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Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC). The androgen receptor (AR) remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD). Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD). Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S)-niphatenone had significantly better activity against the AR NTD compared to (R)-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR) activity and covalently bound to GR activation function-1 (AF-1) region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

The use of fluorescence-polarized microscopy, combined with competitive binding with matched fluorescence companion imaging probes, enable target engagement measurements of covalent and reversible small molecule inhibitors in a single cell. Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo .

Cancer-associated fibroblasts (CAFs) in the stroma of solid tumors promote an immunosuppressive tumor microenvironment (TME) that drives resistance to therapies. The expression of the protease fibroblast activation protein (FAP) on the surface of CAFs has made FAP a target for the development of therapies to mitigate immunosuppression. Relatively few biologics have been developed for FAP and none have been developed that exploit the unique properties of Variable New Antigen Receptors (VNARs) from shark immunoglobulins. Through the direct immunization of a nurse shark with FAP, we created a large anti-FAP VNAR phage display library. This library allowed us to identify a suite of anti-FAP VNARs through traditional biopanning and by an in silico approach that did not require any affinity maturation. We investigated four VNAR-Fc fusion proteins for theranostic properties and found that all four recognized FAP with high affinity and were rapidly internalized by FAP-positive cells. As a result, the VNAR-Fc constructs were effective antibody-drug conjugates in vitro when attached to an anti-mitotic payload and were able to localize to FAP-positive xenografts in vivo by positron emission tomography. Our findings establish VNAR-Fc constructs as a versatile platform for theranostic development that could yield innovative cancer therapies targeting the TME. Through the direct immunization of a nurse shark, we identified a suite of VNAR domains that were effective at targeting FAP.

Therapies against cell-surface targets (CSTs) represent an emerging treatment class in solid malignancies. However, high-throughput investigations of CST expression across cancer types have been reliant on data sets of mostly primary tumors, despite therapeutic use most commonly in metastatic disease. We identified a total of 818 clinical trials of CST therapies with 78 CSTs. We assembled a data set spanning RNA-seq and microarrays in 7,927 benign samples, 16,866 primary tumor samples, and 6,124 metastatic tumor samples. We also utilized single-cell RNA-seq data from 36 benign tissues and 558 primary and metastatic tumor samples, and matched RNA versus protein expression in 29 benign tissue samples, 1,075 tumor samples, and 942 cell lines. High RNA expression accurately predicted high protein expression across CST therapies in benign tissues, tumor samples, and cell lines. We compared metastatic versus primary tumor expression, identified potential opportunities for repositioning, and matched cell lines to tumor types based on CST and global RNA expression. We evaluated single-cell heterogeneity across tumors, and identified rare normal cell subpopulations that may contribute to toxicity. Finally, we identified combinations of CST therapies for which bispecific approaches could improve tumor specificity. This study helps better define the landscape of CST expression in metastatic and primary cancers.

Cancer-associated fibroblasts (CAFs) in the stroma of solid tumors promote an immunosuppressive tumor microenvironment (TME) that drives resistance to therapies. The expression of the protease fibroblast activation protein (FAP) on the surface of CAFs has made FAP a target for development of therapies to dampen immunosuppression. Relatively few biologics have been developed for FAP and none have been developed that exploit the unique engagement properties of Variable New Antigen Receptors (VNARs) from shark antibodies. As the smallest binding domain in nature, VNARs cleverage unique geometries and recognize epitopes conventional antibodies cannot. By directly immunizing a nurse shark with FAP, we created a large anti-FAP VNAR phage display library. This library allowed us to identify a suite of anti-FAP VNARs through traditional biopanning and also by an approach that did not require any prior affinity-based enrichment . We investigated four VNAR-Fc fusion proteins for theranostic properties and found that all four recognized FAP with high affinity and were rapidly internalized by FAP-positive cells. As a result, the VNAR-Fc constructs were effective antibody-drug conjugates and were able to localize to FAP-positive xenografts . Our findings establish VNAR-Fc constructs as a versatile platform for theranostic development that could yield innovative cancer therapies targeting the TME.