p27-p16 fusion gene inhibits angioplasty-induced neointimal hyperplasia and coronary artery occlusion.

Inhibition of proliferative neointima formed by vascular smooth muscle cells is a potential target in preventing angioplasty-induced restenosis. We have created a potent antiproliferative by fusing the active regions of the p27 and p16 cell cycle inhibitors. Intravascular delivery of a replication-deficient adenoviral vector (AV) encoding this p27-p16 fusion protein, named W9, inhibited balloon injury-induced neointimal hyperplasia in rabbit carotid arteries. In a therapeutically more relevant model, AV-W9 was delivered to balloon-injured porcine coronary arteries in vivo using an infusion catheter. Of the three coronary arteries, two were injured with a 15-mm balloon catheter and either were left untreated or were treated with 10(12) viral particles of either AV-W9 or a control null virus. AV-W9 treatment significantly inhibited neointimal hyperplasia in this porcine arterial balloon injury model compared with untreated or control virus-treated vessels. The average intimal area of the AV-W9-treated group 10 days after balloon injury and treatment was 0.42+/-0.36 mm(2), whereas the AV-null group demonstrated an intimal area of 0.70+/-0.52 mm(2). At day 10 the average intimal thickness of the AV-W9-treated vessels was 9.1 microm (n=5, x 20 magnification) compared with 21.2 microm (n=5, x 20 magnification) in control virus-treated vessels. This trend was also observed at 28 days after balloon injury and gene transfer during which AV-W9-treated vessels demonstrated an average intimal thickness of 4.7 microm (n=8, x 20 magnification) compared with 13.3 microm (n=3, x 20 magnification) in control virus-treated vessels and 7.3 microm (n=5, x 20 magnification) in the sham-treated vessels. The AV-W9 treatment was safe and well tolerated. These data suggest that AV-W9 gene therapy may be useful in preventing angioplasty-induced intimal hyperplasia in the coronary artery.

Factors influencing the development of an anti-factor IX (FIX) immune response following administration of adeno-associated virus-FIX.

The present study sought to determine the impact of the route of administration of an adeno-associated virus (AAV) vector encoding human factor IX (hFIX) on the induction of an immune response against the vector and its xenogenic transgene product, hFIX. Increasing doses of AAV-hFIX were administered by different routes to C57Bl/6 mice, which typically demonstrate significant immune tolerance to hFIX. The route of delivery had a profound impact on serum hFIX levels as well as the induction of an anti-hFIX humoral immune response. At all dose levels tested, delivery of AAV-hFIX by an intramuscular (IM) route induced an antibody response against the human FIX protein and no hFIX was detected in the serum of animals even at doses of 2 x 10(11) DNA viral particles (vp) of AAV-hFIX. This was in stark contrast to the mice that received AAV-hFIX by intraportal vein (IPV) administration. No anti-hFIX inhibitors were observed in any of these mice and therapeutic levels of hFIX were detected in the serum of all mice that received doses of 2 x 10(10) vp AAV-hFIX and higher. When pre-existing neutralizing immunity to AAV was established in mice, AAV-hFIX administration by either the IM or IPV routes did not result in detectable serum hFIX. Although hFIX expression was not observed in mice with pre-existing neutralizing immunity to AAV, an anti-hFIX response was induced in all of the animals that received AAV-hFIX by the IM route. This was not observed in the preimmune mice that received AAV-hFIX by IPV administration. These results suggest that the threshold of inducing an immune response against a secreted transgene product, in this case the xenoprotein hFIX, is lower when the vector is administered by the IM route even in animals with pre-existing immunity to AAV. (Blood. 2001;97:3733-3737)

p27-p16 Chimera: a superior antiproliferative for the prevention of neointimal hyperplasia.

Cyclin-dependent kinase inhibitors (CDKi's) may be useful to treat hyperproliferative vascular disorders, such as restenosis induced following angioplasty or vein engraftment. We have shown that a novel fusion protein of the CDKi's p27 and p16, named W9, significantly reduces proliferation of human coronary smooth muscle cells in vitro, by blocking cell proliferation without inducing apoptosis. We have now evaluated the efficacy of adenovirus-mediated gene transfer of W9 (AV-W9) in a balloon-injury model, in the carotid arteries of cholesterol-fed rabbits. We observed that intravascular delivery of 2 x 10(11) viral particles of AV-W9 3 days following balloon injury inhibited intimal hyperplasia by 60% compared to a control virus (P > 0.001). PCNA expression in the AV-W9-treated vessels, a marker of injury-induced cell proliferation, was also reduced compared to the control virus-treated vessels. Direct comparison of the efficacy of AV-W9 and AV-p16 and AV-p27 in this model indicated that delivery of either of the parental genes was significantly less effective in inhibiting intimal thickening compared to the AV-W9 treatment. We conclude that combining the activities of multiple cell cycle regulatory proteins greatly increases the potency of cytostatic gene therapy in the treatment of balloon injury-induced intimal hyperplasia and represents a promising potential approach to preventing postangioplasty restenosis.

Stability of adenoviral vectors following catheter delivery.

Adenoviral vectors have shown promise in a variety of preclinical vascular disease models. Intravascular infusion is one methodology to introduce the adenoviral vector into the affected area of the blood vessel. The biocompatibility of the infusion catheter with the adenoviral vector is key for successful local transfer. It has been recently suggested that catheter-based delivery of adenoviral vectors may result in the loss of vector infectivity. We demonstrate here a catheter capable of delivering adenoviral vectors without the loss of viral particle or infectious titers. First- (DeltaE1) and second- (DeltaE1/DeltaE4) generation adenoviral vectors were tested for their biocompatibility with the Crescendo microporous infusion catheter, which is designed for local infusion of therapeutic agents to human coronary or peripheral arteries. We found that incubation of either the DeltaE1 or the DeltaE1/DeltaE4 viral vectors for up to 30 min in the catheter at 37 degrees C did not result in a loss of viral particles or of viral infectivity. Here, we show that the Crescendo catheter is biocompatible with adenoviral vectors and suitable for vascular gene therapy.

T-cell killing of heterogenous tumor or viral targets with bispecific chimeric immune receptors.

We have previously described several novel chimeric immune receptors (CIRs) that redirect human T cells to kill malignant or HIV-infected cells. These CIRs comprise a cancer- or virus-specific ligand or single-chain antibody fused to the signaling domain of the T-cell receptor CD3-zeta subunit. Binding of the ligand- or antibody-based CIR to the target antigen (Ag) triggers T-cell-mediated cytolysis of the tumor- or virus-infected cell independent of target cell major histocompatibility complex class I expression. A new type of CIR was developed to mediate the lysis of cells that expressed one or more distinct viral or tumor Ags; three bispecific CIRs (BCIRs) were generated that recognized the carcinoembryonic Ag (CEA) and TAG-72 tumor Ags or, alternatively, distinct epitopes in the HIV envelope (HIVenv). T cells expressing the antitumoral Ag BCIR lysed both CEA- and TAG-72-expressing targets and did not kill Ag-negative targets or target cells expressing other members of the CEA family. Similarly, T cells expressing the anti-HIVenv BCIR lysed target cells expressing both the wild-type HIVenv and a mutant HIVenv that lacked the epitopes recognized by the monospecific CIRs. This approach permits the generation of T cells with a broader spectrum of activity capable of killing virus-infected cells and malignant cells and reduces the potential of progression of disease due to Ag loss variants.

The p53-independent tumoricidal activity of an adenoviral vector encoding a p27-p16 fusion tumor suppressor gene.

We describe here that DE1-adenovirus vectors (AV) expressing a p27-p16 fusion molecule, termed W9, induce tumor cell apoptosis when overexpressed in a wide range of tumor cell types. However, in primary human cells derived from a variety of normal tissues, AV-W9 induced minimal apoptosis. In tumor cells AV-W9 demonstrated 5- to 50-fold greater tumoricidal activity than either of the parental molecules p16 and p27. In these studies, AV-W9 elicited apoptosis independent of the p53 and Rb status of the tumor cells. In several murine tumor models AV-W9 demonstrated p53-independent antitumor activity. It completely prevented tumor formation in two ex vivo models, whereas the parental molecules resulted in partial protection. Furthermore, AV-W9 induced tumor regression or suppressed tumor growth when introduced intratumorally into preestablished tumors in mice. This effect may be mediated through tumor cell apoptosis or antiangiogenic activity of AV-W9. Thus, this novel chimeric molecule is more potent and capable of killing a broader spectrum of tumors than the parental p16 and p27 molecules independent of the tumor cell p53 and phenotype and represents a powerful new therapeutic agent for cancer gene therapy.

Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure.

Recombinant adeno-associated virus type 2 (AAV) is a common vector used in human gene therapy protocols. We characterized the humoral immune response to AAV and observed that 80% of normal human subjects have anti-AAV antibodies and that 18% have neutralizing antibodies. To analyze the effect of neutralizing antibodies on AAV readministration, we attempted to deliver recombinant AAV expressing human factor IX (AAV-hFIX) intraportally into the livers of mice which had been preexposed to AAV and shown to harbor a neutralizing antibody response. While all naive control mice expressed hFIX following administration of AAV-hFIX, none of the mice with preexisting immunity expressed hFIX, even after transient immunosuppression at the time of the second administration with anti-CD4 or anti-CD40L antibodies. This suggests that preexisting immunity to AAV, as measured by a neutralizing antibody response, may limit AAV-mediated gene delivery. Using human sera in an enzyme-linked immunosorbent assay for AAV and a capsid peptide scan library to block antibody binding, we mapped seven regions of the AAV capsid containing immunogenic epitopes. Using pools of these peptides to inhibit the binding of neutralizing antibodies, we have identified a subset of six peptides which potentially reconstitute a single neutralizing epitope. This information may allow the design of reverse genetic approaches to circumvent the preexisting immunity that can be encountered in some individuals.

Anti-Tumor CC49-zeta CD4 T cells possess both cytolytic and helper functions.

The authors report that the nature of the T-cell-receptor--derived signal in normal CD4+ T cells can induce interleukin-2 (IL-2) secretion or perforin-mediated cytolytic activity. Normal human T cells were genetically modified to express the tumor antigen specific chimeric immune receptor, CC49-zeta. The CC49-zeta chimeric immune receptor is comprised of the intracellular signaling domains of the TCR CD3zeta protein fused to the single chain scFv of the humanized CC49 antibody, which binds the pan-adenocarcinoma tumor antigen TAG-72. Patient-specific T cells genetically modified to express the CC49-zeta receptor have been used in patients with colon cancer. The authors report that both CD4 and CD8 T cells expressing the CC49-zeta receptor mediated the major histocompatibility complex-unrestricted lysis of TAG-72--expressing tumor cells with comparable efficiency. However, although the CC49-zeta receptor mediated target cell lysis, it did not support the production of IL-2, even in the presence of CD28 stimulation. Robust IL-2 secretion and T-cell proliferation were observed when the same CD4 CC49-zeta T cells were stimulated through the CD28 receptor and endogenous T-cell receptor. These results indicate that CD4 T lymphocytes possess the capacity to act as both cytolytic and helper T cells and that this difference in effector function is controlled by the nature of the T-Cell receptor--derived signals.

Impact of chimeric immune receptor extracellular protein domains on T cell function.

Chimeric immune receptors (CIR) encompass tumor- or virus-specific ligands or antibodies fused to the signaling domains of either the T cell receptor or Fc receptor. T cells expressing these receptors recapitulate the cytopathic effects mediated by the T cell receptor and allow the targeting of tumor or virus infected cells in an MHC-independent manner. With this technology, large numbers of T cells with redirected target specificity can be generated. To define the structural features of recombinant CIRs required for optimal function, a panel of five closely related CIRs with identical target specificity were generated. These receptors recognized HIVenv through the single chain Fv (scFv) of an anti-gp 120 antibody. These scFv-zeta receptors were constructed to include alternative extracellular spacer and transmembrane protein domains derived from members of the immunoglobulin supergene family. The effect of these alternative extracellular protein domains on receptor stability, antigen affinity and T cell activity was assessed. We demonstrate that modifying the extracellular protein domains of the anti-HIVenv CIRs significantly impacted receptor stability and substrate binding affinity and that these effects, and not simply the level of cell surface expression, correlated most strongly with changes in CIR-mediated killing. These studies will aid in the rationale design of recombinant CIRs for the immunotherapy of viral infections, cancer and other diseases.

Anti-tumor activity of human T cells expressing the CC49-zeta chimeric immune receptor.

A chimeric immune receptor consisting of an extracellular antigen-binding domain derived from the CC49 humanized single-chain antibody, linked to the CD3zeta signaling domain of the T cell receptor, was generated (CC49-zeta). This receptor binds to TAG-72, a mucin antigen expressed by most human adenocarcinomas. CC49-zeta was expressed in CD4+ and CD8+ T cells and induced cytokine production on stimulation. Human T cells expressing CC49-zeta recognized and killed tumor cell lines and primary tumor cells expressing TAG-72. CC49-zeta T cells did not mediate bystander killing of TAG-72-negative cells. In addition, CC49-zeta T cells not only killed FasL-positive tumor cells in vitro and in vivo, but also survived in their presence, and were immunoprotective in intraperitoneal and subcutaneous murine tumor xenograft models with TAG-72-positive human tumor cells. Finally, receptor-positive T cells were still effective in killing TAG-72-positive targets in the presence of physiological levels of soluble TAG-72, and did not induce killing of TAG-72-negative cells under the same conditions. This approach is being currently being utilized in a phase I clinical trial for the treatment of colon cancer.

Selective uptake and sustained expression of AAV vectors following subcutaneous delivery.

BACKGROUND: Recombinant adeno-associated viral (rAAV) vectors are capable of long-term expression of secreted and intracellular proteins following delivery to muscle, liver, and the central nervous system. In this study, we have evaluated subcutaneous injection of rAAV encoding a variety of transgenes as an alternative route of administration for the systemic delivery of therapeutic proteins. METHODS: rAAV vectors encoding the human factor IX, human interferon-alpha 2a, murine erythropoietin (epo), and Escherichia coli lacZ genes were used for subcutaneous delivery into mature immunocompetent mice. Expression of factor IX and interferon in mouse serum was measured by ELISA. Expression of Epo was monitored by an increase in hemotocrit and by RIA. The tissue tropism of AAV transduction was determined by histochemistry following administration of the lacZ vector. RESULTS: Long-term protein expression (at least one year) is demonstrated in the serum of immunocompetent mice following subcutaneous delivery of AAV vectors encoding the human factor IX and interferon genes. The murine epo gene delivered via this route resulted in levels of Epo that correlate with increased hematocrits of up to 90% for a duration of nine months. rAAV encoding the lacZ gene revealed that the panniculus carnosus, a skeletal muscle layer of the skin, was transduced upon subcutaneous administration. CONCLUSIONS: This study shows that long-term expression of secreted proteins can be achieved using rAAV vectors injected subcutaneously as a single administration. The observation that the panniculus carnosus is the primary tissue transduced by rAAV illustrates the high tropism of rAAV for skeletal muscle.

Induction of protective anti-tumor immunity by gene-modified dendritic cells.

The context in which an antigen is presented shapes the nature of the immune response to that antigen and can result in B cell activation, T cell activation, or immune tolerance. To elicit anti-tumor immune responses, various cell types have been employed as cellular adjuvants with tumor antigens, and recently several groups have shown that dendritic cells (DCs), cultured with tumor lysates, tumor antigens, or peptides eluted from tumor cells, induced significant anti-tumor immunity in vivo. In all of these approaches, the DCs were pulsed with an exogenous source of antigen. An alternative method is to engineer DCs to express tumor antigens. We genetically modified DCs to express beta-galactosidase (beta-gal) as a surrogate tumor antigen and then tested the anti-tumor activity of the beta-gal+ DCs in mice against a beta-gal+ murine melanoma cell line. A single vaccination with the gene-modified DCs protected mice against a lethal dose of beta-gal-B16 melanoma cells and induced beta-gal-specific cytotoxic T lymphocytes. These results demonstrate that expression of tumor antigens by DCs is a potent method of inducing tumor antigen-specific responses in vivo.

A comparison of budesonide and beclomethasone dipropionate sprays in the treatment of seasonal allergic rhinitis.

Intranasal budesonide and beclomethasone dipropionate (BDP), each administered as aqueous, aerosol formulations at dosages of 200 micrograms twice a day, morning and evening, were compared over a 3-week period in a randomized, parallel group study of 88 adults with seasonal allergic rhinitis. Budesonide treatment produced significantly lower mean symptom scores for the whole study compared with BDP for runny nose, itchy nose and sneezing (P < 0.05). The difference in nasal symptom scores produced by budesonide in comparison with BDP was particularly great towards the end of the treatment period. The budesonide-treated group also had lower scores for nasal blockage and two eye symptoms (runny and sore eyes), but the differences noted were not significant. Adverse events recorded by both groups were mild and transient. In conclusion, aqueously administered budesonide is likely to be of more clinical value than BDP for the control of seasonal allergic rhinitis.

CD28-induced costimulation of T helper type 2 cells mediated by induction of responsiveness to interleukin 4.

Type 1 and type 2 cloned T helper (Th) cells are believed to require different antigen-presenting cell (APC)-derived costimuli for proliferation. In the case of Th1-cloned T cells, CD28 signaling costimulates production of autocrine interleukin 2 (IL-2). Th2 cells produce their autocrine growth factor, IL-4, without costimulation, but require APC-derived costimuli, or IL-1, to respond to IL-4. Here we demonstrate that engagement of CD28 on Th2 cells with anti-CD28 antibody or with APC-associated B7 costimulates Th2 responsiveness to IL-4 but does not affect IL-4 or IL-2 production by Th2 cells. Costimulation of Th2 cells via CD28 appears to involve the induction of IL-1 production by Th2 cells, which acts in an autocrine fashion to induce IL-4 responsiveness. These results suggest that CD28-induced costimulation plays an important role in responses mediated by both types of Th cells.

CD28-mediated signalling co-stimulates murine T cells and prevents induction of anergy in T-cell clones.

Occupancy of the T-cell antigen receptor is insufficient to induce T-cell activation optimally; a second co-stimulatory signal is required. Exposure of T-cell clones to complexes of antigen with major histocompatibility complex molecules in the absence of the co-stimulatory signal induces a state of clonal anergy. This requirement for two stimuli for T-cell activation could have an important role in vivo in establishing peripheral tolerance to antigens not encountered in the thymus. The receptor on T cells required for the co-stimulatory stimulus involved in the prevention of anergy has not been identified. The human T-cell antigen CD28 provides a signal that can synergize with T-cell antigen receptor stimulation in activating T cells to proliferate and secrete lymphokines. Here we report that a monoclonal antibody against the murine homologue of CD28 (ref. 7; J.A.G. et al., manuscript in preparation) can provide a co-stimulatory signal to naive CD4+ T cells and to T-cell clones. Moreover, we demonstrate that this co-stimulatory signal can block the induction of anergy in T-cell clones.

Sequence requirements for the stimulation of gene amplification by a mammalian genomic element.

HSAG-1 is a 3.4-kb genomic element from a human chronic lymphocytic leukemia--Chinese hamster ovary (CHO) hybrid cell line shown to stimulate the amplification of expression vectors in cis when transfected into a variety of cell lines [McArthur and Stanners, J. Biol. Chem. 266 (1991) 6000-6005]. Subfragments of HSAG-1 were tested for amplification activity by insertion into the vector, pSV2DHFR. The results suggest that multiple positive- and negative-acting elements were present that influenced amplification activity. The deletion of regions believed to contain positive-acting elements decreased or abolished the amplification stimulatory activity of the most active 1.45-kb fragment, supporting this hypothesis. The construction of composite sequences containing multiple positive elements and lacking negative elements, however, failed to enhance the activity; maximum activity was obtained only with the original intact configuration of elements. Two of two CHO HSAG-1-like elements tested had an activity equivalent to HSAG-1, while one of 24 random CHO genomic fragments tested had an activity as high as HSAG-1. The combination of sequence and structural features needed to affect the frequency of gene amplification may therefore be quite common in the mammalian genome.

Elements which stimulate gene amplification in mammalian cells: role of recombinogenic sequences/structures and transcriptional activation.

HSAG-1 is a 3.4 kb mammalian genomic element which has been shown to stimulate the amplification of the pSV2DHFR expression vector in cis when transfected into a variety of cell lines (1). This amplification stimulatory activity requires the interaction of multiple positive acting elements that include sequence features associated with recombination 'hotspots', such as Alu-like repetitive sequences and A/T rich regions (2). We demonstrate here that two other members of the HSAG family of elements, HSAG-2 and HSAG-5, also stimulate vector amplification. By analysis of the HSAG-2 nucleotide sequence and of the amplification activity of HSAG-2 and HSAG-5 subfragments, we show that this activity also involves the interaction of multiple positive acting elements. The autonomous replication of the HSAG containing vectors is not responsible for this effect. We also show that the orientation of HSAG elements in pSV2DHFR has a profound effect on their amplification stimulatory activity, and present evidence that the transcription of these elements in pSV2DHFR is necessary for the effect.

A genetic element that increases the frequency of gene amplification.

A repetitive mammalian genetic element, HSAG-1, has been shown to promote the amplification of the vector, pSV2-DHFR, containing the functional cDNA for dihydrofolate reductase (DHFR). LR-73 cells, a line of Chinese hamster ovary cells, were transfected with this recombinant construct or with the parent vector, then subjected to culture in selective medium containing steadily increasing concentrations of methotrexate, a drug which specifically inhibits DHFR. Cultures transfected with the HSAG-1-containing construct acquired drug resistance faster than those transfected with the parent vector. This acceleration of acquisition of drug resistance was due to an increased probability of the generation and subsequent selection of cellular variants with increased copy numbers of the vector. The effect has also been observed in CHO(DHFR-) and HeLa cell lines. Possible mechanisms for the effect of the HSAG-1 element on gene amplification are discussed.