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The remarkably high prevalence of Ras driver mutations in tumors of diverse origins and stages indicates that Ras neoantigens are poor substrates for HLA-mediated detection by the native immune system. Nevertheless, immunotherapies based upon tumor-infiltrating lymphocytes targeting Ras neoantigens have been reported, indicating that such mutations are subject to HLA display and immune surveillance. Antibodies capable of recognizing specific peptide-HLA complexes at the cell surface have the potential to target intracellular tumor neoantigens for therapeutic intervention. However, such reagents have proved to be challenging to develop. Using a novel antibody discovery platform in combination with a proprietary human germline scFv library, we have developed a rapid method for the isolation of antibodies against peptide-HLA complexes. Applying this technology to KRAS G12V, we have isolated two scFvs that recognize the peptide VVVGAVGVGK in complex with HLA-A*11:01. This peptide-HLA combination forms functional complexes both in vitro and at the cell surface and has been reported to elicit CTL responses in vivo. In contrast, the native peptide is a poor substrate for binding to HLA-A*11:01. Binding affinities of the two scFv binders for the target HLA complex are in the sub-100nM range. Both show highly specific, peptide-dependent recognition of the KRAS-HLA-A*11:01 complex, as demonstrated by the failure to bind complexes containing wild-type KRAS peptides or other control peptides. Alanine scanning mutagenesis reveals multiple residues within the neoepitope peptide that are critical for antibody binding, including the mutated valine residue. The extended binding footprint encompassing the neoepitope and the poor presentation of the unmutated peptide are likely to contribute to the on-target specificity of these antibodies. Incorporation of the scFvs into a bispecific T-cell engager (BiTE) format has demonstrated the simultaneous engagement of both the KRAS HLA-A*11:01 complex and the CD3 surface marker of T cells. Ongoing work is aimed to demonstrate BiTE-dependent CTL-mediated lysis of target cells displaying the KRAS HLA-A*11:01 complex. We are currently developing CAR-T cells carrying peptide-HLA specific scFvs and are screening further mutant Ras peptides in the context of other HLA-A subtypes. This abstract is also being presented as Poster B48. Citation Format: Avisa Maserati, Dahna Makris, Ben Kiefel, Matt Beasley, Brad McColl, Ben Clifton. Anti peptide-HLA (TCR-like) antibodies specific for the KRAS G12V neoantigen [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 PR04.

Summary Rnd proteins are atypical Rho family proteins that do not hydrolyse GTP and are instead regulated by expression levels and post-translational modifications. Rnd1 and Rnd3/RhoE induce loss of actin stress fibres and cell rounding in multiple cell types, whereas responses to Rnd2 are more variable. Here we report the responses of endothelial cells to Rnd proteins. Rnd3 induces a very transient decrease in stress fibres but subsequently stimulates a strong increase in stress fibres, in contrast to the reduction observed in other cell types. Rnd2 also increases stress fibres whereas Rnd1 induces a loss of stress fibres and weakening of cell–cell junctions. Rnd3 does not act through any of its known signalling partners and does not need to associate with membranes to increase stress fibres. Instead, it acts by increasing RhoB expression, which is then required for Rnd3-induced stress fibre assembly. Rnd2 also increases RhoB levels. These data indicate that the cytoskeletal response to Rnd3 expression is dependent on cell type and context, and identify regulation of RhoB as a new mechanism for Rnd proteins to affect the actin cytoskeleton.

Summary Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins., Graphical Abstract, Highlights • Rnd small G proteins bind to 14-3-3 via C-terminal phosphorylation and lipid groups • This interaction negatively regulates Rnd proteins by inducing membrane extraction • Structural analysis shows 14-3-3 binding to a hybrid lipid-phosphorylation motif • This motif identifies new 14-3-3-binding proteins, including Rap1A, 14-3-3 inhibit Rnd proteins by extracting them from their site of action on membranes, which is regulated by Rnd phosphorylation.

Angiogenesis and lymphangiogenesis are important contributors to the growth and metastasis of a wide variety of tumor types. Therapeutic targeting of the angiogenic vascular endothelial growth factor (VEGF) is now an FDA-approved treatment for several cancers. Vascular endothelial growth factor-C (VEGF-C) has also been demonstrated to stimulate angiogenesis and lymphangiogenesis in a number of experimental systems, and is essential for development of the lymphatic system during embryogenesis. In addition, expression of VEGF-C drives tumor progression and metastasis in mouse models of cancer, and is associated with poor prognosis in many human cancers. Targeting VEGF-C is therefore a highly promising strategy for novel therapeutics, with the potential to disrupt both angiogenesis and lymphangiogenesis. VGX-100 is a highly specific, fully human monoclonal antibody for VEGF-C that does not bind to other members of the VEGF family. In vitro testing demonstrates that VGX-100 blocks binding of VEGF-C to both VEGF receptor-2 and VEGF receptor-3, and inhibits the proliferation of blood vascular (HUVEC) and lymphatic endothelial cells in response to VEGF-C stimulation. Here we demonstrate anti-tumor efficacy through the use of xenograft models of human cancer, with VGX-100 treatment significantly inhibiting tumor growth in several models. These findings demonstrate that VGX-100 has great potential for development as a new cancer therapeutic, with potential clinical utility in combination with existing therapies or in patients refractory to existing anti-angiogenic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2442.