Differential healing after sirolimus, paclitaxel, and bare metal stent placement in combination with peroxisome proliferator-activator receptor gamma agonists: requirement for mTOR/Akt2 in PPARgamma activation.

RATIONALE: Sirolimus-eluting coronary stents (SESs) and paclitaxel-eluting coronary stents (PESs) are used to reduce restenosis but have different sites of action. The molecular targets of sirolimus overlap with those of the peroxisome proliferator-activated receptor (PPAR)gamma agonist rosiglitazone (RSG) but the consequence of this interaction on endothelialization is unknown. OBJECTIVE: Using the New Zealand white rabbit iliac model of stenting, we examined the effects of RSG on SESs, PESs, and bare metal stents endothelialization. METHODS AND RESULTS: Animals receiving SESs, PESs, or bare metal stents and either RSG (3 mg/kg per day) or placebo were euthanized at 28 days, and arteries were evaluated by scanning electron microscopy. Fourteen-day organ culture and Western blotting of iliac arteries and tissue culture experiments were conducted. Endothelialization was significantly reduced by RSG in SESs but not in PESs or bare metal stents. Organ culture revealed reduced vascular endothelial growth factor in SESs receiving RSG compared to RSG animals receiving bare metal stent or PESs. Quantitative polymerase chain reaction in human aortic endothelial cells (HAECs) revealed that sirolimus (but not paclitaxel) inhibited RSG-induced vascular endothelial growth factor transcription. Western blotting demonstrated that inhibition of molecular signaling in SES+RSG-treated arteries was similar to findings in HAECs treated with RSG and small interfering RNA to PPARgamma, suggesting that sirolimus inhibits PPARgamma. Transfection of HAECs with mTOR (mammalian target of rapamycin) short hairpin RNA and with Akt2 small interfering RNA significantly inhibited RSG-mediated transcriptional upregulation of heme oxygenase-1, a PPARgamma target gene. Chromatin immunoprecipitation assay demonstrated sirolimus interferes with binding of PPARgamma to its response elements in heme oxygenase-1 promoter. CONCLUSIONS: mTOR/Akt2 is required for optimal PPARgamma activation. Patients who receive SESs during concomitant RSG treatment may be at risk for delayed stent healing.

Pre-clinical validation of B cell maturation antigen (BCMA) as a target for T cell immunotherapy of multiple myeloma.

Multiple myeloma has a continued need for more effective and durable therapies. B cell maturation antigen (BCMA), a plasma cell surface antigen and member of the tumor necrosis factor (TNF) receptor superfamily, is an attractive target for immunotherapy of multiple myeloma due to its high prevalence on malignant plasma cells. The current work details the pre-clinical evaluation of BCMA expression and development of a chimeric antigen receptor (CAR) targeting this antigen using a fully human single chain variable fragment (scFv). We demonstrate that BCMA is prevalently, but variably expressed by all MM with expression on 25-100% of malignant plasma cells. Extensive Immunohistochemical analysis of normal tissue expression using commercially available polyclonal antibodies demonstrated expression within B-lineage cells across a number of tissues as expected. Based upon the highly restricted expression of BCMA within normal tissues, we generated a set of novel, fully human scFv binding domains to BCMA by screening a naïve B-cell derived phage display library. Using a series of in vitro and pre-clinical in vivo studies, we identified a scFv with high specificity for BCMA and robust anti-myeloma activity when used as the binding domain of a second-generation CAR bearing a CD137 costimulatory domain. This BCMA-specific CAR is currently being evaluated in a Phase 1b clinical study in relapsed and refractory MM patients (NCT02546167).

Long-acting protein drugs for the treatment of ocular diseases.

Protein drugs that neutralize vascular endothelial growth factor (VEGF), such as aflibercept or ranibizumab, rescue vision in patients with retinal vascular diseases. Nonetheless, optimal visual outcomes require intraocular injections as frequently as every month. Here we report a method to extend the intravitreal half-life of protein drugs as an alternative to either encapsulation or chemical modifications with polymers. We combine a 97-amino-acid peptide of human origin that binds hyaluronan, a major macromolecular component of the eye's vitreous, with therapeutic antibodies and proteins. When administered to rabbit and monkey eyes, the half-life of the modified proteins is increased ∼3-4-fold relative to unmodified proteins. We further show that prototype long-acting anti-VEGF drugs (LAVAs) that include this peptide attenuate VEGF-induced retinal changes in animal models of neovascular retinal disease ∼3-4-fold longer than unmodified drugs. This approach has the potential to reduce the dosing frequency associated with retinal disease treatments.

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).

A novel microtubule-modulating agent induces mitochondrially driven caspase-dependent apoptosis via mitotic checkpoint activation in human prostate cancer cells.

Hormone-refractory prostate cancer, its skeletal metastasis and complications remain a therapeutic challenge. Here we show that treatment with (S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyiso-benzofuran-1(3H)-one (EM011), the brominated analogue of a plant-derived non-toxic antitussive alkaloid, noscapine, achieved significant inhibition of hormone-refractory human prostate cancer implanted intratibially in the bone as shown by non-invasive, real-time bioluminescent imaging of tumour growth in nude mice. Mechanistically, in vitro data suggested that the antiproliferative and proapoptotic effects of EM011 in human prostate cancer cell lines were through blockade of cell-cycle progression by impairing the formation of a bipolar spindle apparatus. The G2/M arrest was accompanied by activation of the mitotic checkpoint, a pre-requisite for induction of optimal apoptosis. Attenuation of mitotic checkpoint by siRNA duplexes led to a reduction in mitotic arrest and subsequent apoptosis. Our results further demonstrated participation of an intrinsic mitochondrially mediated apoptotic pathway that ultimately triggered caspase-driven EM011-induced apoptosis. EM011 did not exert any detectable toxicity in normal tissues with frequently dividing cells such as the gut and bone marrow. Thus, these data warrant further evaluation of EM011 for the management of prostate cancer.