Coxsackie And Adenovirus Receptor Research Articles

Coxsackievirus B3 Infection of Human iPSC Lines and Derived Primary Germ-Layer Cells Regarding Receptor Expression.

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.

Telomerase-specific oncolytic immunotherapy for promoting efficacy of PD-1 blockade in osteosarcoma.

Immune checkpoint inhibitors including anti-programmed cell death 1 (PD-1) antibody have recently improved clinical outcome in certain cancer patients; however, osteosarcoma (OS) patients are refractory to PD-1 blockade. Oncolytic virotherapy has emerged as novel immunogenic therapy to augment antitumor immune response. We developed a telomerase-specific replication-competent oncolytic adenovirus OBP-502 that induces lytic cell death via binding to integrins. In this study, we assessed the combined effect of PD-1 blockade and OBP-502 in OS cells. The expression of coxsackie and adenovirus receptor (CAR), integrins αvβ3 and αvβ5, and programmed cell death ligand 1 (PD-L1) was analyzed in two murine OS cells (K7M2, NHOS). The cytopathic activity of OBP-502 in both cells was analyzed using the XTT assay. OBP-502-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). Subcutaneous tumor models for K7M2 and NHOS cells were used to evaluate the antitumor effect and number of tumor-infiltrating CD8+ cells in combination therapy. K7M2 and NHOS cells showed high expression of integrins αvβ3 and αvβ5, but not CAR. OBP-502 significantly suppressed the viability of both cells, in which PD-L1 expression and the release of ATP and HMGB1 were significantly increased. Intratumoral injection of OBP-502 significantly augmented the efficacy of PD-1 blockade on subcutaneous K2M2 and NHOS tumor models via enhancement of tumor-infiltrating CD8+ T cells. Our results suggest that telomerase-specific oncolytic virotherapy is a promising antitumor strategy to promote the efficacy of PD-1 blockade in OS.

Combination of rAd-p53 in situ gene therapy and anti-PD-1 antibody immunotherapy induced anti-tumor activity in mouse syngeneic urogenital cancer models

In this study we undertook a novel combination therapy using rAd-p53 in situ gene therapy and immunotherapy with immune checkpoint inhibitor (ICI) anti-PD-1 antibody for urogenital cancers. Three mouse syngeneic tumor cell lines, TRAMP-C2 (prostate cancer derived from C57BL/6 mice), MBT-2 (bladder cancer derived from C3H mice) and Renca (kidney cancer derived from BALB/c mice) were used in this study. The highest coxsackie and adenovirus receptor (CAR) mRNA expression was observed in TRAMP-C2 cells, followed by Renca and then MBT-2 cells. Consistent with the CAR expressions, rAd-p53 at 160 multiplicity of infection (MOI) significantly inhibited the cell proliferation of TRAMP-C2 and Renca cells, but not MBT-2 cells. In in vivo experiments, the combination of intratumoral injections of rAd-p53 (1 × 109 plaque-forming units) every other day and intraperitoneal injections of anti-mouse PD-1 antibody (200 μg) twice a week suppressed tumor growth and prolonged survival compared to rAd-p53 or anti-PD-1 antibody monotherapy in both the TRAMP-C2 and Renca models. Our results encourage the clinical development of combination therapy comprised of in situ gene therapy with rAd-p53 and immunotherapy with an ICI anti-PD-1 antibody for urogenital cancers.

Extracellular Vesicles-Mimetic Encapsulation Improves Oncolytic Viro-Immunotherapy in Tumors With Low Coxsackie and Adenovirus Receptor.

The oncolytic adenovirus (Adv) exhibited poor infection efficiency in tumor cells with low coxsackie and adenovirus receptor (CAR) on the cell surface, which limits the therapeutic efficacy of the Adv-mediated cancer gene therapy. In addition, the abundant adenovirus neutralizing antibodies also abrogate the viral infection of cancer cells. Therefore, novel strategies are required to overcome these two major hurdles to improve the Adv-mediated cancer virotherapy. We constructed a recombinant adenovirus expressing the extracellular domain of PD1 (Ad5-P). The 293T cells expressing VSV-G protein on the cell surface (293T-VSV-G) were infected with Ad5-P. Then Ad5-P infected 293T-VSV-G cells were harvested and squeezed stepwisely through a serial of polycarbonate membranes. Next, the extracellular vesicles-mimetic (EVM) encapsulated Ad5-P (EVM/VSV-G Ad5-P) were collected by density gradient centrifugation. In cell lines with low CAR expression, EVM/VSV-G Ad5-P showed a significantly improved infection efficiency, oncolytic ability, and soluble PD-1 production. In passively immunized mice with Ad5 neutralizing antibody, EVM/VSV-G Ad5-P successfully escaped from antibodies, and the soluble PD-1expression of Ad5-P was significantly prolonged. Finally, EVM/VSV-G Ad5-P treatment significantly improved the antitumor immune responses and prolonged survival of mice with HCC ascites. The EVM/VSV-G Ad5-P not only bypasses the limitation of low CAR expression in tumor cells to improve the viral entry, but also significantly protects the virus from the neutralization antibodies. The EVM encapsulation technology can be successfully used for loading of non-enveloped viruses to generate the extracellular vesicle-mimetic encapsulated viral particles. Our results provide a novel strategy in OVs manufacture to improve the efficacy of tumor oncolytic virotherapy.

The role of JAML-CAR costimulation in gamma delta T cell-mediated repair of the intestinal epithelium

Abstract Tissue-resident γδ T cells are key players in the maintenance of the body’s epithelial barrier surfaces and perform an essential role in promoting tissue repair. Although γδ IELs are important coordinators of repair in the intestinal epithelium, relatively little is known about how these cells recognize and respond to damage. Recognition of skin wounds by γδ T cells requires costimulation through the junctional adhesion molecule-like protein (JAML) by ligation of coxsackie and adenovirus receptor (CAR), raising the possibility that these molecules may have a similar function in the gut. Here we evaluated the role of JAML-CAR costimulation in the activation and function of γδ IELs using the dextran sulfate sodium (DSS) model of intestinal injury and repair. Following DSS treatment we found that γδ IELs upregulated their surface expression of JAML and observed a shift in the isoforms of CAR expressed by intestinal epithelial cells. In addition, JAML−/− mice exhibited more severe weight loss and epithelial damage than wildtype animals in response to DSS treatment. While we saw that JAML−/− mice had lower numbers of IELs in the colon, this difference did not account for the more severe response to DSS-induced injury in these animals. Importantly, the activation of γδ IELs following DSS treatment was reduced in the absence of JAML-CAR costimulation. In addition, we found evidence of impaired γδ IEL production of IL-17, a cytokine known to protect against DSS-induced epithelial injury. Taken together our results suggest that JAML-CAR costimulation may be needed for the activation of tissue repair functions in γδ T cells across barrier tissues and supports targeting this pathway for therapeutic approaches for the acceleration of healing.

Naturally occurring variants in the transmembrane and cytoplasmic domains of the human Coxsackie- and adenovirus receptor have no impact on virus internalisation

The Coxsackie- and adenovirus receptor (CAR) mediates homophilic cell-cell contacts and susceptibility to both human pathogenic viruses through its membrane-distal immunoglobulin domain. In the present study, we screened five missense variants of the human CAR gene for their influence on adenovector or Coxsackievirus entry into Chinese hamster ovary cells. The CAR variants facilitated virus internalisation to a similar extent as wild type CAR. This underlines CAR’s presumed invariance and essential physiological role in embryogenesis.

Prospective virome analyses in young children at increased genetic risk for type 1 diabetes.

Viruses are implicated in the autoimmune destruction of the pancreatic islet β-cells that results in insulin deficiency and type 1 diabetes (T1D)1–4. Certain enteroviruses can infect β-cells in vitro5, have been detected in pancreatic islets of T1D patients6 and shown an association with T1D in meta-analyses4. However, establishing consistency of findings across studies has proved difficult. Obstacles to convincingly link RNA viruses to islet autoimmunity may be attributed to rapid viral mutation rates, cyclical periodicity of viruses7 and the selection of variants with altered pathogenicity and ability to spread in populations. β-cells strongly express cell surface Coxsackie and adenovirus receptor (CXADR) genes, which can facilitate enterovirus infection8. Studies of human pancreata and cultured islets have shown significant variation in enteroviral virulence to β-cells between serotypes and within the same serotype9–10. In this large-scale study of known eukaryotic DNA and RNA viruses in stools from children, we evaluated fecally shed viruses in relation to islet autoimmunity and T1D. This study demonstrated prolonged Enterovirus B (EV-B) rather than independent, short duration EV-B infections may be involved in the development of islet autoimmunity, but not T1D, in some young children. Furthermore, we found fewer early life Human mastadenovirus C infections and CXADR rs6517774 independently correlated with islet autoimmunity.

Abstract 3231: Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma

Abstract Background: Osteosarcoma (OS) is the most frequent primary malignant tumor of bone in children and adolescents. Although immune checkpoint inhibitor, anti-programmed death protein 1 (PD-1) antibody, has dramatically improved the clinical outcome in some cancer patients, OS patients are less sensitive to PD-1 blockade due to poor immune responses. Recently, oncolytic virotherapy has been shown to stimulate the immune system through induction of immunogenic cell death (ICD). We recently developed a RGD fiber-modified telomerase-specific oncolytic adenovirus OBP-502, which can enter into tumor cells by binding to cell surface integrin and induce oncolytic cell death in a telomerase-dependent manner. In this study, we assessed the in vitro and in vivo antitumor efficacy of combination therapy with PD-1 blockade and OBP-502 in OS cells. Methods: We used 2 murine OS cell lines, K7M2 and NHOS. The expression of PD-L1, coxsackie and adenovirus receptor (CAR), and integrin on the cell surface was analyzed by flow cytometric analysis. We analyzed the in vitro antitumor effect of OBP-502 using XTT assay and western blot analysis. Virus-induced ICD was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). To evaluate the therapeutic potential of oncolytic immunotherapy, we investigated the in vivo antitumor effect of combination therapy with anti-PD-1 antibody and OBP-502 using a subcutaneous K7M2 xenograft tumor model. Moreover, the number of tumor-infiltrating CD8+, CD4+ and Foxp3+ T cells was analyzed by immunohistochemistry. Results: Flow cytometric analysis demonstrated that K7M2 and NHOS cells had the expression of PD-L1 and integrin, but not CAR. XTT assay showed that OBP-502 efficiently suppressed the viability of K7M2 and NHOS cells in a dose-dependent manner. Western blot analysis revealed that OBP-502 induced the apoptosis-related cell death in K7M2 and NHOS cells. ELISA assay demonstrated that OBP-502 significantly increased the level of extracellular ATP and HMGB1 in K7M2 and NHOS cells. In vivo experiment using a subcutaneous K7M2 xenograft tumor model showed that combination of anti-PD-1 antibody and OBP-502 significantly reduced tumor growth compared to mock treatment or monotherapy. Immunohistochemical analysis revealed that OBP-502 significantly increased the number of tumor-infiltrating CD8+ T cells compared to mock treatment or monotherapy. By contrast, OBP-502 did not affect the number of CD4+ and Foxp3+ T cells in tumor tissues. Conclusion: These results suggest that oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus is a promising antitumor strategy to promote the therapeutic potential of PD-1 blockade in OS through stimulation of antitumor immune response. Citation Format: Koji Demiya, Hiroshi Tazawa, Yusuke Mochizuki, Miho Kure, Joe Hasei, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Oncolytic immunotherapy with PD-1 blockade and telomerase-specific oncolytic adenovirus in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3231.

Abstract 4776: Characterizing an oncolytic adenovirus modified with the CXCL12 ligand for breast cancer therapy

Abstract Breast cancer is the most commonly diagnosed cancer in women, making up nearly 30% of all diagnosed cancer cases each year. While localized breast cancer is easily treated, advanced cases are difficult to treat resulting in poor five-year survival rates. Thus, new therapies capable of increasing treatment efficacy are needed. Oncolytic virotherapy using human adenovirus is a novel therapeutic approach capable of specifically targeting cancer cells while sparing normal tissue. The adenovirus vector is well-characterized and is uniquely suited for oncolytic virotherapy due to its systemic stability, good safety profile and the ability to infect a broad range of dividing and non-dividing cells. Replication of oncolytic adenoviruses within cancer cells causes the lysis of the cells and subsequent spread of progeny virions within the surrounding tumor stroma. One challenge in targeting cancer cells with adenovirus vectors has been the low expression of the endogenous receptor, the Coxsackie and adenovirus receptor (CXADR), prompting the search for new receptor targets. In this study, we engineered an adenovirus to contain the CXCL12 ligand to target the chemokine receptors CXCR4 and CXCR7 overexpressed in a variety of tumors. Altered expression of these receptors drives tumor progression, migration, invasion and metastasis. Previously, we developed a bispecific adaptor molecule containing the CXCL12 ligand to redirect a replication-deficient wild-type adenovirus to breast cancer cells overexpressing CXCR4. In the current study, we engineered a replication-competent oncolytic adenovirus modified with the CXCL12 ligand to target cancer cells overexpressing CXCR4 and CXCR7. A recombinant gene was constructed using the tail domain of the adenovirus fiber gene fused to the trimerization domain of the T4 fibritin gene followed by the mature CXCL12 sequence. After viral production, the presence of a modified fiber and the CXCL12 ligand were confirmed. Subsequently, the vector was tested in a panel of breast cancer cells for infection and cell killing efficacy in vitro. Receptor knock down and overexpression studies confirmed the specificity of the virus. Together, these studies address the hypothesis that constructing a retargeted oncolytic adenovirus using the CXCL12 ligand provides selective infection and killing of breast cancer cells overexpressing CXCR4 and CXCR7. These results support the rationale for further development of retargeted adenovirus vectors for oncolytic virotherapy in patients. Future experiments will be conducted to assess tumor targeting using a xenograft mouse model of human breast cancer. Citation Format: Samia M. O'Bryan, J. Michael Mathis. Characterizing an oncolytic adenovirus modified with the CXCL12 ligand for breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4776.

Abstract 2873: Elimination of MYCN-amplified neuroblastoma cells by telomerase-targeted oncolytic virotherapy as novel precision medicine

Abstract Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Recent whole-genome sequencing studies have demonstrated that high-risk NB with unfavorable clinical outcome is characterized by amplification of MYCN oncogene and rearrangement of human telomerase reverse transcriptase (hTERT) gene. Since hTERT expression is transcriptionally and commonly activated in high-risk NB, hTERT activation would be an attractive therapeutic target for the treatment of high-risk NB. To eliminate hTERT-activated malignant tumor cells, we have developed two types of hTERT-driven oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702. In this study, we investigated the therapeutic potential of hTERT-driven oncolytic adenoviruses OBP-301 and OBP-702 in MYCN-amplified NB cells. Methods: We used 4 human NB cell lines with MYCN amplification (IMR-32, CHP-134, NB-1, LA-N-5). The expression of cell surface coxsackie and adenovirus receptor (CAR) and hTERT was analyzed using flow cytometric analysis and real-time RT-PCR. We assessed the in vitro antitumor effect of OBP-301 and OBP-702 using a XTT assay. The molecular mechanism of virus-mediated antitumor effect was investigated in aspect of apoptosis, autophagy, and MYCN expression. To address the underlying mechanism of virus-mediated MYCN modulation, we analyzed the role of E2F1 and p53 in the virus-infected NB cells. The in vivo antitumor effect of OBP-301 and OBP-702 was assessed using a subcutaneous CHP-134 xenograft tumor model. Results: All MYCN-amplified NB cells showed CAR and hTERT expression. OBP-301 and OBP-702 showed profound antitumor effect in association with autophagy-related cell death in all MYCN-amplified NB cells. In the virus-infected NB cells, E2F1 expression was inversely correlated with MYCN expression. Virus-induced E2F1 and p53 expression suppressed the MYCN expression in the MYCN-amplified NB cells. OBP-301 and OBP-702 significantly suppressed tumor growth in a subcutaneous CHP-134 xenograft tumor model. Conclusion: Our results suggest that hTERT-driven oncolytic adenoviruses OBP-301 and OBP-702 are promising antitumor agents for eliminating the MYCN-amplified NB cells through E2F1- and p53-mediated suppression of oncogenic MYCN protein. Further clinical study is warranted to evaluate the safety and feasibility of hTERT-driven oncolytic virotherapy as novel precision medicine for high-risk NB patients. Citation Format: Morimichi Tani, Hiroshi Tazawa, Terutaka Tanimoto, Hiroshi Nouso, Yasuo Urata, Shunsuke Kagawa, Takuo Noda, Toshiyoshi Fujiwara. Elimination of MYCN-amplified neuroblastoma cells by telomerase-targeted oncolytic virotherapy as novel precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2873.

Abstract 4774: Engineering an oncolytic adenovirus to target CEA in colorectal cancer

Abstract Colorectal cancer is the second leading cause of cancer-related deaths in the United States and the third most commonly diagnosed cancer overall. Screening methods can identify precancerous polyps before they progress, leading to five-year survival rates of 92%. However, if diagnosed in later stages the survival rates decrease to 12%. While treatment options are available for advanced stages, they remain inadequate to affect overall survival. Thus, new therapies are needed to improve treatment outcomes. Oncolytic virotherapy is a unique therapeutic approach in which a virus is genetically modified to target cancer cells while sparing normal tissue. Oncolytic virotherapy takes advantage of the lytic properties of the viral life cycle. These viruses are used replicate inside cancer cells, lyse them and spread to adjacent cells throughout the tumor. The adenovirus is a well-characterized vector that is easily manipulated and produced and is capable of infecting a wide range of cells. However, the endogenous receptor for adenovirus, the Coxsackie and adenovirus receptor (CXADR), is often downregulated in cancers, thus decreasing the ability to selectively infect cancer cells. In this study, an adenovirus was genetically engineered to present the single variable domain (VHH) derived from a camelid heavy chain antibody B2. The B2 antibody recognizes the human carcinoembryonic antigen (CEA), which is often overexpressed in a variety of cancers, including colorectal cancer. Here, the wild-type fiber and knob domain of the adenovirus fiber gene was replaced with a modified T4 fibritin fiber linked to the B2 VHH. To provide for visual tracking of adenovirus infection and biodistribution, a red fluorescent protein (RFP) marker gene was also fused to the capsid protein IX (pIX). This virus was rescued and amplified in the HEK293 F28 packaging cell line with a final round of amplification in HEK293 cells. The virus was purified by CsCl ultracentrifugation and dialysis. The resulting viral stock was titered and characterized for the presence of the modified fiber and knob. Subsequently, the vector was modified to express the human sodium-iodide symporter (NIS) transgene, to allow for noninvasive imaging of the adenovirus uptake and replication as well as radiotherapy using 131I. Further experiments will be completed to confirm viral selectivity for CEA and subsequent lysis of colorectal cancer cells. The results of this study will advance targeting oncolytic adenoviruses to metastatic colorectal cancer. Note: This abstract was not presented at the meeting. Citation Format: Jessica C. Schaumburg. Engineering an oncolytic adenovirus to target CEA in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4774.

A survivin-responsive, conditionally replicating adenovirus induces potent cytocidal effects in adult T-cell leukemia/lymphoma

BackgroundAdult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy caused by long-term human T-cell leukemia virus type I (HTLV-1) infection. Survivin-responsive, conditionally replicating adenoviruses regulated by multiple tumor-specific factors (Surv.m-CRAs), in which the expression of the adenoviral early region 1A gene is regulated by the survivin (BIRC5) promoter, can be used to treat several cancers. As survivin is overexpressed in ATL, we examined the effects of Surv.m-CRAs on ATL-selective replication and survival.MethodsWe tested two ATL cell lines and four HTLV-1-infected T-cell lines. The cells were subjected to infection with either E1-deleted, replication-defective adenoviruses or Surv.m-CRAs at various multiplicities of infection.ResultsStrong activation of survivin promoter was observed in all six cell lines. Moreover, the expression of the coxsackie and adenovirus receptor (CAR), which is important for adenoviral infection, was high in the cell lines. In contrast, we observed the absence of survivin promoter activity and a low expression of CAR in activated peripheral blood lymphocytes (PBLs) from healthy subjects. Surv.m-CRAs actively replicated and induced cytocidal effects in five out of six cell lines; conversely, we observed minimal viral replication and no marked cytotoxicity in normal activated PBLs.ConclusionsThis is the first report demonstrating that Surv.m-CRAs constitute attractive potential anti-ATL agents.

CEACAM-1 promotes myocardial injury following coxsackievirus infection by regulating the coxsackievirus-adenovirus receptor.

Objective:To determine the effects and mechanism of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1, CC1)-mediated regulation of the Coxsackie and Adenovirus Receptor (CAR) after Coxsackievirus B3 (CVB3) infection.Methods:A mouse CC1 overexpression recombinant virus was constructed, followed by insertion of a pLVX-CEACAM 1-zsgreen-puro (rLV-CEACAM 1) plasmid into the recombinant retrovirus. Cardiac myocytes were assigned into different groups according to various treatments. The apoptosis rate and cell activity in each group were observed. Further, CAR expression and SYK, IL-1β, and p-SYK levels were measured.Results:The recombinant retrovirus titer was measured as 1.5 × 108 TUs/ml. The apoptosis rate of cardiac myocytes in the CC1 overexpression plus CVB3 group was significantly elevated, and the relative expression of the CAR gene was the highest in the CC1 overexpression plus CVB3 group. TNF-α and IL-1β levels increased due to CC1 overexpression and further increased after CVB3 infection. CAR protein expression also changed along with the levels of CC1, SYK, and TNF-α after infection.Conclusion:CC1 may promote CAR expression after CVB3 infection and regulate CAR protein expression by activating the CC1-SYK-TNF-α signaling axis during the infection process.

JAML-CAR – A Mechanism of T Cell Antitumor Immunity

Abstract The junctional adhesion molecule-like protein (JAML) is expressed by a variety of immune cell subsets and binds the Coxsackie and adenovirus receptor (CAR), an adhesion molecule expressed in epithelial tissues. Previous work from our lab demonstrated that JAML-CAR interactions are a unique costimulatory mechanism that controls activation of tissue-resident γδ T cells. High expression of JAML within tumors is associated with improved patient survival in a variety of epithelial cancers, yet the role of JAML-CAR interactions in antitumor immunity has not been characterized. Based on this evidence and the fact that CAR is expressed in epithelial tissues where many tumors arise, we hypothesized that JAML-CAR interactions are critical for antitumor immunity. Initial results utilizing the B16F10 melanoma model show that JAML−/− mice are more susceptible to tumor growth and have fewer γδ and CD8 T cells within tumors compared to WT mice. In WT mice, JAML is upregulated on γδ and CD8 T cells following activation, and a large fraction of γδ and CD8 tumor-infiltrating lymphocytes express JAML. Expression of CAR in mouse B16F10 melanoma is detectable within tumor margins but not within the tumor interior, and in human melanoma, CAR expression decreases with increased malignancy. This data suggests that tumors can downregulate CAR to escape immune responses. In support of this hypothesis, overexpression of CAR on B16F10 melanoma results in slower tumor formation and growth in WT mice but not RAG−/− or JAML−/− mice, which indicates that expression of CAR sensitives tumors to immune detection. These results show that JAML-CAR interactions are critical for T cell antitumor immunity and may be a novel therapeutic target to treat epithelial cancers.

Novel Junctional Adhesion Molecule‐Like (JAML) Regulates Neutrophil Transepithelial Migration During Intestinal Inflammation

Neutrophil (PMN) migration in the intestine is an essential component of host defense and wound repair. However, dysregulated PMN recruitment is a hallmark of inflammatory bowel disease (IBD). Disease flares in IBD are associated with robust PMN migrating across the intestinal mucosa resulting in crypt abscess formation and mucosal ulceration. Despite strong association of PMN infiltration into the gut with patient symptoms in IBD, the molecular basis of PMN transepithelial migration (TEpM) represents a significantly understudied area. It is now appreciated that PMN migration into intestinal mucosa requires multiple adhesive interactions between migrating PMN and intestinal epithelial cells (IECs), that represents a multi‐step process facilitated by complex interactions between PMN and IEC expressed proteins including members of the Junctional Adhesion Molecules (JAMs) family. We have reported that the novel JAM‐like protein termed JAML expressed on PMN, associates with the coxsackie and adenovirus receptor (CAR) expressed on epithelial cells. Binding of soluble JAML ectodomains to CAR has been shown to disrupt JAML‐CAR interactions and reduce PMN TEpM in‐vitro. To define the molecular mechanism of JAML regulation of PMN TEpM during intestinal inflammation in‐vivo, we generated JAML knockout mice (JAML−/−). PMN TEpM was evaluated using a small intestinal loop model, and PMN migration in response to the chemoattractant LTB4 (1nM) quantified by flow cytometry. While WT and JAML−/− mice had similar numbers of circulating leukocytes, we observed reduced PMN TEpM in‐vivo in JAML−/− mice compared to WT animals. This decrease in PMN traffic into the lumen was associated with increased retention of PMN within the intestinal epithelium in JAML−/− mice. In addition, epithelial accumulation of PMN in JAML−/− mice paralleled an increase in intestinal permeability to a 4KDa FITC‐dextran as detected by fluorescence intensity in serum. Finally, we studied the role of JAML on susceptibility to DSS induced colitis. Loss of JAML markedly increased disease activity index, inflammation and mucosal ulceration compared to WT mice. Overall, these observations suggest that JAML is an important adhesion molecule that regulates PMN TEpM in the intestinal mucosa. JAML thus represents a new therapeutic target to dampen excessive PMN migration during intestinal inflammation as seen in IBD.Support or Funding InformationCCFA CDA454814 and NIH 3R01 DK072564‐23S1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Diversity within the adenovirus fiber knob hypervariable loops influences primary receptor interactions

Adenovirus based vectors are of increasing importance for wide ranging therapeutic applications. As vaccines, vectors derived from human adenovirus species D serotypes 26 and 48 (HAdV-D26/48) are demonstrating promising efficacy as protective platforms against infectious diseases. Significant clinical progress has been made, yet definitive studies underpinning mechanisms of entry, infection, and receptor usage are currently lacking. Here, we perform structural and biological analysis of the receptor binding fiber-knob protein of HAdV-D26/48, reporting crystal structures, and modelling putative interactions with two previously suggested attachment receptors, CD46 and Coxsackie and Adenovirus Receptor (CAR). We provide evidence of a low affinity interaction with CAR, with modelling suggesting affinity is attenuated through extended, semi-flexible loop structures, providing steric hindrance. Conversely, in silico and in vitro experiments are unable to provide evidence of interaction between HAdV-D26/48 fiber-knob with CD46, or with Desmoglein 2. Our findings provide insight into the cell-virus interactions of HAdV-D26/48, with important implications for the design and engineering of optimised Ad-based therapeutics.

P147 NOVEL JUNCTIONAL ADHESION MOLECULE-LIKE (JAML) REGULATES NEUTROPHIL TRANSEPITHELIAL MIGRATION DURING INTESTINAL INFLAMMATION

Neutrophil (PMN) migration in the intestine is an essential component of host defense and wound repair. However, dysregulated PMN recruitment is a hallmark of chronic active inflammatory bowel disease (IBD) characterized by persistent inflammation. Disease flares in IBD are associated with marked recruitment of PMN into the intestinal mucosa that results in crypt abscess formation and mucosal ulcerations. Despite the strong association with IBD, the process of PMN transepithelial migration (TEpM) represents a significantly understudied area. PMN migration into the intestinal mucosa requires multiple adhesive interactions between migrating PMN and intestinal epithelial cells (IECs). PMN TEpM is a multi-step process facilitated by complex interactions between PMN and IEC expressed proteins including members of the Juntional Adhesion Molecules (JAMs) family. We have reported that the novel JAM-like protein termed JAML expressed on PMN, associates with the coxsackie and adenovirus receptor (CAR) on epithelial cells. Binding of recombinant JAML ectodomains disrupts JAML-CAR interactions and reduces PMN TEpM. To define the molecular mechanism of JAML regulation of PMN TEpM during intestinal inflammation in-vivo, we generated JAML knockout mice (JAML-/-). PMN TEpM was evaluated using a small intestinal loop model, and PMN migration into the intestinal lumen in response to the chemoattractant LTB4(1nM) for 1h was quantified by flow cytometry. While WT and JAML-/- mice had similar numbers of circulating leukocytes, we observed reduced PMN migration into the intestinal lumen in response to LTB4 in JAML-/- mice compared to WT animals. In addition, there were increased PMN in the epithelial cell fractions derived from intestines of JAML-/- mice suggesting that JAML regulates PMN TEpM in vivo. We also assessed the role of JAML on susceptibility to DSS induced colitis and recovery. Markedly increased disease activity index, inflammation and mucosal ulceration were observed in JAML-/- mice compared to controls. These observations suggest that JAML represents an important adhesion molecule that regulates PMN TEpM. Furthermore, loss of JAML increases susceptibility to colitis. JAML may thus represent a new therapeutic target to dampen excessive PMN migration during intestinal inflammation as seen in IBD.

CXADR-Mediated Formation of an AKT Inhibitory Signalosome at Tight Junctions Controls Epithelial-Mesenchymal Plasticity in Breast Cancer.

Tight junctions (TJ) act as hubs for intracellular signaling pathways controlling epithelial cell fate and function. Deregulation of TJ is a hallmark of epithelial-mesenchymal transition (EMT), which contributes to carcinoma progression and metastasis. However, the signaling mechanisms linking TJ to the induction of EMT are not understood. Here, we identify a TJ-based signalosome, which controls AKT signaling and EMT in breast cancer. The coxsackie and adenovirus receptor (CXADR), a TJ protein with an essential yet uncharacterized role in organogenesis and tissue homeostasis, was identified as a key component of the signalosome. CXADR regulated the stability and function of the phosphatases and AKT inhibitors PTEN and PHLPP2. Loss of CXADR led to hyperactivation of AKT and sensitized cells to TGFβ1-induced EMT. Conversely, restoration of CXADR stabilized PHLPP2 and PTEN, inhibited AKT, and promoted epithelial differentiation. Loss of CXADR in luminal A breast cancer correlated with loss of PHLPP2 and PTEN and poor prognosis. These results show that CXADR promotes the formation of an AKT-inhibitory signalosome at TJ and regulates epithelial-mesenchymal plasticity in breast cancer cells. Moreover, loss of CXADR might be used as a prognostic marker in luminal breast cancer. SIGNIFICANCE: The tight junction protein CXADR controls epithelial-mesenchymal plasticity in breast cancer by stabilizing the AKT regulators PTEN and PHLPP2.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/47/F1.large.jpg.

Expression of coxsackie and adenovirus receptor is correlated with inferior prognosis in liver cancer patients.

The coxsackie and adenovirus receptor (CAR), a tumor suppressor, is vital for the effectiveness of therapies which utilize the adenovirus. However, studies on CAR expression in hepatocellular carcinoma (HCC) are conflicting and its clinical significance requires exploration. In this study, immunohistochemistry has been carried out on tissue microarrays consisting of 198 pairs of HCC and neighboring healthy tissue specimens from Chinese Han patients to evaluate CAR expression. Relative to normal tissues, decreased CAR expression (56% vs. 57%; P>0.05) was detected in HCC samples. CAR immunopositivity in tumors was not dependent upon sex, age, tumor dimensions, differentiation, TNM stage or metastasis in HCC patients; however, positive expression was observed in 56% of the samples from patients with hepatic metastasis, which was the same as those devoid of metastasis (56%; P=0.042). Furthermore, survival analysis confirmed that the expression of CAR revealed no correlation with the prognosis. It was established that CAR exerted complex effects during liver tumorigenesis, potentially based on the stage of the cancer. Therefore, CAR expression analysis has to be carried out prior to adenoviral oncolytic therapy to stratify the patients.

Ad5NULL-A20: A Tropism-Modified, αvβ6 Integrin-Selective Oncolytic Adenovirus for Epithelial Ovarian Cancer Therapies.

Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvβ6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues.Experimental Design: Ad5NULL-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvβ6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity in vitro and in vivo was assessed.Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5NULL-A20 via coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins, and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and selectively transduced αvβ6+ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of preexisting anti-Ad5 immunity. In vivo biodistribution of Ad5NULL-A20 following systemic delivery in non-tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 107-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5NULL-A20-treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5.Conclusions: Oncolytic Ad5NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvβ6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. Clin Cancer Res; 24(17); 4215-24. ©2018 AACR.