Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma : preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D (KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.Peer reviewe

Endothelium-Derived Netrin-4 Supports Pancreatic Epithelial Cell Adhesion and Differentiation through Integrins α2β1 and α3β1

BACKGROUND: Netrins have been extensively studied in the developing central nervous system as pathfinding guidance cues, and more recently in non-neural tissues where they mediate cell adhesion, migration and differentiation. Netrin-4, a distant relative of Netrins 1-3, has been proposed to affect cell fate determination in developing epithelia, though receptors mediating these functions have yet to be identified. METHODOLOGY/PRINCIPAL FINDINGS: Using human embryonic pancreatic cells as a model of developing epithelium, here we report that Netrin-4 is abundantly expressed in vascular endothelial cells and pancreatic ductal cells, and supports epithelial cell adhesion through integrins α2β1 and α3β1. Interestingly, we find that Netrin-4 recognition by embryonic pancreatic cells through integrins α2β1 and α3β1 promotes insulin and glucagon gene expression. In addition, full genome microarray analysis revealed that fetal pancreatic cell adhesion to Netrin-4 causes a prominent down-regulation of cyclins and up-regulation of negative regulators of the cell cycle. Consistent with these results, a number of other genes whose activities have been linked to developmental decisions and/or cellular differentiation are up-regulated. CONCLUSIONS/SIGNIFICANCE: Given the recognized function of blood vessels in epithelial tissue morphogenesis, our results provide a mechanism by which endothelial-derived Netrin-4 may function as a pro-differentiation cue for adjacent developing pancreatic cell populations expressing adhesion receptors α2β1 and α3β1 integrins

Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma

The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an ironmimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/ phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors

Enzyme and integrin expression by high and low metastatic melanoma cell lines

Dissemination of a malignant tumour is the result of a cascade of events beginning with detachment of cells from primary tumour followed by extravasation and growth at secondary sites. the differences in metastatic ability could be attributed to properties intrinsic to the various tumour types. Thus the clonal selection of tumour cells from successive metastases apparently results in cells better equipped for survival and formation of colonies in secondary sites, indicating that survival is not a random phenomenon. Many studies of malignant cells have correlated the overexpression of adhesion receptors such as integrins and the production of cysteine proteases and glycosidases with the progression of malignancy. the interaction of cysteine proteases with basement membrane components has been implicated in tumour invasion, activation of hormones and growth factors. On the other hand, the expression of the heparanase gene and its protein has been associated with the metastatic potential of several human and mouse tumour cell lines. This study aimed to investigate the correlations between the metastatic properties of clones with high and low metastatic potential and their ability to adhere to the extracellular matrix and to degrade proteins and sulphated glycosaminoglycans present there. Clonal selection of the B16F10 cell line was performed, and the clones were examined for the expression of an integrin-type laminin receptor. A significantly higher level was detected in a high metastatic clone. Enzymatic assays showed higher activity for alpha-D-N-acetylglucosaminidase, beta-D-N-acetylgalactosaminidase and beta-D-glucuronidase in conditioned medium from low metastatic clones compared with that from high metastatic clones. However, higher endopeptidase activity was observed in conditioned medium from high metastatic clones. in summary, these results showed a positive correlation between high metastatic potential and endopeptidase secretion. Similarly, a positive correlation was observed between low metastatic cells and the secretion of glycosaminoglycan-degrading glycosidases.Universidade Federal de São Paulo, Disciplina Imunol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Mol Biol, BR-04023062 São Paulo, BrazilUniv Mogi Cruzes, Interdisciplinary Ctr Biochem Invest, Mogi Das Cruzes, BrazilUniversidade Federal de São Paulo, Disciplina Imunol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Mol Biol, BR-04023062 São Paulo, BrazilWeb of Scienc

Interleukin-11 receptor is a candidate target for ligand-directed therapy in lung cancer: analysis of clinical samples and BMTP-11 pre-clinical activity

We previously isolated an IL-11–mimic motif (CGRRAGGSC) that binds to IL-11 receptor (IL-11R) in vitro and accumulates in IL-11R–expressing tumors in vivo. This synthetic peptide ligand was used as a tumor-targeting moiety in the rational design of BMTP-11, which is a drug candidate in clinical trials. Here, we investigated the specificity and accessibility of IL-11R as a target and the efficacy of BMTP-11 as a ligand-targeted drug in lung cancer. We observed high IL-11R expression levels in a large cohort of patients (n = 368). In matching surgical specimens (i.e., paired tumors and nonmalignant tissues), the cytoplasmic levels of IL-11R in tumor areas were significantly higher than in nonmalignant tissues (n = 36; P = 0.003). Notably, marked overexpression of IL-11R was observed in both tumor epithelial and vascular endothelial cell membranes (n = 301; P < 0.0001). BMTP-11 induced in vitro cell death in a representative panel of human lung cancer cell lines. BMTP-11 treatment attenuated the growth of subcutaneous xenografts and reduced the number of pulmonary tumors after tail vein injection of human lung cancer cells in mice. Our findings validate BMTP-11 as a pharmacologic candidate drug in preclinical models of lung cancer and patient-derived tumors. Moreover, the high expression level in patients with non-small cell lung cancer is a promising feature for potential translational applications

Networks of gold nanoparticles and bacteriophage as biological sensors and cell-targeting agents

Biological molecular assemblies are excellent models for the development of nanoengineered systems with desirable biomedical properties. Here we report an approach for fabrication of spontaneous, biologically active molecular networks consisting of bacteriophage (phage) directly assembled with gold (Au) nanoparticles (termed Au–phage). We show that when the phage are engineered so that each phage particle displays a peptide, such networks preserve the cell surface receptor binding and internalization attributes of the displayed peptide. The spontaneous organization of these targeted networks can be manipulated further by incorporation of imidazole (Au–phage–imid), which induces changes in fractal structure and near-infrared optical properties. The networks can be used as labels for enhanced fluorescence and dark-field microscopy, surface-enhanced Raman scattering detection, and near-infrared photon-to-heat conversion. Together, the physical and biological features within these targeted networks offer convenient multifunctional integration within a single entity with potential for nanotechnology-based biomedical applications

A preclinical model for predicting drug response in soft-tissue sarcoma with targeted AAVP molecular imaging

Human sarcomas are rare but diverse malignant tumors derived from mesenchymal tissue. Clinical response to therapy is currently determined by the modified World Health Organization (WHO) criteria or the Response Evaluation Criteria in Solid Tumors (RECIST), but these standards correlate poorly with sarcoma patient outcome. We introduced ligand-directed particles with elements of AAV and phage (AAVP) to enable integration of tumor targeting to molecular imaging. We report drug-response monitoring and prediction in a nude rat model of human sarcoma by AAVP imaging. As a proof-of-concept, we imaged Herpes simplex thymidine kinase in a clinic-ready setting with PET to show that one can a priori predict tumor response to a systemic cytotoxic. Given the target expression in patient-derived sarcomas, this platform may be translated in clinical applications. Sarcoma-specific ligands and promoters may ultimately lead to an imaging transcriptome