Gla-domain mediated targeting of externalized phosphatidylserine for intracellular delivery.

Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging, and targeted delivery of therapeutic agents. While an array of PS-binding molecules exist, their utility has been limited by their inability to internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS-binding motif comprised of a carboxylated glutamic acid (GLA) residue domain that both recognizes and binds cell surface-exposed PS, and then unlike other PS-binding molecules is internalized into these cells. Internalization is independent of the traditional endosomal-lysosomal pathway, directly entering the cytosol of the target cell rapidly. We demonstrate that this PS recognition extends to stem cells and that GLA-domain-conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offer new opportunities for specifically targeting cells with surface-exposed PS for imaging and delivery of therapeutics.

Phosphatidylserine-Exposing Annexin A1-Positive Extracellular Vesicles: Potential Cancer Biomarkers.

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1+) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

Long-Acting IL-33 Mobilizes High-Quality Hematopoietic Stem and Progenitor Cells More Efficiently Than Granulocyte Colony-Stimulating Factor or AMD3100.

Mobilization of hematopoietic stem and progenitor cells (HSPCs) has become increasingly important for hematopoietic cell transplantation. Current mobilization approaches are insufficient because they fail to mobilize sufficient numbers of cells in a significant fraction of patients and are biased toward myeloid immune reconstitution. A novel, single drug mobilization agent that allows a more balanced (myeloid and lymphoid) reconstitution would therefore be highly favorable to improve transplantation outcome. In this present study, we tested commercially available IL-33 molecules and engineered novel variants of IL-33. These molecules were tested in cell-based assays in vitro and in mobilization models in vivo. We observed for the first time that IL-33 treatment in mice mobilized HSPCs and common myeloid progenitors more efficiently than clinical mobilizing agents granulocyte colony-stimulating factor (G-CSF) or AMD3100. We engineered several oxidation-resistant IL-33 variants with equal or better in vitro activity. In vivo, these variants mobilized HSPCs and, interestingly, also hematopoietic stem cells, common lymphoid progenitor cells, and endothelial progenitor cells more efficiently than wild-type IL-33 or G-CSF. We then engineered an IL-33-Fc fusion molecule, a single dose of which was sufficient to significantly increase the mobilization of HSPCs after 4 days. In conclusion, our findings suggest that long-acting, oxidation-resistant IL-33 may be a novel approach for HSPC transplantation. IL-33-mobilized HSPCs differ from cells mobilized with G-CSF and AMD3100, and it is possible that these differences may result in better transplantation outcomes.

A macaque model of HIV-1 infection.

The lack of a primate model that utilizes HIV-1 as the challenge virus is an impediment to AIDS research; existing models generally employ simian viruses that are divergent from HIV-1, reducing their usefulness in preclinical investigations. Based on an understanding of species-specific variation in primate TRIM5 and APOBEC3 antiretroviral genes, we constructed simian-tropic (st)HIV-1 strains that differ from HIV-1 only in the vif gene. We demonstrate that such minimally modified stHIV-1 strains are capable of high levels of replication in vitro in pig-tailed macaque (Macaca nemestrina) lymphocytes. Importantly, infection of pig-tailed macaques with stHIV-1 results in acute viremia, approaching the levels observed in HIV-1-infected humans, and an ensuing persistent infection for several months. stHIV-1 replication was controlled thereafter, at least in part, by CD8+ T cells. We demonstrate the potential utility of this HIV-1-based animal model in a chemoprophylaxis experiment, by showing that a commonly used HIV-1 therapeutic regimen can provide apparently sterilizing protection from infection following a rigorous high-dose stHIV-1 challenge.

Antibodies neutralizing hepsin protease activity do not impact cell growth but inhibit invasion of prostate and ovarian tumor cells in culture.

Hepsin is a type II transmembrane serine protease that is expressed in normal liver, and at lower levels in kidney, pancreas, and testis. Several studies have shown that hepsin mRNA is significantly elevated in most prostate tumors, as well as a significant fraction of ovarian and renal cell carcinomas and hepatomas. Although the overexpression of mRNA in these tumors has been extensively documented, there has been conflicting literature on whether hepsin plays a role in tumor cell growth and progression. Early literature implied a role for hepsin in human tumor cell proliferation, whereas recent studies with a transgenic mouse model for prostate cancer support a role for hepsin in tumor progression and metastases. To evaluate this issue further, we have expressed an activatable form of hepsin, and have generated a set of monoclonal antibodies that neutralize enzyme activity. The neutralizing antibodies inhibit hepsin enzymatic activity in biochemical and cell-based assays. Selected neutralizing and nonneutralizing antibodies were used in cell-based assays with tumor cells to evaluate the effect of antibodies on tumor cell growth and invasion. Neutralizing antibodies failed to inhibit the growth of prostate, ovarian, and hepatoma cell lines in culture. However, potent inhibitory effects of the antibodies were seen on invasion of ovarian and prostate cells in transwell-based invasion assays. These results support a role for hepsin in tumor cell progression but not in primary tumor growth. Consistent with this, immunohistochemical experiments with a mouse monoclonal antibody reveal progressively increased staining of prostate tumors with advanced disease, and in particular, extensive staining of bone metastatic lesions.

Identification of a novel prostate tumor target, mindin/RG-1, for antibody-based radiotherapy of prostate cancer.

Gene expression analysis showed that a human mindin homologue, mindin/RG-1, is expressed selectively in prostate tissues and that its expression level is elevated in some prostate tumors. Mindin/RG-1 protein expression is maintained in >80% of prostate cancers metastatic to bone or lymph nodes as well as in locally recurrent tumors in androgen-unresponsive patients. In contrast, mindin/RG-1 expression in other normal tissues is significantly lower than that seen in the prostate. A fully human antibody, 19G9, was generated against mindin/RG-1 protein and was shown to accumulate at high abundance in LNCaP tumor xenografts. Conjugates of this antibody with the chelator CHX-A''-DTPA were generated and radiolabeled with either 111In, 90Y, or 86Y. Small animal positron emission tomography imaging with the 86Y-radiolabeled conjugate showed very specific accumulation of the antibody in LNCaP tumor xenografts with clear tumor delineation apparent at 4 hours. The therapeutic efficacy of [90Y]-CHX-A''-DTPA-19G9 was evaluated in mice bearing LNCaP xenografts. A dose-finding study identified a nontoxic therapeutic dose to be approximately 75 microCi. Significant antitumor effects were seen with a single administration of radiolabeled antibody to animals bearing 200 to 400 mm3 tumors. Inhibition of tumor growth was observed in all treated animals over a 49-day period. At 49 days posttreatment, slow tumor growth recurred but this could be prevented for an additional 40-day period by a second administration of a 75 microCi dose at day 49. We conclude that [90Y]-CHX-A''-DTPA-19G9 is a novel antibody conjugate that has considerable promise for therapy of metastatic prostate cancer in androgen-unresponsive patients.

Targeting tomoregulin for radioimmunotherapy of prostate cancer.

Radiotherapy is an effective approach for the treatment of local prostate cancer. However, once prostate cancer metastasizes, radiotherapy cannot be used due to the distribution of multiple metastases to lymph nodes and bones. In contrast, radioimmunotherapy should still be efficacious in metastatic prostate cancer as radioisotopes are brought to tumor cells by targeting antibodies. Here we identify and validate a prostate-expressed molecule, tomoregulin, as a target for radioimmunotherapy of prostate cancer. Tomoregulin is a transmembrane protein selectively expressed in the brain, prostate, and prostate cancer, but not expressed in other normal tissues. Immunohistochemical studies of tomoregulin protein in clinical samples show its location in the luminal epithelium of normal prostate, benign prostatic hyperplasia, and prostatic intraepithelial neoplasia. More importantly, the tomoregulin protein is expressed in primary prostate tumors and in their lymph node and bone metastases. The nature of tomoregulin as a transmembrane protein and its tissue-specific expression make tomoregulin an attractive target for radioimmunotherapy, in which tomoregulin-specific antibodies will deliver a radioisotope to prostate tumor cells and metastases. Indeed, biodistribution studies using a prostate tumor xenograft model showed that the (111)In-labeled anti-tomoregulin antibody 2H8 specifically recognizes tomoregulin protein in vivo, leading to a strong tumor-specific accumulation of the antibody. In efficacy studies, a single i.p. dose of 150 microCi (163 microg) (90)Y-labeled 2H8 substantially inhibits the growth rate of established LNCaP human prostate tumor xenograft in nude mice but produces no overt toxicity despite cross-reactivity of 2H8 with mouse tomoregulin. Our data clearly validate tomoregulin as a target for radioimmunotherapy of prostate cancer.