A dynamical systems model for combinatorial cancer therapy enhances oncolytic adenovirus efficacy by MEK-inhibition

Oncolytic adenoviruses, such as ONYX-015, have been tested in clinical trials for currently untreatable tumors, but have yet to demonstrate adequate therapeutic efficacy. The extent to which viruses infect targeted cells determines the efficacy of this approach but many tumors down-regulate the Coxsackievirus and Adenovirus Receptor (CAR), rendering them less susceptible to infection. Disrupting MAPK pathway signaling by pharmacological inhibition of MEK up-regulates CAR expression, offering possible enhanced adenovirus infection. MEK inhibition, however, interferes with adenovirus replication due to resulting G1-phase cell cycle arrest. Therefore, enhanced efficacy will depend on treatment protocols that productively balance these competing effects. Predictive understanding of how to attain and enhance therapeutic efficacy of combinatorial treatment is difficult since the effects of MEK inhibitors, in conjunction with adenovirus/cell interactions, are complex nonlinear dynamic processes. We investigated combinatorial treatment strategies using a mathematical model that predicts the impact of MEK inhibition on tumor cell proliferation, ONYX-015 infection, and oncolysis. Specifically, we fit a nonlinear differential equation system to dedicated experimental data and analyzed the resulting simulations for favorable treatment strategies. Simulations predicted enhanced combinatorial therapy when both treatments were applied simultaneously; we successfully validated these predictions in an ensuing explicit test study. Further analysis revealed that a CAR-independent mechanism may be responsible for amplified virus production and cell death. We conclude that integrated computational and experimental analysis of combinatorial therapy provides a useful means to identify treatment/infection protocols that yield clinically significant oncolysis. Enhanced oncolytic therapy has the potential to dramatically improve non-surgical cancer treatment, especially in locally advanced or metastatic cases where treatment options remain limited.     

Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape

Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.     

Expression of coxsackie and adenovirus receptor distinguishes transitional cancer states in therapy-induced cellular senescence

Therapy-induced cellular senescence describes the phenomenon of cell cycle arrest that can be invoked in cancer cells in response to chemotherapy. Sustained proliferative arrest is often overcome as a contingent of senescent tumor cells can bypass this cell cycle restriction. The mechanism regulating cell cycle re-entry of senescent cancer cells remains poorly understood. This is the first report of the isolation and characterization of two distinct transitional states in chemotherapy-induced senescent cells that share indistinguishable morphological senescence phenotypes and are functionally classified by their ability to escape cell cycle arrest. It has been observed that cell surface expression of coxsackie and adenovirus receptor (CAR) is downregulated in cancer cells treated with chemotherapy. We show the novel use of surface CAR expression and adenoviral transduction to differentiate senescent states and also show in vivo evidence of CAR downregulation in colorectal cancer patients treated with neoadjuvant chemoradiation. This study suggests that CAR is a candidate biomarker for senescence response to antitumor therapy, and CAR expression can be used to distinguish transitional states in early senescence to study fundamental regulatory events in therapy-induced senescence.     

An Adenovirus Vector with Genetically Modified Fibers Demonstrates Expanded Tropism via Utilization of a Coxsackievirus and Adenovirus Receptor-Independent Cell Entry Mechanism

Recombinant adenoviruses (Ad) have become the vector system of choice for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to cells expressing marginal levels of the coxsackievirus and adenovirus receptor (CAR). In order to achieve CAR-independent gene transfer by Ad vectors in clinically important contexts, we proposed modification of viral tropism via genetic alterations to the viral fiber protein. We have shown that incorporation of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob domain results in the ability of the virus to utilize an alternative receptor during the cell entry process. We have also demonstrated that due to its expanded tissue tropism, this novel vector is capable of efficient transduction of primary tumor cells. An increase in gene transfer to ovarian cancer cells of 2 to 3 orders of magnitude was demonstrated by the vector, suggesting that recombinant Ad containing fibers with an incorporated RGD peptide may be of great utility for treatment of neoplasms characterized by deficiency of the primary Ad type 5 receptor.     

Proteasome inhibitor MG-132 modifies coxsackie and adenovirus receptor expression in colon cancer cell line lovo

The efficacy of adenovirus vector-based cancer gene therapy is controversial. Its uptake by cells in many cases requires the major receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR). Low transduction is believed to be one of the main barriers as the expression of CAR on tumor cells is frequently reduced. Increasing CAR expression on tumor cells thus offers a promising opportunity for more effective adenovirus based treatment. Expression of CAR in 62 cases of colon tumor specimens were examined with immunohistochemistry. To modify the CAR expression, the effects of proteasome inhibitor MG132 on CAR expression of colon cancer cell lines were determined by flow cytometry, RT-PCR, and western blot. To evaluate adenovirus transfer, we further used rAd.EGFP, rAd.p53, and oncolytic adenovirus to infect target cells. The CAR expression was significantly decreased in colon carcinomas, both in primary tumors and lymphonode metastasis. Though the deregulation of CAR occurred in early disease and showed no relationship with TNM stage, when primary tumors are more than 5 cm in diameter, this deregulation becomes more frequent. More importantly, proteasome inhibitor MG-132 could enhance CAR expression in colon carcinoma cell line lovo, accompanied with enhanced adenovirus transfer, target gene expression, and oncolysis. These data provide a rational basis for evaluation of CAR expression in tumors and pretreatment with CAR conditioner prior to adenovirus vector-based gene therapy.     

Enhancement of anti-DIII antibodies by the C3d derivative P28 results in lower viral titers and augments protection in mice

Background

Viruses bind to specific cellular receptors in order to infect their hosts. The specific receptors a virus uses are important factors in determining host range, cellular tropism, and pathogenesis. For adenovirus, the existing model of entry requires two receptor interactions. First, the viral fiber protein binds Coxsackie and Adenovirus Receptor (CAR), its primary cellular receptor, which docks the virus to the cell surface. Next, viral penton base engages cellular integrins, coreceptors thought to be required exclusively for internalization and not contributing to binding. However, a number of studies reporting data which conflicts with this simple model have been published. These observations have led us to question the proposed two-step model for adenovirus infection.

Results

In this study we report that cells which express little to no CAR can be efficiently transduced by adenovirus. Using competition experiments between whole virus and soluble viral fiber protein or integrin blocking peptides, we show virus binding is not dependent on fiber binding to cells but rather on penton base binding cellular integrins. Further, we find that binding to low CAR expressing cells is inhibited specifically by a blocking antibody to integrin αvβ5, demonstrating that in these cells integrin αvβ5 and not CAR is required for adenovirus attachment. The binding mediated by integrin αvβ5 is extremely high affinity, in the picomolar range.

Conclusions

Our data further challenges the model of adenovirus infection in which binding to primary receptor CAR is required in order for subsequent interactions between adenovirus and integrins to initiate viral entry. In low CAR cells, binding occurs through integrin αvβ5, a receptor previously thought to be used exclusively in internalization. We show for the first time that integrin αvβ5 can be used as an alternate binding receptor.     

Adenoviruses use lactoferrin as a bridge for CAR-independent binding to and infection of epithelial cells

Most adenoviruses bind to the coxsackie- and adenovirus receptor (CAR). Surprisingly, CAR is not expressed apically on polarized cells and is thus not easily available to viruses. Consequently, alternative mechanisms for entry of coxsackievirus and adenovirus into cells have been suggested. We have found that tear fluid promotes adenovirus infection, and we have identified human lactoferrin (HLf) as the tear fluid component responsible for this effect. HLf alone was found to promote binding of adenovirus to epithelial cells in a dose-dependent manner and also infection of epithelial cells by adenovirus. HLf was also found to promote gene delivery from an adenovirus-based vector. The mechanism takes place at the binding stage and functions independently of CAR. Thus, we have identified a novel binding mechanism whereby adenovirus hijacks HLf, a component of the innate immune system, and uses it as a bridge for attachment to host cells.     

嵌合型E1B 55-kDa蛋白缺陷型腺病毒载体治疗肿瘤的评价

ONYX-015和H101为可复制E1B 55-kDa蛋白缺陷的C族腺病毒,它们正作为抗癌药物进行临床研究. 然而它们在癌症基因治疗中的应用却受到了C族腺病毒天然特性的制约,部分原因是由于在恶性肿瘤中C族腺病毒受体(coxsackievirus-adenovirus receptor,CAR) 的表达量较低. 构建了一个以H101为骨架的含有编码35型腺病毒鞭毛区域的基因,替代5型腺病毒鞭毛基因的嵌合型腺病毒载体. 这一改动使得腺病毒载体可以通过一种在肿瘤中高表达的膜蛋白CD46感染肿瘤细胞. 应用RT-PCR方法检测不同肿瘤细胞株中CAR和CD46表达量的区别. 在CAR受体低表达的细胞株中(MDA-MB-435和MCF-7),H101-F35表现出比H101和ONYX-015更强的细胞杀伤效果;在CAR受体高表达的细胞株中(A549,NCI-H446,Hep3B,LNCaP,ZR-75-30和Bcap-37),H101-F35、 H101和ONYX-015的细胞杀伤效果则相似. 在荷MDA-MB-435肿瘤的裸鼠模型中,注射H101-F35的抑瘤效果比注射H101的抑瘤效果更明显. 这些结果表明嵌合型溶瘤腺病毒载体H101-F35在肿瘤基因治疗中将有很好的应用前景.     

Adenovirus Serotype 30 Fiber Does Not Mediate Transduction via the Coxsackie-Adenovirus Receptor

Prior work by members of our laboratory and others demonstrated that adenovirus serotype 30 (Ad30), a group D adenovirus, exhibited novel transduction characteristics compared to those of serotype 5 (Ad5, belonging to group C). While some serotype D adenoviruses bind to the coxsackie-adenovirus receptor (CAR), the ability of Ad30 fiber to bind CAR is unknown. We amplified and purified Ad30 and cloned the Ad30 fiber by overlap PCR. Alignment of Ad30 fiber with Ad3, Ad35, Ad5, Ad9, and Ad17 revealed that Ad30, like Ad9 and Ad17, has a shortened fiber sequence relative to that of Ad5. The knob region of fiber was 45% identical to that of the Ad5 knob regions. We made a chimeric recombinant virus (Ad5GFPf30) in which the Ad5 fiber (amino acids [aa]47 to 582) was replaced with Ad30 fiber sequences (aa 46 to 372), and CAR-mediated viral entry was determined on CAR-expressing Chinese hamster ovary (CHO) cells. While CAR expression significantly increased Ad5GFP-mediated transduction in CHO cells (from 1 to 36%), it did not enhance Ad5GFPf30 gene transfer. Binding of radiolabeled Ad5GFPf30 or Ad30 wild-type virus was also not improved by the expression of CAR. These results suggest that Ad30 fiber is distinct from Ad5, Ad9, and Ad17 fibers in its inability to direct transduction via CAR.     

The PDZ1 and PDZ3 Domains of MAGI-1 Regulate the Eight-Exon Isoform of the Coxsackievirus and Adenovirus Receptor

Epithelial integrity is essential for homeostasis and poses a formidable barrier to pathogen entry. Major factors for viral entry into epithelial cells are the localization and abundance of the primary receptor. The coxsackievirus and adenovirus receptor (CAR) is a primary receptor for these two pathogenic groups of viruses. In polarized epithelia, a low-abundance, alternatively spliced eight-exon isoform of CAR, CAR(Ex8), is localized apically where it can support viral infection from the air-exposed surface. Using biochemical, cell biology, genetic, and spectroscopic approaches, we show that the levels of apical CAR(Ex8) are negatively regulated by the PDZ domain-containing protein MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1) and that two MAGI-1 PDZ domains, PDZ1 and PDZ3, regulate CAR(Ex8) levels in opposing ways. Similar to full-length MAGI-1, expression of the isolated PDZ3 domain significantly reduces cell surface CAR(Ex8) abundance and adenovirus infection. In contrast, the PDZ1 domain is able to rescue CAR(Ex8) and adenovirus infection from MAGI-1-mediated suppression. These data suggest a novel cell-based strategy to either suppress viral infection or augment adenovirus-based gene therapy.     

Construction of a Pseudoreceptor That Mediates Transduction by Adenoviruses Expressing a Ligand in Fiber or Penton Base

Modification of adenovirus to achieve tissue specific targeting for the delivery of therapeutic genes requires both the ablation of its native tropism and the introduction of specific, novel interactions. Inactivation of the native receptor interactions, however, would cripple the virus for growth in production cells. We have developed an alternative receptor, or pseudoreceptor, for the virus which might allow propagation of viruses with modified fiber proteins that no longer bind to the native adenovirus receptor (coxsackievirus/adenovirus receptor [CAR]). We have constructed a membrane-anchored single-chain antibody [m-scFv(HA)] which recognizes a linear peptide epitope (hemagglutinin [HA]). Incorporation of HA within the HI loop of the fiber protein enabled the modified virus to transduce pseudoreceptor expressing cells under conditions where fiber-CAR interaction was blocked or absent. The pseudoreceptor mediated virus transduction with an efficiency similar to that of CAR. In addition, the HA epitope mediated virus transduction through interaction with the m-scFv(HA) when it was introduced into penton base. These findings indicate that cells expressing the pseudoreceptor should support production of HA-tagged adenoviruses independent of retaining the fiber-CAR interaction. Moreover, they demonstrate that high-affinity targeting ligands may function following insertion into either penton base or fiber.     

CD46 is a cellular receptor for group B adenoviruses

Group B adenoviruses, a subgenus of human Adenoviridae, are associated with a variety of often-fatal illnesses in immunocompromised individuals, including bone marrow transplant recipients and cancer and AIDS patients. Recently, group B adenovirus derivatives have gained interest as attractive gene therapy vectors because they can transduce target tissues, such as hematopoietic stem cells, dendritic cells and malignant tumor cells, that are refractory to infection by commonly used adenoviral vectors. Whereas many adenoviruses infect cells through the coxsackievirus and adenovirus receptor (CAR), group B adenoviruses use an alternate, as-yet-unidentified cellular attachment receptor. Using mass spectrometric analysis of proteins interacting with a group B fiber, we identified human CD46 as a cellular attachment receptor for most group B adenoviruses. We show that ectopic expression of human CD46 rendered nonhuman cells susceptible to infection with group B viruses in vitro and in vivo. In addition, both siRNA-mediated knockdown of CD46 and a soluble form of CD46 blocked infection of human cell lines and primary human cells. The discovery that group B adenoviruses use CD46, a ubiquitously expressed complement regulatory protein, as a cellular attachment receptor elucidates the diverse clinical manifestation of group B virus infections, and bears directly on the application of these vectors for gene therapy.     

Dilated cardiomyopathy alters the expression patterns of CAR and other adenoviral receptors in human heart

Gene therapy trials for heart failure have demonstrated the key role of efficient gene transfer in achieving therapeutic efficacy. An attractive approach to improve adenoviral gene transfer is to use alternative virus serotypes with modified tropism. We performed a detailed analysis of cardiac expression of receptors for several adenovirus serotypes with a focus on differential expression of CAR and CD46, as adenoviruses targeting these receptors have been used in various applications. Explanted hearts from patients with DCM and healthy donors were analyzed using Q-RT-PCR, western blot and immunohistochemistry. Q-RT-PCR and Western analyses revealed robust expression of all receptors except CD80 in normal hearts with lower expression levels in DCM. Immunohistochemical analyses demonstrated that CD46 expression was somewhat higher than CAR both in normal and DCM hearts with highest levels of expression in intramyocardial coronary vessels. Total CAR expression was upregulated in DCM. Triple staining on these vessels demonstrated that both CAR and CD46 were confined to the subendothelial layer in normal hearts. The situation was clearly different in DCM, where both CAR and CD46 were expressed by endothelial cells. The induction of expression of CAR and CD46 by endothelial cells in DCM suggests that viruses targeting these receptors could more easily gain entry to heart cells after intravascular administration. This finding thus has potential implications for the development of targeted gene therapy for heart failure.     

Expression of a human coxsackie/adenovirus receptor transgene permits adenovirus infection of primary lymphocytes

Replication-defective adenoviruses are effective vehicles for gene transfer, both for the repair of defective genes and for studies of gene function in primary cells. Many cell types, including lymphocytes, are refractory to adenovirus infection because they lack the Coxsackie/adenovirus receptor (CAR) needed for virus attachment. To extend the advantages of adenovirus-mediated gene transfer to primary lymphoid populations and other cell types lacking endogenous CAR, we produced a mouse that expresses human (h) CAR as a transgene under control of a murine MHC class I promoter. hCAR protein is expressed on T and B lymphocytes from a variety of organs (spleen, lymph node, bone marrow, thymus, and peritoneum). These lymphocytes are susceptible to adenovirus infection, as demonstrated by reporter green fluorescent protein gene expression, with the fraction of expressing cells as high as 70%. Some lymphocyte subpopulations required stimulation subsequent to adenovirus infection for reporter expression. This activation requirement is a restriction imposed by the promoter used in the adenovirus construct. In subpopulations requiring activation, the elongation factor 1 promoter was far superior to a hCMV promoter for directing green fluorescent protein production. We also find that hCAR mRNA is produced in nonlymphoid tissues from all founder lines, including tissues that do not express endogenous murine CAR, suggesting the opportunity for effecting gene delivery to and testing gene function in a wide variety of primary cell types previously resistant to gene transfer.     

Altering the Ad5 packaging domain affects the maturation of the Ad particles

We have previously described a new family of mutant adenoviruses carrying different combinations of attB/attP sequences from bacteriophage PhiC31 flanking the Ad5 packaging domain. These novel helper viruses have a significantly delayed viral life cycle and a severe packaging impairment, regardless of the presence of PhiC31 recombinase. Their infectious viral titers are significantly lower (100-1000 fold) than those of control adenovirus at 36 hours post-infection, but allow for efficient packaging of helper-dependent adenovirus. In the present work, we have analyzed which steps of the adenovirus life cycle are altered in attB-helper adenoviruses and investigated whether these viruses can provide the necessary viral proteins in trans. The entry of attB-adenoviral genomes into the cell nucleus early at early timepoints post-infection was not impaired and viral protein expression levels were found to be similar to those of control adenovirus. However, electron microscopy and capsid protein composition analyses revealed that attB-adenoviruses remain at an intermediate state of maturation 36 hours post-infection in comparison to control adenovirus which were fully mature and infective at this time point. Therefore, an additional 20-24 hours were found to be required for the appearance of mature attB-adenovirus. Interestingly, attB-adenovirus assembly and infectivity was restored by inserting a second packaging signal close to the right-end ITR, thus discarding the possibility that the attB-adenovirus genome was retained in a nuclear compartment deleterious for virus assembly. The present study may have substantive implications for helper-dependent adenovirus technology since helper attB-adenovirus allows for preferential packaging of helper-dependent adenovirus genomes.     

Virus-host coevolution in a persistently coxsackievirus B3-infected cardiomyocyte cell line

Coevolution of virus and host is a process that emerges in persistent virus infections. Here we studied the coevolutionary development of coxsackievirus B3 (CVB3) and cardiac myocytes representing the major target cells of CVB3 in the heart in a newly established persistently CVB3-infected murine cardiac myocyte cell line, HL-1(CVB3). CVB3 persistence in HL-1(CVB3) cells represented a typical carrier-state infection with high levels (10(6) to 10(8) PFU/ml) of infectious virus produced from only a small proportion (approximately 10%) of infected cells. CVB3 persistence was characterized by the evolution of a CVB3 variant (CVB3-HL1) that displayed strongly increased cytotoxicity in the naive HL-1 cell line and showed increased replication rates in cultured primary cardiac myocytes of mouse, rat, and naive HL-1 cells in vitro, whereas it was unable to establish murine cardiac infection in vivo. Resistance of HL-1(CVB3) cells to CVB3-HL1 was associated with reduction of coxsackievirus and adenovirus receptor (CAR) expression. Decreasing host cell CAR expression was partially overcome by the CVB3-HL1 variant through CAR-independent entry into resistant cells. Moreover, CVB3-HL1 conserved the ability to infect cells via CAR. The employment of a soluble CAR variant resulted in the complete cure of HL-1(CVB3) cells with respect to the adapted virus. In conclusion, this is the first report of a CVB3 carrier-state infection in a cardiomyocyte cell line, revealing natural coevolution of CAR downregulation with CAR-independent viral entry in resistant host cells as an important mechanism of induction of CVB3 persistence.     

Activation of adenovirus early promoters and lytic phase in differentiated strata of organotypic cultures of human keratinocytes

Human oncolytic adenoviruses have been used in clinical trials targeting cancers of epithelial origin. To gain a better understanding of the infectious cycle of adenovirus in normal human squamous tissues, we examined the viral infection process in organotypic cultures of primary human keratinocytes. We show that for the infection to occur, wounding of the epithelium is required. In addition, infection appears to initiate at the basal or parabasal cells that express the high-affinity coxsackievirus-adenovirus receptor, CAR, whereas the productive phase takes place in differentiated cells. This is due, at least in part, to the differentiation-dependent activation of the E1A and E2A early promoters and E4 promoters. We also show that adenovirus infection triggers a response mediated by the abnormal accumulation of cyclin E and p21cip1 proteins similar to the one previously observed in human papillomavirus-infected tissues. However, the virus seems to be able to overcome it, at least partially.