L-DOS47 Elevates Pancreatic Cancer Tumor pH and Enhances Response to Immunotherapy.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds to CEACAM6, a cell-surface protein that is highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) to measure the tumor extracellular pH (pHe), we confirmed that L-DOS47 raises the tumor pHe from 4 h to 96 h post injection in acidic tumors (average increase of 0.13 units). Additional studies showed that combining L-DOS47 with anti-PD1 significantly increases the efficacy of the anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

In vivo and in silico pharmacokinetics and biodistribution of a melanocortin receptor 1 targeted agent in preclinical models of melanoma.

The melanocortin 1 receptor (MC1R) is overexpressed in most melanoma metastases, making it a promising target for imaging of melanomas. In this study, the expression of MC1R in a large fraction of patients with melanoma was confirmed using mRNA and tissue microarray. Here, we have characterized the in vivo tumor and tissue distribution and pharmacokinetics (PK) of uptake and clearance of a MC1R specific peptidomimetic ligand conjugated to a near-infrared fluorescent dye. We propose an interdisciplinary framework to bridge the different time and space scales of ligand-tumor-host interactions: intravital fluorescence microscopy to quantify probe internalization at the cellular level, a xenograft tumor model for whole body pharmacokinetics, and a computational pharmacokinetic model for integration and interpretation of experimental data. Administration of the probe into mice bearing tumors with high and low MC1R expression demonstrated normalized image intensities that correlated with expression levels (p < 0.05). The biodistribution study showed high kidney uptake as early as 30 min postinjection. The PK computational model predicted the presence of receptors in the kidneys with a lower affinity, but at higher numbers than in the tumors. As the mouse kidney is known to express the MC5R, this hypothesis was confirmed by both coinjection of a ligand with higher MC5R affinity compared to MC1R and by injection of lower probe concentrations (e.g., 1 nmol/kg), both leading to decreased kidney accumulation of the MC1R ligand. In addition, through this interdisciplinary approach we could predict the rates of ligand accumulation and clearance into and from organs and tumors, and the amount of injected ligand required to have maximum specific retention in tumors. These predictions have potential to aid in the translation of a targeted agent from lab to the clinic. In conclusion, the characterized MC1R-specific probe has excellent potential for in vivo detection of melanoma metastases. The process of cell-surface marker validation, targeted imaging probe development, and in vitro, in vivo, and in silico characterization described in this study can be generally applied to preclinical development of targeted agents.

L-DOS47 enhances response to immunotherapy in pancreatic cancer tumor.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds CEACAM6, a cell surface protein highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Our results demonstrate that combining L DOS47 with anti-PD1 significantly increases the efficacy of anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

Targeting Ligand Specificity Linked to Tumor Tissue Topological Heterogeneity via Single-Cell Micro-Pharmacological Modeling.

Targeted therapy has held promise to be a successful anticancer treatment due to its specificity towards tumor cells that express the target receptors. However, not all targeting drugs used in the clinic are equally effective in tumor eradication. To examine which biochemical and biophysical properties of targeted agents are pivotal for their effective distribution inside the tumor and their efficient cellular uptake, we combine mathematical micro-pharmacological modeling with in vivo imaging of targeted human xenograft tumors in SCID mice. The mathematical model calibrated to experimental data was used to explore properties of the targeting ligand (diffusion and affinity) and ligand release schemes (rates and concentrations) with a goal to identify the properties of cells and ligands that enable high receptor saturation. By accounting for heterogeneities typical of in vivo tumors, our model was able to identify cell- and tissue-level barriers to efficient drug uptake. This work provides a base for utilizing experimentally measurable properties of a ligand-targeted agent and patient-specific attributes of the tumor tissue to support the development of novel targeted imaging agents and for improvement in their delivery to individual tumor cells.

Lysophosphatidic acid induction of urokinase plasminogen activator secretion requires activation of the p38MAPK pathway.

Lysophosphatidic acid (LPA) is an important intercellular signaling molecule involved in a myriad of biological responses. Elevated concentrations of LPA are present in the ascites and plasma of ovarian cancer patients suggesting a role for LPA in the pathophysiology of ovarian cancer. We have demonstrated previously that oleoyl (18:1) LPA at concentrations present in ascites induces the secretion of urokinase plasminogen activator (uPA) from ovarian cancer cells, possibly linking LPA to cellular invasion. In this study we sought to elucidate which signaling pathway(s) are involved in LPA-mediated secretion of uPA from ovarian cancer cells. Specific inhibitors were utilized to determine if interference with the p38(MAPK), p42/44(MAPK), and PI3K pathways functionally blocked LPA-mediated uPA secretion. LPA stimulation of ovarian cancer cells markedly increased the phosphorylation and activity of p38(MAPK), p42/p44(MAPK), and PI3K. Both tyrosine phosphorylation and Src kinase activity were required for optimal activation of signaling by LPA including phosphorylation of p38(MAPK). Inhibition of p38(MAPK) signaling by SB202190 completely abrogated LPA-induced uPA secretion, while inhibition of the p42/44(MAPK) or PI3K pathways with PD98059 or wortmannin and LY294002, respectively, decreased but did not completely block uPA secretion. In contrast, inhibitors of phospholipase D or the p70S6 kinase pathway did not alter LPA-induced uPA secretion. Further, tyrosine phosphorylation and functional Src were required for optimal uPA secretion. Finally, LPA induces uPA secretion from ovarian cancer cells predominantly through the LPA2 receptor, with LPA3 contributing to this process. These results indicate that the p38(MAPK) signaling pathway is required for optimal LPA-dependent uPA secretion from ovarian cancer cells.

Activated SRC protein tyrosine kinase is overexpressed in late-stage human ovarian cancers.

OBJECTIVE: The objective of this study was to determine if the Src tyrosine kinase is overexpressed and activated in late-stage human ovarian cancers. METHODS: Western analysis and immune complex kinase assays were performed on a panel of human ovarian cancer cell lines and normal ovarian epithelial cell cultures, and immunohistochemical analysis for Src and activated Src were performed on a panel of late-stage human ovarian tumors. RESULTS AND CONCLUSIONS: Src is overexpressed and activated in a majority of late-stage ovarian tumors as well as in a panel of cultured malignant human ovarian epithelium grown in vitro, but not in normal ovarian epithelium (NOE) or immortalized NOE. Src overexpression was found to be frequently, but not always, associated with HER-2/neu overexpression, but no statistical association between Src and Her-2/neu overexpression could be demonstrated.