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Background: ZVex is a novel, integration-deficient, dendritic cell-targeting lentiviral vector platform currently being evaluated in clinical trials in sarcoma patients. The vector is capable of delivering multiple full-length genes simultaneously. Here, we describe ZVex preparations encoding the human cancer testis antigens MAGE-A1, 3, 4, 10 and Interleukin 12. Because lentiviral vectors package two RNA molecules each, this ZVex approach results in the generation of 15 possible permutations of homozygous or heterozygous vector genotypes. The homologous nature of the MAGEA family members (50-80% sequence identity) poses significant challenges to their specific detection and quantification in a MAGEA gene pool. Here, we describe highly specific qPCR assays for detection of the 4 MAGEA genes and IL12 for characterization and identity testing of multigenome ZVex preparations. Materials and Methods: Genes were codon-modified to enable specific detection of vector-expressed genes over cellular sequences. The entire IL12B and short, non-overlapping, divergent target regions in each of the 4 MAGEA genes were carefully selected as templates for primer/probe design. Primer-blast was used to predict absence of off-target detection of cross-reacting genomic sequences. To further enhance the specificity of the primers/probes to each of their target MAGEA, potential off-target amplicons that can be generated in the other 3 MAGEA genes due to base pair mismatch were further sequence-modified to reduce sequence identities. ZVex vectors were generated by transfecting 4 or 5 different plasmids encoding the codon modified full-length MAGEA and/or IL12 genes in a producer cell line along with essential vector packaging plasmids. Single/multiplex qPCR assays were performed to analyze vector RNA/reverse-transcribed DNA. Results: The primer/probe combinations were highly specific for each of the 4 MAGEA genes and IL12 as indicated by melt curve analysis and producing a single qPCR product at the expected size. The primer/probes specifically detected from 10 to 107 copies of their target MAGEA with >=93% efficiency and specifically detected reverse-transcribed vector DNA in a cell-based vector transduction assay. The assays demonstrated relative gene specific titers that correlate with transfected plasmid ratios. MAGEA/IL12 specific qPCR assays benchmark well against previously established “gold standard” qPCR assays used for quantifying total ZVex vector genome titers. Conclusions: Component-specific qPCR assays were developed for the specific assessment of constituent genes in multigenome ZVex preparations. These assays demonstrate inter/intragene specificity, are amenable to multiplexing, and can be used for quality control, titration, characterization, and identity testing of ZVex lots in a cGMP setting. Citation Format: Anshika Bajaj, Tsai-Yu Lin, Lisa Y. Ngo, Michele Murphy, Brenna Kelley-Clarke, Wayne R. Gombotz, Jan H. ter Meulen, Peter Berglund. Component-specific qPCR assays for characterization and identity testing of multigenome ZVex®, a dendritic cell-targeting lentiviral vector platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5919.

BACKGROUND: The toxicity of chemotherapeutic drugs is often the limiting factor in patient dosing and can be the source of severe discomfort and suffering for patients undergoing chemotherapy. For example, the anthracycline antibiotic doxorubicin (DOX) is a potent chemotherapeutic but prolonged use can also lead to permanent heart damage. Tumors can also develop resistance to DOX by increased expression of drug efflux pumps. Nanoparticle formulations of DOX have been reported to improve tumor-specific delivery via the enhanced permeability and retention (EPR) effect. METHODS: In this work, we have encapsulated DOX in polymeric micelles formed from mixtures of amphiphilic triblock copolymers. The copolymers are composed of a hydrophobic polymer segment flanked by two hydrophilic blocks. Two different copolymers were used to form the mixed micelles: a copolymer of poly(ethylene oxide) and poly(hydroxybutyrate) (PEO-PHB-PEO), and a copolymer of poly(ethylene oxide) and poly(propylene oxide) (Pluronic F127). We hypothesize that forming mixed micelles will improve drug loading and enable preparation at room temperature (where Pluronic F127 micelles are highly unstable), while facilitating drug delivery by utilizing the stability of Pluronic F127 micelles at physiological temperatures. RESULTS: We have demonstrated that the micelles formed from the mixtures of PEO-PHB-PEO and Pluronic F127 triblock copolymers provide a combination of high drug loading content and drug delivery capabilities. DOX loading for mixed micelles was greater than that for Pluronic F127-only micelles (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4770. doi:1538-7445.AM2012-4770

The goal of this study is to develop formulations for non-invasive, near-infrared detection of tumors. Indocyanine green (ICG) is an FDA-approved near-infrared (NIR) imaging agent that is clinically used for angiography and evaluation of liver function. ICG could potentially be used as a NIR fluorescence imaging agent for shallow tumors such as breast cancers. To fulfill this potential, formulations that improve the stability of ICG and direct its accumulation in tumors are necessary. We have previously described Pluronic F127 copolymer micelles that stabilize ICG, enhance its stability, and facilitate tumor accumulation in vivo after systemic administration. A major advantage of Pluronic F127 is its temperature-sensitive behavior that results in more stable micelle formulations at body temperature. However, Pluronic F127 micelles are not stable at room temperature, making micelle preparation and handling challenging. We hypothesized that mixed micelles formed from both Pluronic F127 and a temperature insensitive triblock copolymer composed of poly(ethylene oxide) and poly(3-hydroxybutyrate) (PEO-PHB-PEO), would result in micelles that are both easy to formulate at room temperature and that demonstrate increased stability at body temperature. Micelle formulations with various ratios of Pluronic F127: PEO-PHB-PEO were prepared and loaded with ICG. The micelle stability was determined by assessment of critical micelle concentration measurements, and time-course fluorescence assays were conducted to assess the ability of each micelle formulation to stabilize ICG fluorescence in buffer conditions. The ICG-loaded micelles were administered to tumor-bearing mice by tail vein injection and tumor localization was monitored by non-invasive fluorescence imaging in live mice. The polymer ratio used in micelle formulation was found to play a significant role in the ability of each preparation to stabilize ICG fluorescence Mixed micelles were found to enhance the stability of ICG fluorescence in buffer conditions, and CMC was shown to be dependent on polymer ratios. In vivo, mixed micelles were found to be more effective at maintaining ICG fluorescence in the bloodstream and achieved higher fluorescence signals in the tumor tissues compared with either PEO-PHB-PEO or Pluronic 127 micelles. These results suggest that the mixed micelle formulations offer a promising route to stabilizing these nanoparticles for improved delivery of NIR fluorescent contrast agents to tumors for imaging applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5293. doi:10.1158/1538-7445.AM2011-5293