventional antibody regimens owing to the intracellular location restricting conventional-antibody accessibility. Here, we report a pan-RAS–targeting IgG antibody, inRas37, which directly targets the intracellularly activated form of various RAS^MUT subtypes after tumor cell–specific internalization into the cytosol to block the interactions with effector proteins, thereby suppressing the downstream signaling. Systemic administration of inRas37 exerted a potent antitumor activity in a subset of RAS^MUT tumor xenografts in mice, but little efficacy in RAS^MUT tumors with concurrent downstream PI3K mutations, which were overcome by combination with a PI3K inhibitor. The YAP1 protein was up-regulated as an adaptive resistance-inducing response to inRas37 in RAS^MUT-dependent colorectal tumors; accordingly, a combination of inRas37 with a YAP1 inhibitor manifested synergistic antitumor effects in vitro and in vivo. Our study offers a promising pan-RAS–targeting antibody and the corresponding therapeutic strategy against RAS^MUT tumors.

Immunotoxins (ITs), which are toxin-fused tumor antigen-specific antibody chimeric proteins, have been developed to selectively kill targeted cancer cells. The epidermal growth factor receptor (EGFR) is an attractive target for the development of anti-EGFR ITs against solid tumors due to its overexpression on the cell surface of various solid tumors. However, the low basal level expression of EGFR in normal tissue cells can cause undesirable on-target/off-tumor toxicity and reduce the therapeutic window of anti-EGFR ITs. Here, based on an anti-EGFR monobody with cross-reactivity to both human and murine EGFR, we developed a strategy to tailor the anti-EGFR affinity of the monobody-based ITs carrying a 24-kDa fragment of Pseudomonas exotoxin A (PE24), termed ER-PE24, to distinguish tumors that overexpress EGFR from normal tissues. Five variants of ER-PE24 were generated with different EGFR affinities (K_D ≈ 0.24 nM to 104 nM), showing comparable binding activity for both human and murine EGFR. ER/0.2-PE24 with the highest affinity (K_D ≈ 0.24 nM) exhibited a narrow therapeutic window of 19 pM to 93 pM, whereas ER/21-PE24 with an intermediate affinity (K_D ≈ 21 nM) showed a much broader therapeutic window of 73 pM to 1.5 nM in in vitro cytotoxic assays using tumor model cell lines. In EGFR-overexpressing tumor xenograft mouse models, the maximum tolerated dose (MTD) of intravenous injection of ER/21-PE24 was found to be 0.4 mg/kg, which was fourfold higher than the MTD (0.1 mg/kg) of ER/0.2-PE24. Our study provides a strategy for the development of IT targeting tumor overexpressed antigens with basal expression in broad normal tissues by tailoring tumor antigen affinities.

Oncogenic Ras mutants, and most frequently KRas mutants (86% of Ras-driven cancers), are found in approximately 25% of human cancers and are high-priority anticancer drug targets. Despite 30 years of effort to develop drugs that directly target oncogenic Ras mutants, no effective pharmacologic inhibitors for these mutants are clinically available, mainly because of the lack of suitable surface binding pockets for small molecules. More than 50 therapeutic antibodies have been clinically approved against many extracellular proteins. However, such antibodies do not have the capacity to localize in intracellular cytosolic regions after receptor-mediated endocytosis, restricting their therapeutic application for targeting cytosolic proteins. Our group recently developed a platform technology of cytosol-penetrating antibody, which in the IgG format can reach the cytosolic space of living cells owing to its endosomal escaping ability after receptor-mediated endocytosis. Exploiting the cytosol-penetrating antibody technology, we have engineered a human IgG1 format antibody, named iMab (internalizing and protein-protein interaction [PPI] interfering monoclonal antibody), which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of oncogenic Ras mutants. iMab specifically binds to the PPI interfaces of activated Ras with effector proteins to block the associations, thereby inhibiting the Ras downstream oncogenic signaling and exerting antiproliferation effects on oncogenic Ras mutant tumor cells. For in vivo antitumor efficacy assessment, we further engineer iMab to have tumor tissue-homing ability by fusion of tumor-associated integrin αvβ3/αvβ5 binding cyclic peptide to the N-terminus of light chain. When systemically administered, the iMab variant significantly inhibited the in vivo growth of oncogenic Ras-mutated tumor xenografts in mice, but not wild-type Ras-harboring tumors. Our results demonstrate the feasibility of developing antibody therapeutics that directly target cytosolic proteins involved in disease-associated PPIs, such as oncogenic Ras mutants, by systemic administration, similar to conventional therapeutic antibody regimens. Because the oncogenic Ras targeting antibody holds many desirable features of the conventional IgG antibody, it shows great potential for development as a first-in-class anticancer antibody. Citation Format: Seung-Min Shin, Ji-Sun Kim, Jin-Sun Hong, Seong-wook Park, Sei-Yong Jun, Hye-Jin Kweon, Dong-Ki Choi, Yong-Sung Kim. Direct targeting oncogenic Ras mutants by IgG-format cytosol-penetrating antibody [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B28.