COX2/mPGES1/PGE2 pathway regulates PD-L1 expression in tumor-associated macrophages and myeloid-derived suppressor cells.

In recent years, it has been established that programmed cell death protein ligand 1 (PD-L1)-mediated inhibition of activated PD-1+ T lymphocytes plays a major role in tumor escape from immune system during cancer progression. Lately, the anti-PD-L1 and -PD-1 immune therapies have become an important tool for treatment of advanced human cancers, including bladder cancer. However, the underlying mechanisms of PD-L1 expression in cancer are not fully understood. We found that coculture of murine bone marrow cells with bladder tumor cells promoted strong expression of PD-L1 in bone marrow-derived myeloid cells. Tumor-induced expression of PD-L1 was limited to F4/80+ macrophages and Ly-6C+ myeloid-derived suppressor cells. These PD-L1-expressing cells were immunosuppressive and were capable of eliminating CD8 T cells in vitro. Tumor-infiltrating PD-L1+ cells isolated from tumor-bearing mice also exerted morphology of tumor-associated macrophages and expressed high levels of prostaglandin E2 (PGE2)-forming enzymes microsomal PGE2 synthase 1 (mPGES1) and COX2. Inhibition of PGE2 formation, using pharmacologic mPGES1 and COX2 inhibitors or genetic overexpression of PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH), resulted in reduced PD-L1 expression. Together, our study demonstrates that the COX2/mPGES1/PGE2 pathway involved in the regulation of PD-L1 expression in tumor-infiltrating myeloid cells and, therefore, reprogramming of PGE2 metabolism in tumor microenvironment provides an opportunity to reduce immune suppression in tumor host.

Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9.

Serum deficiency diseases such as alpha-1-antitrypsin deficiency are characterized by reduced function of serum proteins, caused by deleterious genetic mutations. These diseases are promising targets for genetic interventions. Gene therapies using viral vectors have been used to introduce correct copies of the disease-causing gene in preclinical and clinical studies. However, these studies highlighted that disease-alleviating gene expression is lost over time. Integration into a specific chromosomal site could provide lasting therapeutic expression to overcome this major limitation. Additionally, targeted integration could avoid detrimental mutagenesis associated with integrative vectors, such as tumorigenesis or functional gene perturbation. To test if adenoviral vectors can facilitate long-term gene expression through targeted integration, we somatically incorporated the human alpha-1-antitrypsin gene into the ROSA26 "safe harbor" locus in murine livers, using CRISPR/Cas9. We found adenoviral-mediated delivery of CRISPR/Cas9 achieved gene editing outcomes persisting over 200 days. Furthermore, gene knock-in maintained greater levels of the serum protein than provided by episomal expression. Importantly, our "knock-in" approach is generalizable to other serum proteins and supports in vivo cDNA replacement therapy to achieve stable gene expression.

Functional Characterization of ATP-Binding Cassette Transporter A3 Mutations from Infants with Respiratory Distress Syndrome.

Mutations in the ATP-binding cassette transporter A3 gene (ABCA3) result in severe neonatal respiratory distress syndrome and childhood interstitial lung disease. As most ABCA3 mutations are rare or private, determination of mutation pathogenicity is often based on results from in silico prediction tools, identification in unrelated diseased individuals, statistical association studies, or expert opinion. Functional biologic studies of ABCA3 mutations are needed to confirm mutation pathogenicity and inform clinical decision making. Our objective was to functionally characterize two ABCA3 mutations (p.R288K and p.R1474W) identified among term and late-preterm infants with respiratory distress syndrome with unclear pathogenicity in a genetically versatile model system. We performed transient transfection of HEK293T cells with wild-type or mutant ABCA3 alleles to assess protein processing with immunoblotting. We used transduction of A549 cells with adenoviral vectors, which concurrently silenced endogenous ABCA3 and expressed either wild-type or mutant ABCA3 alleles (p.R288K and p.R1474W) to assess immunofluorescent localization, ATPase activity, and organelle ultrastructure. Both ABCA3 mutations (p.R288K and p.R1474W) encoded proteins with reduced ATPase activity but with normal intracellular localization and protein processing. Ultrastructural phenotypes of lamellar body-like vesicles in A549 cells transduced with mutant alleles were similar to wild type. Mutant proteins encoded by ABCA3 mutations p.R288K and p.R1474W had reduced ATPase activity, a biologically plausible explanation for disruption of surfactant metabolism by impaired phospholipid transport into the lamellar body. These results also demonstrate the usefulness of a genetically versatile, human model system for functional characterization of ABCA3 mutations with unclear pathogenicity.

Adenoviral targeting using genetically incorporated camelid single variable domains.

The unique ability of human adenovirus serotype 5 (Ad5) to accomplish efficient transduction has allowed the use of Ad5-based vectors for a range of gene therapy applications. Several strategies have been developed to alter tropism of Ad vectors to achieve a cell-specific gene delivery by using fiber modifications via genetic incorporation of targeting motifs. In this study, we have explored the utility of novel anti-human carcinoembryonic antigen (hCEA) single variable domains derived from heavy chain (VHH) camelid family of antibodies to achieve targeted gene transfer. To obtain anti-CEA VHHs, we produced a VHH-display library from peripheral blood lymphocytes RNA of alpacas at the peak of immune response to the hCEA antigen (Ag). We genetically incorporated an anti-hCEA VHH into a de-knobbed Ad5 fiber-fibritin chimera and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of target cells. We report that the anti-hCEA VHH used in this study retains Ag recognition functionality and provides specificity for gene transfer of capsid-modified Ad5 vectors. These studies clearly demonstrated the feasibility of retargeting of Ad5-based gene transfer using VHHs.

The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

Altered expression of 15-hydroxyprostaglandin dehydrogenase in tumor-infiltrated CD11b myeloid cells: a mechanism for immune evasion in cancer.

Many cancers are known to produce high amounts of PGE(2), which is involved in both tumor progression and tumor-induced immune dysfunction. The key enzyme responsible for the biological inactivation of PGE(2) in tissue is NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). It is well established that cancer cells frequently show down-regulated expression of 15-PGDH, which plays a major role in catabolism of the PGE(2). Here we demonstrate that tumor-infiltrated CD11b cells are also deficient for the 15-PGDH gene. Targeted adenovirus-mediated delivery of 15-PGDH gene resulted in substantial inhibition of tumor growth in mice with implanted CT-26 colon carcinomas. PGDH-mediated antitumor effect was associated with attenuated tumor-induced immune suppression and substantially reduced secretion of immunosuppressive mediators and cytokines such as PGE(2), IL-10, IL-13, and IL-6 by intratumoral CD11b cells. We show also that introduction of 15-PGDH gene in tumor tissue is sufficient to redirect the differentiation of intratumoral CD11b cells from immunosuppressive M2-oriented F4/80(+) tumor-associated macrophages (TAM) into M1-oriented CD11c(+) MHC class II-positive myeloid APCs. Notably, the administration of the 15-PGDH gene alone demonstrated a significant therapeutic effect promoting tumor eradication and long-term survival in 70% of mice with preestablished tumors. Surviving mice acquired antitumor T cell-mediated immune response. This study for the first time demonstrates an important role of the 15-PGDH in regulation of local antitumor immune response and highlights the potential to be implemented to enhance the efficacy of cancer therapy and immunotherapy.

Molecular chemotherapy of pancreatic cancer using novel mutant bacterial cytosine deaminase gene.

The combination of molecular chemotherapy with radiation therapy has the potential to become a powerful approach for treatment of pancreatic cancer. We have developed an adenoviral vector (AdbCD-D314A) encoding a mutant bacterial cytosine deaminase (bCD) gene, which converts the prodrug 5-fluorocytosine (5-FC) into the active drug 5-fluorouracil. The aim of this study was to investigate AdbCD-D314A/5-FC-mediated cytotoxicity in vitro and therapeutic efficacy in vivo alone and in combination with radiation against human pancreatic cancer cells and xenografts. AdbCD-D314A/5-FC-mediated cytotoxicity alone and in combination with radiation was analyzed using crystal violet inclusion and clonogenic survival assays. CD enzyme activity was determined by measuring conversion of [3H]5-FC to [3H]5-fluorouracil after adenoviral infection of pancreatic cancer cells in vitro and pancreatic tumor xenografts by TLC. S.c. pancreatic tumor xenografts were used to evaluate the therapeutic efficacy of AdbCD-D314A/5-FC molecular chemotherapy in combination with radiation therapy. AdbCD-D314A infection resulted in increased 5-FC-mediated pancreatic cancer cell killing that correlated with significantly enhanced CD enzyme activity compared with AdbCDwt encoding wild-type of bCD. Animal studies showed significant inhibition of growth of human pancreatic tumors treated with AdbCD-D314A/5-FC in comparison with AdbCDwt/5-FC. Also, a significantly greater inhibition of growth of Panc2.03 and MIA PaCA-2 tumor xenografts was produced by the combination of AdbCD-D314A/5-FC with radiation compared with either agent alone. The results indicate that the combination of AdbCD-D314A/5-FC molecular chemotherapy with radiation therapy significantly enhanced cytotoxicity of pancreatic cancer cells in vitro and increased therapeutic efficacy against human pancreatic tumor xenografts.

A new model of multi-visceral and bone metastatic prostate cancer with perivascular niche targeting by a novel endothelial specific adenoviral vector.

While modern therapies for metastatic prostate cancer (PCa) have improved survival they are associated with an increasingly prevalent entity, aggressive variant PCa (AVPCa), lacking androgen receptor (AR) expression, enriched for cancer stem cells (CSCs), and evidencing epithelial-mesenchymal plasticity with a varying extent of neuroendocrine transdifferentiation. Parallel work revealed that endothelial cells (ECs) create a perivascular CSC niche mediated by juxtacrine and membrane tethered signaling. There is increasing interest in pharmacological metastatic niche targeting, however, targeted access has been impossible. Here, we discovered that the Gleason 7 derived, androgen receptor negative, IGR-CaP1 cell line possessed some but not all of the molecular features of AVPCa. Intracardiac injection into NOD/SCID/IL2Rg -/- (NSG) mice produced a completely penetrant bone, liver, adrenal, and brain metastatic phenotype; noninvasively and histologically detectable at 2 weeks, and necessitating sacrifice 4-5 weeks post injection. Bone metastases were osteoblastic, and osteolytic. IGR-CaP1 cells expressed the neuroendocrine marker synaptophysin, near equivalent levels of vimentin and e-cadherin, all of the EMT transcription factors, and activation of NOTCH and WNT pathways. In parallel, we created a new triple-targeted adenoviral vector containing a fiber knob RGD peptide, a hexon mutation, and an EC specific ROBO4 promoter (Ad.RGD.H5/3.ROBO4). This vector was expressed in metastatic microvessels tightly juxtaposed to IGR-CaP1 cells in bone and visceral niches. Thus, the combination of IGR-CaP1 cells and NSG mice produces a completely penetrant metastatic PCa model emulating end-stage human disease. In addition, the metastatic niche access provided by our novel Ad vector could be therapeutically leveraged for future disease control or cure.

Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein.

Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses.

Transcriptional targeting of primary and metastatic tumor neovasculature by an adenoviral type 5 roundabout4 vector in mice.

New approaches targeting metastatic neovasculature are needed. Payload capacity, cellular transduction efficiency, and first-pass cellular uptake following systemic vector administration, motivates persistent interest in tumor vascular endothelial cell (EC) adenoviral (Ad) vector targeting. While EC transductional and transcriptional targeting has been accomplished, vector administration approaches of limited clinical utility, lack of tumor-wide EC expression quantification, and failure to address avid liver sequestration, challenged prior work. Here, we intravenously injected an Ad vector containing 3 kb of the human roundabout4 (ROBO4) enhancer/promoter transcriptionally regulating an enhanced green fluorescent protein (EGFP) reporter into immunodeficient mice bearing 786-O renal cell carcinoma subcutaneous (SC) xenografts and kidney orthotopic (KO) tumors. Initial experiments performed in human coxsackie virus and adenovirus receptor (hCAR) transgenic:Rag2 knockout mice revealed multiple ECs with high-level Ad5ROBO4-EGFP expression throughout KO and SC tumors. In contrast, Ad5CMV-EGFP was sporadically expressed in a few tumor vascular ECs and stromal cells. As the hCAR transgene also facilitated Ad5ROBO4 and control Ad5CMV vector EC expression in multiple host organs, follow-on experiments engaged warfarin-mediated liver vector detargeting in hCAR non-transgenic mice. Ad5ROBO4-mediated EC expression was undetectable in most host organs, while the frequencies of vector expressing intratumoral vessels and whole tumor EGFP protein levels remained elevated. In contrast, AdCMV vector expression was only detectable in one or two stromal cells throughout the whole tumor. The Ad5ROBO4 vector, in conjunction with liver detargeting, provides tractable genetic access for in-vivo EC genetic engineering in malignancies.

Retargeting of gene expression using endothelium specific hexon modified adenoviral vector.

Adenovirus serotype 5 (Ad5) vectors are well suited for gene therapy. However, tissue-selective transduction by systemically administered Ad5-based vectors is confounded by viral particle sequestration in the liver. Hexon-modified Ad5 expressing reporter gene under transcriptional control by the immediate/early cytomegalovirus (CMV) or the Roundabout 4 receptor (Robo4) enhancer/promoter was characterized by growth in cell culture, stability in vitro, gene transfer in the presence of human coagulation factor X, and biodistribution in mice. The obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Substitution of the hypervariable region 7 (HVR7) of the Ad5 hexon with HVR7 from Ad serotype 3 resulted in decreased liver tropism and dramatically altered biodistribution of gene expression. The results of these studies suggest that the combination of liver detargeting using a genetic modification of hexon with an endothelium-specific transcriptional control element produces an additive effect in the improvement of Ad5 biodistribution.

Experimental cancer therapy using restoration of NAD+ -linked 15-hydroxyprostaglandin dehydrogenase expression.

Preclinical and clinical evidence shows that cyclooxygenase-2 (Cox-2)-mediated prostaglandin E(2) (PGE(2)) overexpression plays an important role in tumor growth, metastasis, and immunosuppression. It has been shown that expression of NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme responsible for PGE(2) inactivation, is suppressed in the majority of cancers, including breast and colon carcinoma. We have developed adenoviral vectors (Ad) encoding the 15-PGDH gene under control of the vascular endothelial growth factor receptor 1 (VEGFR1/flt-1; Adflt-PGDH) and the Cox-2 (Adcox-PGDH) promoters. The purpose of this study was to investigate cytotoxicity in vitro and therapeutic efficacy in vivo of 15-PGDH-mediated cancer therapy. The levels of PGE(2) and VEGF expression were correlated with PGE(2) receptor and Cox-2 and flt-1 expression in cancer cells. The in vitro study showed that Ad-mediated 15-PGDH expression significantly decreased proliferation and migration of cancer cells. Animal breast and colon tumor therapy studies showed that 15-PGDH gene therapy produced a significant delay in 2LMP and LS174T tumor growth. Combined therapy using 15-PGDH and anti-VEGF antibody (bevacizumab) significantly increased inhibition of growth of LS174T tumor xenografts in comparison with agents alone. These results suggest that 15-PGDH-mediated regulation of PGE(2) catabolism in the tumor microenvironment represents a novel approach for therapy of human breast and colon cancer.

Adenovirus-mediated FLT1-targeted proapoptotic gene therapy of human prostate cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is of particular interest in the development of prostate carcinoma therapeutics as it preferentially induces apoptosis of tumor cells. To employ adenoviral vectors for highly efficient and specific TRAIL gene transfer into cancer cells could overcome some potential problems for recombinant TRAIL. The vascular endothelial growth factor receptor FLT-1 is involved in regulation of angiogenesis and tumor growth, invasion, and metastasis of prostate carcinoma. FLT-1 expression is observed in both tumor endothelial cells and prostate cancer cells. We developed an adenoviral vector encoding the TRAIL gene under control of the FLT1 promoter (AdFlt-TRAIL), which produced endothelial and prostate cancer cell death. The combination of ionizing radiation and adenovirus-driven TRAIL expression overcame human prostate cancer cell resistance to TRAIL. Furthermore, in vivo administration of AdFlt-TRAIL at the site of tumor growth in combination with radiation treatment produced significant suppression of the growth of DU145 human prostate tumor xenografts in athymic nude mice. Our results suggest that specific TRAIL delivery employing the FLT1 promoter can effectively inhibit tumor growth and demonstrate the advantage of combination radiotherapy and gene therapy for the treatment of prostate cancer.