Mechanisms of action underlying the immunotherapeutic activity of Allovectin in advanced melanoma.

Allovectin (velimogene aliplasmid) is a cancer immunotherapeutic currently completing a pivotal phase 3 study for metastatic melanoma. Consisting of a bicistronic plasmid encoding both major histocompatibility complex (MHC) class I heavy and light chains (HLA-B7 and ß2-microglobulin, respectively) formulated with a cationic lipid-based system, it is designed for direct intratumoral administration. Following injection into a single lesion, the product is intended to induce anti-tumor immune responses against both treated and distal lesions. Both the plasmid and lipid components of Allovectin contribute to the biological activity of the drug product, and its therapeutic activity is hypothesized to derive from multiple mechanisms of actions (MOAs). These include the induction of both cytotoxic T-cell and innate immune responses directed against allogeneic as well as tumor-derived targets, consequences of both an increased MHC class I expression on tumor cells and the induction of a localized immune/inflammatory response. In this paper, we review Allovectin's proposed MOAs, placing their contributions in the context of anti-tumor immunity and highlighting both preclinical and clinical supporting data.

Isoform-selective PI3K inhibitors as novel therapeutics for the treatment of acute myocardial infarction.

In the present paper, we review the preclinical development of TG100-115, a PI3K (phosphoinositide 3-kinase) gamma/delta isoform-specific inhibitor currently in clinical trials for the reduction of acute MI (myocardial infarction). An overview is presented outlining the pathogenesis of acute MI and the rationale for clinical use of PI3K gamma/delta-specific inhibitors in this indication. TG100-115's broad anti-inflammatory activities are described, as well as its ability to discriminate between cellular signalling pathways downstream of receptor tyrosine kinase ligands such as vascular endothelial growth factor. Finally, we review TG100-115's potent cardioprotective activities as revealed in rigorous animal models of acute MI, and, based on these data, this compound's potential for clinical utility.

Matrix immobilization enhances the tissue repair activity of growth factor gene therapy vectors.

Although growth factor proteins display potent tissue repair activities, difficulty in sustaining localized therapeutic concentrations limits their therapeutic activity. We reasoned that enhanced histogenesis might be achieved by combining growth factor genes with biocompatible matrices capable of immobilizing vectors at delivery sites. When delivered to subcutaneously implanted sponges, a platelet-derived growth factor B-encoding adenovirus (AdPDGF-B) formulated in a collagen matrix enhanced granulation tissue deposition 3- to 4-fold (p < or = 0.0002), whereas vectors encoding fibroblast growth factor 2 or vascular endothelial growth factor promoted primarily angiogenic responses. By day 8 posttreatment of ischemic excisional wounds, collagen-formulated AdPDGF-B enhanced granulation tissue and epithelial areas up to 13- and 6-fold (p < 0.009), respectively, and wound closure up to 2-fold (p < 0.05). At longer times, complete healing without excessive scar formation was achieved. Collagen matrices were shown to retain both vector and transgene products within delivery sites, enabling the transduction and stimulation of infiltrating repair cells. Quantitative PCR and RT-PCR demonstrated both vector DNA and transgene mRNA within wound beds as late as 28 days posttreatment. By contrast, aqueous formulations allowed vector seepage from application sites, leading to PDGF-induced hyperplasia in surrounding tissues but not wound beds. Finally, repeated applications of PDGF-BB protein were required for neotissue induction approaching equivalence to a single application of collagen-immobilized AdPDGF-B, confirming the utility of this gene transfer approach. Overall, these studies demonstrate that immobilizing matrices enable the controlled delivery and activity of tissue promoting genes for the effective regeneration of injured tissues.

Uptake of adenoviral vectors via fibroblast growth factor receptors involves intracellular pathways that differ from the targeting ligand.

Target-specific delivery of adenoviral gene therapy vectors has been achieved by introducing basic fibroblast growth factor (FGF2) onto viral capsids. FGF2-retargeted vectors enter the cell through high-affinity FGF receptors while normal adenoviral receptor interactions are ablated. In addition, FGF2-mediated targeting permits a higher level of transgene expression and in vivo efficacy. We now present studies on the intracellular pathways and mechanisms of transduction by FGF2-retargeted adenovirus. FGF2 retargeting results in increased virion entry. Nuclear delivery is also increased, but to a level that is directly proportional to virion entry. In addition, after entry, the retargeted particle rapidly localizes to the nucleus in a time frame similar to that of adenovirus alone. Transgene expression is always enhanced with FGF2-mediated delivery, whether overall transduction of the population is increased, equivalent, or decreased relative to nontargeted adenoviral vectors. However, the increase in transgene expression does not correlate quantitatively with enhanced cellular entry, indicating that other factors may influence transgene expression levels. The increase in transgene expression occurs only when the FGF2-retargeting moiety is physically complexed with the adenoviral vector, indicating a requirement for a spatial link between the ligand and the virus particle. The FGF2-adenoviral complex activates the FGF receptor-mediated proliferative signaling cascade, but this signal transduction is not required for the enhanced level of gene expression observed after FGF2-mediated delivery. These findings emphasize that, in addition to altering receptor tropism, the influence of FGF2 retargeting extends to intracellular adenoviral trafficking pathways. Although the increased delivery of virions into the cell and nucleus contributes to the enhanced transgene expression observed with FGF2 retargeting, other as yet undefined cellular mechanisms also contribute to this process.

Functional comparison of transmyocardial revascularization by mechanical and laser means.

BACKGROUND: As a result of the clinical benefit observed in angina patients treated by transmyocardial revascularization (TMR) with a laser, interest in mechanical TMR has been renewed. Although the injury induced by mechanical TMR is similar to laser TMR, the resultant impact on myocardial contractility is unknown. The purpose of this study was to determine whether mechanical TMR improves ventricular function as compared with laser TMR in chronically ischemic myocardium. METHODS: After establishing an area of chronic myocardial ischemia, 25 domestic pigs were randomized to treatment by: excimer laser (group I), a hot needle (50 degrees C) (group II), a normothermic needle (group III), an ultrasonic needle (40 KHz) (group IV), or no treatment (group V). All devices create a transmural channel of the same diameter; 22 +/- 1 transmural channels were created in each animal. Regional myocardial contractility was assessed by measuring ventricular wall thickening at rest and with dobutamine stress echocardiography. Six weeks after revascularization, the animals were restudied at rest and with stress. Postsacrifice and histologic analysis of angiogenesis and TMR effects was then assessed. RESULTS: Laser TMR provided significant recovery of ischemic myocardial function. This improvement in contractility after laser TMR was a 75% increase over the baseline function of the ischemic zone (p < 0.01). Mechanical TMR provided no significant improvement in function posttreatment. In fact, TMR achieved with an ultrasonic needle demonstrated a 40% worsening of the contractility versus the pretreatment baseline (p < 0.05). Histologic analysis demonstrated a significant increase in new blood vessels in the ischemic zone after laser TMR, which was not demonstrated for any of the other groups (p < 0.05). Additionally, evaluation of the mechanical TMR channels demonstrated significant scarring, which correlated with the functional results. CONCLUSIONS: Using devices to create an injury analogous to the laser, mechanical TMR failed to improve the function of chronically ischemic myocardium. Only laser TMR significantly improved myocardial function.

FGF2-Targeted adenovirus encoding platelet-derived growth factor-B enhances de novo tissue formation.

Gene therapy has yet to achieve reproducible clinical efficacy, due to inadequate gene delivery, inadequate gene expression, or dose-limiting toxicity. We have developed a gene therapy technology for tissue repair and regeneration that employs a structural matrix for DNA delivery. The matrix holds the DNA vector at the treatment site and provides a scaffolding for in-growth and accumulation of repair cells and efficient DNA transfection. We now report, for the first time, matrix-mediated delivery of targeted DNA vectors for soft tissue repair. A collagen matrix was used to deliver an adenoviral vector encoding platelet-derived growth factor-B (AdPDGF-B), resulting in efficient transgene expression in vitro and in vivo. Increases in the overall levels of expression and in the relative amounts of secreted PDGF-BB were achieved when AdPDGF-B was conjugated to fibroblast growth factor (FGF2) such that the virus was targeted for cellular uptake via FGF receptors. Matrix-mediated delivery of AdPDGF-B enhanced wound healing responses in vivo, and FGF2 targeting generated effects comparable to nontargeted vectors at significantly lower doses. Therefore, matrix-mediated delivery in combination with FGF2 targeting overcomes some of the safety and efficacy limitations of current gene therapy strategies and is an attractive therapeutic approach for tissue repair and regeneration.

Matrix-enabled gene transfer for cutaneous wound repair.

Several growth factor proteins have been evaluated as therapeutic agents for the treatment of chronic dermal wounds. Unfortunately, most have failed to produce significant improvements in wound healing, in part due to ineffective delivery and poor retention in the wound defect. It has been proposed that gene therapy might overcome the limitations of protein therapy via ongoing transcription and translation, thus prolonging the availability of the therapeutic protein. Reasoning that it would be of further benefit to ensure retention of the DNA vector as well as the therapeutic protein within the wound defect, we have evaluated matrix-enabled gene transfer for cutaneous wound repair (Gene Activated Matrix). Formulations consisting of bovine type I collagen mixed with adenoviral or plasmid gene vectors have been evaluated in 3 in vivo models. The therapeutic transgenes employed encode human platelet-derived growth factor-A or -B, proteins key to each phase of normal wound repair. Increased granulation tissue formation, vascularization, and reepithelialization have been shown compared to controls treated with collagen alone or collagen containing a reporter gene vector. Further enhancements of the tissue repair response have been achieved by combining matrix-enabled gene transfer with molecular targeting, in which the DNA vector is conjugated to a growth factor ligand (basic fibroblast growth factor). These promising results support the clinical evaluation of gene activated matrices for the treatment of chronic dermal wounds.

Retargeted delivery of adenoviral vectors through fibroblast growth factor receptors involves unique cellular pathways.

A major goal of gene therapy is to improve target specificity by delivering vectors through alternative cellular receptors. We previously reported that adenoviral vector delivery through basic fibroblast growth factor (FGF2) receptors enhances both cellular transduction and in vivo efficacy. We now present studies addressing the cellular pathways and mechanisms underlying these events. Cellular receptors for adenoviruses are not required for transduction by FGF2-retargeted vectors. Moreover, alpha(V) integrins can antagonize FGF2 retargeting, in contrast to their obligatory role in non-retargeted vector delivery. By contrast, high-affinity FGF receptors, which are overexpressed on potential tumor targets, are required for FGF2-retargeted transduction. Low-affinity heparan sulfate proteoglycan interactions, however, are not a prerequisite, in marked contrast to their obligatory role in FGF2 mitogenic signaling. By comparing receptor expression and ligand binding with transgene expression, we also demonstrate that FGF2 retargeting enhances transduction by mechanisms other than increasing the number of targeted cells. Rather, the use of alternative targeting ligands supports the conclusion that specific receptor interactions and intracellular events serve to enhance transgene expression. Together, these studies highlight the unique delivery and transduction pathways used by FGF2-retargeted adenoviruses, and help define the basis for their enhanced in vivo efficacy.

Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice.

Adenovirus (Ad) have been used as vectors to deliver genes to a wide variety of tissues. Despite achieving high expression levels in vivo, Ad vectors display normal tissue toxicity, transient expression, and antivector immune responses that limit therapeutic potential. To circumvent these problems, several retargeting strategies to abrogate native tropism and redirect Ad uptake through defined receptors have been attempted. Despite success in cell culture, in vivo results have generally not shown sufficient selectivity for target tissues. We have previously identified (C. K. Goldman et al., Cancer Res., 57: 1447-1451, 1997) the fibroblast growth factor (FGF) ligand and receptor families as conferring sufficient specificity and binding affinity to be useful for targeting DNA in vivo. In the present studies, we retargeted Ad using basic FGF (FGF2) as a targeting ligand. Cellular uptake is redirected through high-affinity FGF receptors (FGFRs) and not the more ubiquitous lower-affinity Ad receptors. Initial in vitro experiments demonstrated a 10- to 100-fold increase in gene expression in numerous FGFR positive (FGFR+) cell lines using FGF2-Ad when compared with Ad. To determine whether increased selectivity could be detected in vivo, FGF2-Ad was administered i.v. to normal mice. FGF2-Ad demonstrates markedly decreased hepatic toxicity and liver transgene expression compared with Ad treatment. Importantly, FGF2-Ad encoding the herpes simplex virus thymidine kinase (TK) gene transduces Ad-resistant FGFR+ tumor cells both ex vivo and in vivo, which results in substantially enhanced survival (180-260%) when the prodrug ganciclovir is administered. Because FGFRs are up-regulated on many types of malignant or injured cells, this broadly useful method to redirect native Ad tropism and to increase the potency of gene expression may offer significant therapeutic advantages.

FGF2-targeted adenoviral vectors for systemic and local disease.

Adenoviral vectors have proven useful for transducing a variety of cell types. However, both adenoviral resistant cell types and vector-related toxicities (due to non-specific tropism), limit their widespread clinical utility. These limitations can be greatly reduced by targeting adenoviral vectors to alternative cell surface receptors. One ligand family, highly effective at targeting adenoviral vectors, is the family of fibroblast growth factors (FGFs). The FGFs allow a high degree of targeting specificity due to their cognate high affinity FGF receptors, which are expressed on cells undergoing repair and regeneration. Recent publications, reviewed herein, demonstrate that FGF-targeted adenoviruses result in enhanced potency and reduced toxicity, and thus substantially increase the therapeutic index in vivo. As a result, vector delivery through FGF receptors provides the targeting specificity required for successful local and systemic clinical applications of gene therapy.

Targeted delivery of DNA encoding cytotoxic proteins through high-affinity fibroblast growth factor receptors.

Nonviral DNA delivery strategies for gene therapy have generally been limited by a lack of specificity and efficacy. However, ligand-mediated endocytosis can specifically deliver DNA in vitro to cells bearing the appropriate cognate receptors. Similarly, in order to circumvent problems related to efficacy, DNA must encode proteins with high intrinsic activities. We show here that the ligand basic fibroblast growth factor (FGF2) can target FGF receptor-bearing cells with DNA encoding therapeutic proteins. Delivery of genes encoding saporin, a highly potent ribosomal inactivating protein, or the conditionally cytotoxic herpes simplex virus thymidine kinase, a protein that can kill cells by activating the prodrug ganciclovir, is demonstrated. The saporin gene was codon optimized for mammalian expression and demonstrated to express functional protein in a cell-free assay. FGF2-mediated delivery of saporin DNA or thymidine kinase DNA followed by ganciclovir treatment resulted in a 60 and 75% decrease in cell number, respectively. Specificity of gene delivery was demonstrated in competition assays with free FGF2 or with recombinant soluble FGF receptor. Alternatively, when histone H1, a ligand that binds to cell surface heparan sulfate proteoglycans ("low-affinity" FGF receptors), was used to deliver DNA encoding thymidine kinase, no ganciclovir sensitivity was observed. These findings establish the feasibility of using ligands such as FGF2 to specifically deliver genes encoding molecular chemotherapeutic agents to cells.

Diethylcarbamazine (DEC) does not induce nitric oxide (NO) synthesis.

Diethylcarbamazine (DEC) was discovered in 1947 as a potent therapeutic agent in lymphatic filariasis and has been a mainstay of antifilarial therapy over the past five decades (R. I. Hewitt, et al., 1947, Journal of Laboratory and Clinical Medicine 32, 1304-1313). Several hundred million doses of this drug have been administered to people. Despite its widespread and successful use over this prolonged time scale, its mechanism of action remains obscure (R. M. Maizels and D. A. Denham, 1992, Parasitology 105 Suppl. 549-560). Numerous studies suggest that DEC has no direct effect on the parasite (F. Hawking and W. Laurie, 1949, Lancet 2, 146-147) and that it exerts its action by stimulating host immune defense mechanisms (F. Hawking et al., 1948, Lancet 2, 730-731), or by activating host platelets to become microfilaricidal (J. Y. Cesbron et al., 1987, Nature 325(6104) 533-536). Recent data from two different laboratories suggest that NO may be involved in host defense against filarial parasites (T. V. Rajan et al., 1996, Infection and Immunity 64(8), 3351-3353; M. J. Taylor et al., 1996, Parasitology 112, 315-322). We investigated whether DEC stimulates the production of NO from murine macrophages or rat endothelial cells. DEC did not stimulate the synthesis or secretion of NO from either, nor did it synergize with interferon-gamma or tumor necrosis factor-alpha in the induction of inducible NO synthase (iNOS). In addition, there was no consistent increase in the output of inorganic nitrate, the end product of NO metabolism, in the urines of rats treated with DEC. These data suggest that DEC does not achieve its therapeutic efficacy through the induction of host iNOS.

Anti-endothelial cell autoantibodies in BB rats with spontaneous and induced IDDM.

Anti-endothelial cell (anti-EC) antibodies occur in several autoimmune diseases, including human IDDM, but the time course of their development and their importance in disease pathogenesis are unknown. To study such antibodies further, we investigated the BB rat model of autoimmunity. Diabetes-prone (DP) BB rats spontaneously develop autoimmune diabetes, whereas coisogenic diabetes-resistant (DR) BB rats are disease free but can be induced to become diabetic by the depletion of T-cells expressing the RT6 alloantigen. Anti-EC autoantibodies were readily detectable in both untreated DP-BB rats and RT6-depleted DR-BB rats before the onset of diabetes. Their concentration increased with time. The anti-EC antibodies in DP-BB rats were almost exclusively of the IgG2b subclass, whereas those in RT6-depleted DR-BB rats included both the IgG1/2a and the IgG2b subclasses. We also found that intravenous injections of purified immunoglobulins from RT6-depleted DR-BB rats induced abnormal pancreatic vascular leakage in mice. The preabsorption of immunoglobulins against cultured ECs abolished this activity. The pretreatment of mice with silica also abolished the ability of immunoglobulins of RT6-depleted DR-BB rats to induce pancreatic leakage, suggesting that monocytes are involved in the mechanism of anti-EC autoantibody-induced vascular leakage. We conclude that anti-EC autoantibodies are present in rat strains that are genetically predisposed to develop autoimmune diabetes. Their presence early in the disease process and their ability to induce pancreatic vascular leakage suggest that they may participate in diabetes pathogenesis.

Thymic epithelial defects and predisposition to autoimmune disease in BB rats.

We report an association between thymic epithelial defects and predisposition to autoimmunity. Diabetes-prone (DP) BB rats develop spontaneous hyperglycemia and are deficient in T cell subsets expressing the RT6 alloantigen. Diabetes resistant (DR) BB rats become diabetic if depleted of RT6+ T cells. The inciting immune system defects are unknown. We made the following observations: 1) Regions of thymic cortex and medulla devoid of thymic epithelium exist in DP-BB, DR-BB, and Lewis rats, all of which are susceptible to autoimmune disorders. Such defects were absent in eight normal rat strains. 2) Thymic epithelial defects are absent at birth, but present in BB rats at 4 weeks of age. 3) The genetic predisposition to thymic epithelial defects is an autosomal dominant trait. 4) The observation of thymic defects in (DP x WF)F1 rats led to the prediction that such animals, which never develop spontaneous autoimmunity, might be susceptible to its induction. Following depletion of RT6+ T cells we observed diabetes in 91%, and thyroiditis in 43%, of treated F1 animals (n = 23). Pancreatic insulitis was uniformly present. Because thymic epithelium participates in the positive and negative selection of developing thymocytes, we propose that thymic epithelial defects may play an important role in the predisposition of BB rats to autoimmunity.

Polyinosinic:polycytidylic acid is a potent activator of endothelial cells.

Polyinosinic:polycytidylic acid (poly I:C) is a synthetic double-stranded polyribonucleotide that elicits immune responses analogous to those observed during viral infection. It is also known to modulate the expression of certain autoimmune disorders including diabetes mellitus in the BB rat and NOD mouse. The mechanism underlying these immunomodulatory effects is not known, but it could involve activation of vascular endothelium. We now report that parenteral poly I:C induces rat pancreatic endothelium to hyperexpress intercellular adhesion molecule 1 (CD54). This is accompanied by a perivascular recruitment of mononuclear cells to the exocrine pancreas. Corollary in vitro studies demonstrated that poly I:C is a potent activator of both rat and human endothelial cells in culture. It upregulates endothelial expression of several leukocyte adhesion molecules, stimulates the release of interleukin-6 and interleukin-8, and antagonizes interferon-gamma induction of major histocompatibility complex class II expression. We conclude that poly I:C activates endothelial cells to express surface molecules and cytokines in a pattern classically associated with leukocyte recruitment. These effects may in part contribute to the immunomodulatory effects of poly I:C in animal models of autoimmunity.

Reversible endothelial cell relaxation induced by oxygen and glucose deprivation. A model of ischemia in vitro.

Endothelial cells (EC) cultured on polymerized silicone deform the underlying substrate, producing microscopically visible wrinkles. This has been interpreted as cellular contraction, and we have previously concluded that EC normally maintain an active contractile tone. Since in ischemic tissues capillaries become "paralyzed" and lose their tone, we decided to examine the effects of glucose and/or oxygen deprivation on EC contractility. Contracting cultures with wrinkled silicone substrates were exposed to complete anoxia with or without exogenous glucose and followed by time-lapse photography. Under either glucose-or oxygen-free conditions, contraction was maintained for up to 4 days. If, however, both oxygen and glucose were removed, cellular contraction was reversed. After a period of 2 to 4 hours substrate wrinkles gradually disappeared, until by 3 to 7 hours, few to no wrinkles remained. Furthermore, within 10 minutes of restoration to normal oxygen (but not glucose) levels, substrate wrinkling reappeared. F-actin microfilament patterns and cell number per unit area were also altered by glucose and oxygen deprivation. Similar results were obtained using large or small vessel EC. We conclude that in the absence of glucose and oxygen EC lose their contractile tone, and that tone can be re-established upon re-exposure to oxygen. These findings should have implications for the pathogenesis of capillary paralysis in ischemia.

T lymphocytes capable of activating endothelial cells in vitro are present in rats with autoimmune diabetes.

Endothelial activation as evidenced by increased expression of leukocyte adhesion molecules occurs during immune-mediated inflammatory processes. One such process is insulitis, the pancreatic islet inflammation that leads to autoimmune insulin-dependent diabetes mellitus (IDDM). To determine if the induction of IDDM correlates with the presence of T lymphocytes capable of activating endothelial cells (EC), we studied the diabetes resistant BB (DR) rat. These animals become diabetic after in vivo depletion of T cells expressing the RT6 alloantigen. Various populations of purified DR T lymphocytes were cocultured with MHC compatible rat EC. We observed: 1) RT6- T cells from diabetic animals induced maximal endothelial MHC Ag expression. 2) The ability of RT6- T cells to activate EC increased with the duration of in vivo RT6 depletion. It was acquired before the onset of insulitis but subsided after the onset of diabetes. 3) In contrast, neither unsorted total T cells nor in vitro-purified RT6- T cells activated EC. 4) Older DR rats depleted of RT6+ T cells did not become diabetic and their RT6- T cells did not activate EC. 5) T cell IFN-gamma production correlated with the intensity of EC activation. 6) direct T cell-EC contact was required for maximal IFN-gamma production and EC activation. We conclude that RT6- T cells capable of activating EC are generated during the induction of IDDM in DR rats. We hypothesize that such T cell activity may lead to endothelial activation in vivo and contribute to immune-mediated insulitis, beta-cell destruction, and IDDM.