Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

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.

Caspase‐11 non‐canonical inflammasome: a critical sensor of intracellular lipopolysaccharide in macrophage‐mediated inflammatory responses

“…In conclusion, given the strong evidence of a critical role of caspase‐11 in the non‐canonical inflammasome response to intracellular LPS in macrophages, selective targeting of the caspase‐11 non‐canonical inflammasome pathway by various useful approaches, including caspase‐11‐targeting small interference RNAs, the agents interfering with the binding between caspase‐11 and intracellular LPS, and caspase‐11‐targeting antibody therapeutics using the recently developed intracellular molecule‐targeting antibody technology could be a novel and promising strategy for the prevention and treatment of a number of infectious and pathogen‐induced inflammatory/autoimmune diseases, such as sepsis, chronic respiratory/lung diseases, coronary artery disease, cystic fibrosis, rheumatoid arthritis, and some intestinal inflammatory diseases, including inflammatory bowel disease, Crohn's disease and ulcerative colitis …”

Competitive Inhibitors of Ras Effector Binding

“…Although the RBDvs are a unique tool for studying Ras-dependent signaling processes, several intracellular affinity reagents targeting GTP-bound Ras have recently been published 6,8,9,10 . This raises the question of how the RBDvs compare to these engineered proteins.…”

“…The cell penetrating TMab4 RT11 antibody targeting the switch 1 site of Ras family GTPases has also been described to require high concentrations to effectively inhibit Ras-mediated signaling events. Whether this observation is due to a comparable avidity effect or incomplete release of the antibody from the endosomes is unclear 10 . In addition to these examples, two designed ankyrin repeat proteins (DARPins) with specificity to either the GDP-bound inactive state (K27) or the GTP-bound active state (K55) of Ras have recently been reported 6 .…”

“…An alternative, more direct strategy for inhibiting Ras-signaling using engineered proteins is to prevent the recruitment of downstream effectors or block the activation of Ras by GEFs. To this end, several affinity reagents have been generated that compete with Ras effectors by selectively binding to the active or inactive state of Ras 6,7,8,9,10,11,12 . Interestingly, recent work showed that a subset of these affinity reagents had only effects in cells at high concentrations 9,10,13 .…”

Where in the Cell Is our Cargo? Methods Currently Used To Study Intracellular Cytosolic Localisation

“…[99] Thist echnique was applied to confirm cytosol delivery of CPPs, [76] protein-CPPf usions [77] or antibodies into cells. [70,100] In the last case, to enhance GFP reconstitution, the antibody-oligopeptide partnera nd the nonfluorescent GFP1-10 can be fused to streptavidin-binding peptide2(SBP2)a nd streptavidin, respectively. [70]…”