Immunologic reactivity to lipid-DNA conjugates has traditionally been viewed as less of an issue than with viral vectors. We performed a dose escalation safety trial of aerosolized cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lower airways of eight adult cystic fibrosis patients, and monitored expression by RT-PCR. The cDNA was complexed to a cationic lipid amphiphile (GL-67) consisting of a cholesterol anchor linked to a spermine head group. CFTR transgene was detected in three patients at 2-7 days after gene administration. Four of the eight patients developed a pronounced clinical syndrome of fever (maximum of 103.3EF), myalgias, and arthralgia beginning within 6 hr of gene administration. Serum IL-6 but not levels of IL-8, IL-1, TNF-alpha, or IFN-gamma became elevated within 1-3 hr of gene administration. No antibodies to the cationic liposome or plasmid DNA were detected. We found that plasmid DNA by itself elicited minimal proliferation of peripheral blood mononuclear cells taken from study patients, but led to brisk immune cell proliferation when complexed to a cationic lipid. Lipid and DNA were synergistic in causing this response. Cellular proliferation was also seen with eukaryotic DNA, suggesting that at least part of the immunologic response to lipid-DNA conjugates is independent of unmethylated (E. coli-derived) CpG sequences that have previously been associated with innate inflammatory changes in the lung.

Cationic lipids show promise as vectors for transfer of CFTR cDNA to airway epithelia of patients with cystic fibrosis (CF). However, previous studies have not compared the effect of DNA-lipid to DNA alone. Recently, we developed a formulation of plasmid encoding CFTR (pCF1-CFTR) and cationic lipid (GL-67:DOPE) that generated greater gene transfer in mouse lung than previously described DNA-lipid vectors. Therefore, we tested the hypothesis that DNA-lipid complexes were more effective than DNA alone at transferring CFTR cDNA to airway epithelia in vivo. We administered complexes of DNA-lipid to one nostril and DNA alone to the other nostril in a randomized, double-blind study. Electrophysiologic measurements showed that DNA-lipid complexes partially corrected the Cl

Provoking a specific cellular immune response against tumor-associated antigens is a promising therapeutic strategy to treat cancers with defined antigens such as melanoma. In recent clinical trials, however, immune responses against melanoma antigens have been elicited without consistent clinical responses, suggesting the need for approaches that potentiate the specific cellular immune response. Since B lymphocytes have been reported to exert a negative effect on the cellular arm of the immune response in certain model systems, the authors compared the protective immunity elicited by melanoma antigens in B cell-deficient microMT mice to that obtained in fully immunocompetent C57BL/6 mice. Immunization with melanoma-associated antigens was accomplished using recombinant adenovirus (Ad) vectors encoding human gp100 (Ad2/gp100) or murine TRP-2 (Ad2/mTRP-2). A single dose of Ad2/gp100 or Ad2/mTRP-2 inhibited the growth of established subcutaneous B16 melanoma tumors in B cell-deficient but not wild-type C57BL/6 mice. The enhanced tumor protection observed in B cell-deficient mice appeared to be associated with potentiation of the magnitude and longevity of the specific cellular immune response. Natural killer (NK) cells were also found to be essential to the protective immune response in microMT mice because NK cell depletion with anti-asialo-GM1 antibody resulted in both the loss of tumor growth suppression and attenuation of the specific cellular immune response. The authors conclude that the protective cell-mediated immunity provoked by Ad-based cancer vaccines is enhanced in the absence of B cells, suggesting that a therapeutic regimen that includes depletion of B lymphocytes may be beneficial to cancer vaccine therapy.

Cystic fibrosis (CF), an autosomal recessive disorder resulting from mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, is the most common lethal genetic illness in the Caucasian population. Gene transfer to airway epithelium, using adenoviruses containing normal CFTR cDNA, leads to transient production of CFTR mRNA and, in some studies, to correction of the airway epithelial ion transport defect caused by dysfunctional CFTR. Inflammatory responses to the adenoviral vector have been reported, particularly at high viral titers. We evaluated the effects of adenovirus-mediated CFTR gene transfer to airway epithelium in 36 subjects with CF (34 individuals, 2 of whom received two separate doses of vector), 20 by lobar instillation and 16 by aerosol administration. Doses ranged from 8 x 10(6) to 2.5 x 10(10) infective units (IU), in 0.5-log increments. After lobar administration of low doses there were occasional reports of cough, low-grade temperature, and myalgias. At the highest lobar dose (2.5 x 10(9) IU) two of three patients had transient myalgias, fever, and increased sputum production with obvious infiltrates on CT scan. After aerosol administration there were no significant systemic symptoms until the 2.5 x 10(10) IU dose, when both patients experienced myalgias and fever that resolved within 24 hr. There were no infiltrates seen on chest CT scans in any of the patients in the aerosol administration group. There were no consistent changes in pulmonary function tests or any significant rise in serum IgG or neutralizing antibodies in patients from either group. Serum, sputum, and nasal cytokines, measured before and after vector administration, showed no correlation with adenoviral dose. Gene transfer to lung cells was inefficient and expression was transient. Cells infected with the vector included mononuclear inflammatory cells as well as cuboidal and columnar epithelial cells. In summary, we found no consistent immune response, no evidence of viral shedding, and no consistent change in pulmonary function in response to adenovirus-mediated CFTR gene transfer. At higher doses there was a mild, nonspecific inflammatory response, as evidenced by fevers and myalgias. Overall, vector administration was tolerated but transfer of CFTR cDNA was inefficient and transgene expression was transient for the doses and method of administration used here.

These studies characterize the depletive effects of mATG in normal mice and provide insight into mechanisms of action of Thymoglobulin.

A phase I clinical trial was conducted in which recombinant adenovirus containing the cystic fibrosis trans-membrane regulator (CFTR) (Ad2/CFTR) was administered by bronchoscopic instillation or aerosolization to the lungs of cystic fibrosis (CF) patients. In this paper, we evaluate the efficiency of Ad2/CFTR-mediated transduction of bronchial airway cells. The ability of an Ad2/CFTR vector to transduce airway cells was first evaluated in patients to whom the vector was administered by bronchoscopic instillation. Cells at the administration site were collected 2 days after treatment by bronchoscopic brushing. Ad2-specific CFTR DNA was detected in four of five individuals by PCR, and Ad2-specific CFTR RNA was detected in three of five individuals by RT-PCR. Ad2/CFTR-mediated transduction of airway epithelial cells was then determined in CF individuals receiving this vector by aerosol inhalation. Ad2-specific CFTR DNA was detected in 13 of 13 individuals 2 days after aerosolization, and in 3 of 5 individuals 7 days after aerosolization. Ad2-specific RNA was detected in 4 of 13 individuals on day 2, but was not detected in the 5 individuals tested on day 7. The percentage of airway epithelial cells containing nuclear-localized vector DNA was < or =2.4% as determined by fluorescence in situ hybridization (FISH). However, in some cases, a high percentage of nonepithelial mononuclear cells or squamous metaplastic epithelial cells was infected with the adenoviral vector. In conclusion, aerosol administration is a feasible means to distribute adenoviral vectors throughout the conducting airways, but improvements in adenovirus-mediated transduction of airway epithelial cells are necessary before gene therapy for CF will be effective.

No abstract

Adenoviral vectors expressing tumor-associated antigens can be used to evoke a specific immune response and inhibit tumor growth. In this study, we tested the efficacy of adenoviral vectors encoding human gp100 (Ad2/hugp100), murine gp100 (Ad2/mugp100), or murine TRP-2 (Ad2/muTRP-2) for their ability to elicit a specific cellular immune response and inhibit the growth of B16 melanoma tumor cells in the mouse. C57BL/6 mice were immunized with Ad2/hugp100, Ad2/mugp100, or Ad2/muTRP-2 either 2 weeks prior to B16-F10 tumor challenge (prophylactic treatment) or 3 days after tumor challenge (active treatment). Ad2/hugp100 and Ad2/muTRP-2 administered to two or more intradermal (i.d.) sites inhibited subsequent subcutaneous tumor growth in > or = 80% of the mice and elicited an antigen-specific cytotoxic T lymphocyte response, whereas other administration routes were not as effective. Ad2/mugp100 administered to two i.d. sites did not inhibit tumor growth or provoke cellular immunity. Immunization was less effective with active treatment where tumor growth was not significantly inhibited by a single dose of either Ad2/muTRP-2 or Ad2/hugp100. However, increasing the number of intradermal immunization sites and the number of doses resulted in progressive improvements in protection from tumor growth in the active treatment model. In conclusion, breaking host tolerance to elicit protective immunity by using adenoviral vectors expressing melanoma-associated antigens is dependent upon the choice of antigen, the site of administration, and the number of doses. These observations provide insights into the clinical applicability of adenoviral vaccines for immunotherapy of malignant diseases.