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Introduction: Epidermal growth receptor (EGFR) is the most expressed membrane oncogenic protein in human cancers. KRAS and BRAF mutations are significant drivers of resistance to EGFR-targeted therapies. Unlike other treatments, EGFR-targeting, CD3 bispecific T cell engagers (TCEs) can potentially retain activity against tumors bearing resistance mutations. However, cytokine release syndrome (CRS), on-target off-tumor toxicities, and poor pharmacokinetics (PK) properties present significant clinical limitations for these potent immunomodulators. To overcome these challenges, Janux has developed JANX008, an EGFR- and CD3-targeted tumor-activated T cell engager (TRACTr). JANX008 is a humanized tri-specific protein that contains EGFR- and CD3-binding domains, an albumin binding domain to extend circulating half-life, and two different peptide masks fused to the molecule through tumor protease cleavable linkers. One peptide mask inhibits EGFR engagement on target cells, and the other inhibits CD3 engagement on T cells. Once the cleavage sequences undergo proteolysis by tumor proteases, the EGFR and CD3 masks are released, and the resulting active molecule can bind EGFR and CD3 on target cells. Methods: Peptide masks against EGFR- and CD3-binding domains were identified via phage display. The efficiency of the masks was evaluated using human EGFR and CD3 ELISAs. JANX008-induced cleavage-dependent T cell killing was evaluated in human PBMC/tumor cell co-culture assays. Anti-tumor efficacy of JANX008 was tested in multiple preclinical models, including EGFR antibody-resistant KRAS- and PIK3CA-mutant mouse colon cancer model (HCT116) and a fully human primary colorectal cancer (CRC) tumoroid system. The pharmacokinetic and safety profile of JANX008 was evaluated in non-human primate studies. Results: JANX008 target engagement was cleavage-dependent where masking reduced EGFR and CD3 binding by >300x and >1,000x, respectively. JANX008 exhibited potent cleavage- and dose-dependent activity in multiple preclinical models, including EGFR antibody-resistant tumor and T cell co-culture assays, humanized mouse CRC model, and a human primary CRC tumoroids with an intact tumor microenvironment. JANX008 showed a significantly enhanced safety profile in NHPs compared to non-masked EGFR-TCE, including decreased CRS-associated cytokines and healthy tissue toxicities at high exposures. Clinical chemistry, hematology, and pathology measurements all supported No-Observed-Adverse-Effect-Level ≥ 0.6 mg/kg/dose. Finally, the cleavable albumin-binding domain extended the half-life of JANX008 to ~94h, relative to the ~1.3h half-life of non-masked TCE, supporting its weekly clinical dosing. Conclusions: Preclinical data demonstrate key characteristics of JANX008, including cleavage-dependent activity, half-life extended PK, the potential for superior safety, and manufacturability properties that could mitigate significant limitations of TCEs and support JANX008 clinical development. Citation Format: Thomas R DiRaimondo, Natalija Budimir, Lina Ma, Simon Shenhav, Vanessa Cicchini, Hu Wu, Renee Jocic, Fabrece Roup, Calvin Campbell, Carolina Caffaro, Hans Aerni, Ugur Eskiocak, Wayne Godfrey, Charles Winter, Marc Nasoff, Neil Gibson, David Campbell, shahram Salek-Ardakani. Preclinical activity and safety profile or JANX008, a novel EGFR-targeting tumor-activated T cell engager for treatment of solid tumors [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B04.

Structural studies of filament-forming biomolecules have classically relied on optical imaging methods such as fluorescence and electron microscopy. While these methods have yielded novel insights into mechanisms of macromolecular assembly, they often require perturbative labeling or staining procedures that can affect the assembly process. In contrast, Atomic Force Microscopy (AFM) has emerged as a powerful imaging tool to directly study native nanostructures at high resolution without labeling. Unlike optical methods, AFM utilizes a nano-scale cantilever to generate a topography map of surface-immobilized molecules in air or in aqueous environments, allowing studies under more physiological or dynamic conditions. However, like all imaging methods, most biological samples require electrostatic or covalent interaction of the biomolecule with a surface for imaging, and care must be taken to avoid surface-specific effects on the sample. Here, we examined the structural organization of the bacterial polar scaffold protein PopZ by AFM, and analyzed the effect of surface-immobilization conditions on PopZ's nanostructural assembly characteristics. We measured the structures and densities of PopZ complexes on positively and negatively charged as well as hydrophobic surfaces, and compared structural organization of these assemblies in aqueous environments. Our results illustrate how choice of surface immobilization conditions can affect structural studies of polymeric assemblies, and demonstrate the tremendous advantages of AFM for directly imaging biomolecules in aqueous, physiological conditions. Finally, our results provide new insight into the structures of multimeric PopZ nano-assemblies that have been thus far unattainable using standard EM methods, providing direct evidence for PopZ self-assembly into organized three-dimensional polymeric networks.

Malaria remains a major health problem especially in tropical and subtropical regions of the world, and therefore developing new antimalarial drugs constitutes an urgent challenge. Lipid metabolism has been attracting a lot of attention as an application for malarial chemotherapeutic purposes in recent years. However, little is known about glycosphingolipid biosynthesis in Plasmodium falciparum. In this report we describe for the first time the presence of an active glucosylceramide synthase in the intraerythrocytic stages of the parasite. Two different experiments, using UDP-[(14)C]glucose as donor with ceramides as acceptors, or UDP-glucose as donor and fluorescent ceramides as acceptors, were performed. In both cases, we found that the parasitic enzyme was able to glycosylate only dihydroceramide. The enzyme activity could be inhibited in vitro with low concentrations of d,l-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP). In addition, de novo biosynthesis of glycosphingolipids was shown by metabolic incorporation of [(14)C]palmitic acid and [(14)C]glucose in the three intraerythrocytic stages of the parasite. The structure of the ceramide, monohexosylceramide, trihexosylceramide and tetrahexosylceramide fractions was analysed by UV-MALDI-TOF mass spectrometry. When PPMP was added to parasite cultures, a correlation between arrest of parasite growth and inhibition of glycosphingolipid biosynthesis was observed. The particular substrate specificity of the malarial glucosylceramide synthase must be added to the already known unique and amazing features of P. falciparum lipid metabolism; therefore this enzyme might represent a new attractive target for malarial chemotherapy.

Many protein-RNA complexes exist within cells and have various functions, including shuttling mRNA from the site of transcription to specific cellular regions. Often, these protein-RNA complexes consist of multiple mRNAs of which some are known and others remain unknown. The traditional method used to isolate protein-mRNA complexes is immunoprecipitation (IP). IP involves lysing a population of cells and isolating the protein-mRNA complexes using antibodies specific to the protein to "pulldown" the corresponding protein-mRNA complex of interest. We demonstrate the use of AFM as a complementary analysis tool for studying protein-RNA complexes in single living cells. While IP homogenizes the composition of protein-mRNA complexes from the cellular population under investigation and represents an "average", the AFM can interrogate not only a single cell but also subcellular regions of a single live cell, allowing stratification of protein-mRNA complexes from different regions of the cell. The result is the ability to spatially discriminate different populations of mRNAs that may be carried by the shuttling protein. To demonstrate extraction of a protein-mRNA complex, we selected the shuttling protein, Zipcode Binding Protein 1 (ZBP1) and its known associated mRNA, beta-actin, as a model system. Rat fibroblasts expressing a fusion protein, ZBP1-mCherry, were visualized using the fluorescent microscope of the AFM and then the cells of interest were punctured with an AFM tip conjugated with antibodies to ZBP1. Subsequently, the AFM tip was collected and analyzed for beta-actin mRNA using RT-PCR. BioAnalyzer results confirmed the extraction of beta-actin mRNA. This work shows that the AFM can be used as a tool to extract protein-RNA complexes from different regions of single living cells, potentially expanding the cell biologist's toolset.