Combination nonviral interleukin 2 and interleukin 12 gene therapy for head and neck squamous cell carcinoma.

OBJECTIVE: To determine the feasibility and efficacy of combination nonviral lipid-formulated murine interleukin 2 (mIL-2) and polymer-formulated murine interleukin 12 (mIL-12) gene therapy for head and neck squamous cell carcinoma (HNSCC) in a murine model. METHODS: Randomized, controlled studies in a murine HNSCC model. Tumors were established in the floor of mouth in C3H/HeJ immunocompetent mice. Established tumors were directly injected with lipid-formulated mIL-2 and polymer-formulated mIL-12 alone and in combination. Antitumor responses, cytokine expression, and natural killer cell and cytolytic T-lymphocyte activity were assayed. RESULTS: The use of combined mIL-2 and mIL-12 gene therapy resulted in significant antitumor effects, compared with each of the single-cytokine and no-treatment (control) groups (P =.01 to P =.02). Tumors treated with the formulated cytokine genes showed an increased level of the corresponding proteins and decreased level of transforming growth factor beta (TGF-beta) expression. Combined mIL-2 and mIL-12 treatment consistently produced the greater activation of cytolytic T-lymphocyte and natural killer cells than did single-cytokine treatment or other controls at all concentrations tested. Augmented immune responses correlated with clinical antitumor effects. CONCLUSIONS: The nonviral gene delivery system was well tolerated, and combined mIL-2 and mIL-12 gene transfer generated potent antitumor immune responses against HNSCC in our murine model. Combined nonviral IL-2 and IL-12 gene therapy may have great potential as a primary or adjuvant treatment for HNSCC in humans.

Combination nonviral cytokine gene therapy for head and neck cancer.

OBJECTIVE: To establish the feasibility and efficacy of combination nonviral murine interferon-alpha (mIFN-alpha)and murine interleukin-2 (mIL-2) or murine interleukin-12 (mIL-12) gene therapy for head and neck squamous cell carcinoma in a murine model. STUDY DESIGN: Randomized controlled studies in a murine head and neck cancer model were performed to assess antitumor responses, secondary cytokine expression, and both natural killer (NK) cell and cytolytic T-cell (CTL) activity. METHODS: Tumors were established in the floor of mouth in C3H/HeJ immunocompetent mice. Established tumors were directly injected with polymer-formulated murine interferon-alpha (mIFN-alpha), lipid-formulated mIL-2, and polymer-formulated mIL-12 alone or in combination. Primary and secondary cytokine expression,NK cell activity, and CTL activity were assayed. RESULTS: The use of mIFN-alpha gene therapy in combination with either mIL-2 or mIL-12 resulted in significant antitumor effects as compared with each of the single cytokine and control treatment groups (P = .002). Increased levels of NK cell activity and tumor specific CD8+ cytotoxic T-lymphocyte activity were found in the combination mIFN-alpha and mIL-2 or mIL-12 groups. Augmented immune responses correlated with clinical antitumor effects. CONCLUSIONS: The present study demonstrates that mIL-2 or mIL-12 augments tumor inhibition from mIFN-alpha and increases activation of NK and CD8+ T cells. These data support further investigation of polymer and lipid mediated delivery of cytokine genes for head and neck cancer.

Antitumoral effect of a nonviral interleukin-2 gene therapy is enhanced by combination with 5-fluorouracil.

Using a novel cationic lipid delivery system consisting of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride and cholesterol, we delivered murine interleukin-2 (IL-2) cDNA directly into an established murine renal cell carcinoma (Renca). Production of IL-2 within the tumor induced rejection of established tumors (62% on average), whereas control plasmid had little or no effect (17% on average). Surviving animals treated with IL-2:lipid were highly resistant to Renca rechallenge, but not to cross-challenge with a syngeneic mammary adenocarcinoma. Experiments on selectively immunosuppressed animals indicated a requirement for CD8+ T, natural killer, and polymorphonuclear cells. By contrast, depletion of CD4+ T cells did not disrupt the ability of IL-2:lipid to induce tumor rejection. A combination of IL-2 gene therapy with 5-fluorouracil treatment increased the antitumoral efficacy and survival of mice bearing primary and metastatic Renca tumors (42% survival with IL-2:lipid compared with 94% survival with IL-2:lipid plus 5-fluorouracil). These data indicate that rejection of primary and metastatic tumors can be achieved after intratumoral delivery of a nonviral IL-2 gene therapy, and is increased in combination with systemic delivery of a conventional chemotherapeutic agent.

Combination of interleukin 12 and interferon alpha gene therapy induces a synergistic antitumor response against colon and renal cell carcinoma.

The antitumor effect and mechanism of action of IL-12 gene therapy combined with IFN-alpha gene therapy were investigated in tumor-bearing mice using renal and colon carcinoma models, Renca and CT26, respectively. Tumors were treated with murine IL-12 plasmid alone or in combination with IFN-alpha plasmid formulated with a polymeric interactive noncondensing (PINC) gene delivery system. Intratumoral injection of IL-12 DNA/polyvinyl pyrrolidone (PVP) alone induced rejection of 58 and 17% of Renca and CT26 tumors, respectively, whereas 25% (Renca) and 0% (CT26) rejection was observed in mice treated with IFN-alpha plasmid/PVP. Combination gene therapy of formulated plasmids, IL-12 with IFN-alpha, synergistically increased the antitumor response against Renca (100% tumor rejection) and CT26 (50%). In vivo depletion of leukocyte subsets indicated that CD8(+) T and NK cells were the primary effectors of the antitumor response induced by the combined cytokine gene therapy. Moreover, mice that rejected the primary tumors after combined treatment with IL-12 and IFN-alpha plasmid formulation developed protective immunity against a subsequent tumor challenge. Analysis of tumor-infiltrating leukocytes from mice treated with the combined IL-12 and IFN-alpha gene therapy showed upregulation of CD40 molecules on antigen-presenting cells (Mac-1(hi) cells). Finally, levels of mRNA for the chemokines IP-10 and TCA-3 were higher in tumors treated with the combination of cytokine plasmids than in tumors treated with either cytokine gene alone. These data provide evidence that IL12 gene therapy combined with IFN-alpha gene therapy synergistically induces regression of established tumors and may represent a novel therapeutic strategy for cancer treatment.

Combination surgery and nonviral interleukin 2 gene therapy for head and neck cancer.

We have developed a novel nonviral interleukin 2 (IL-2) gene therapy that demonstrates significant treatment-specific, antitumor efficacy in combination with subtotal surgical resection in a head and neck cancer murine model. Treatment of established head and neck tumors in immunocompetent mice was performed via direct injection with a cationic liposome composed of DOTMA and cholesterol formulation carrying DNA plasmid for human IL-2 (hIL-2) gene expression. ELISA assays of tumor extracts 24 h after treatment of hIL-2 gene therapy revealed increased local hIL-2 production as well as a formulation-specific secondary induction of murine IFN-gamma and IL-12. We hypothesize that the paracrine production of multiple cytokines after IL-2 single gene transfer is important for generating a therapeutic effect, and that this strategy will be well tolerated and effective in combination with surgery for head and neck cancer. In animal experiments where surgery was performed in conjunction with an operative site injection of hIL-2 plasmid formulation, no pre-, intra-, or postoperative toxicity or compromise to wound healing was identified. In murine experiments combining partial surgical resection with the nonviral gene therapy, significant antitumor efficacy was demonstrated in the hIL-2 plasmid formulation group compared with empty plasmid formulation and lactose-injected controls. In a separate experiment using smaller tumor sizes, we also demonstrated that treatment outcomes were dependent on the technical aspect of the actual treatment injection as well as visualization with surgical access. The hIL-2 plasmid formulation gene therapy induces local expression of multiple cytokines, results in treatment-specific antitumor effects, and circumvents many of the concerns and toxicity encountered with viral gene transfer. These data support the need for continued preclinical investigation and the consideration of human clinical trials for combination nonviral hIL-2 gene therapy and surgery for head and neck cancer.

Intravenous delivery of an endostatin gene complexed in cationic lipid inhibits systemic angiogenesis and tumor growth in murine models.

Inhibition of the neovascularization of tumors has proven efficacious in reducing tumor growth and metastases. Attaining antiangiogenesis through cationic lipid-based systemic gene therapy presents an attractive approach to the treatment of disseminated and primary cancers. Intravenous administration of an endostatin plasmid, complexed with a cationic lipid system, produced significant levels of endostatin in the lung and the circulation. The expressed endostatin blocked systemic angiogenesis and inhibited tumor growth in murine models. Cytokine induction by cationic lipid/DNA complex increased the anti-tumor activities of endostatin. These results demonstrate the possibility of using cationic lipid delivery of an antiangiogenic gene for cancer treatment.

Intramolecular G-quartet motifs confer nuclease resistance to a potent anti-HIV oligonucleotide.

We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5'-g.tggtgggtgggtggg.t-3', where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 microM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with t1/2 of 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (t1/2 from approximately 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.

Novel polyaminolipids enhance the cellular uptake of oligonucleotides.

Two new polyaminolipids have been synthesized for the purpose of improving cellular uptake of oligonucleotides. The amphipathic compounds are conjugates of spermidine or spermine linked through a carbamate bond to cholesterol. The polyaminolipids are relatively nontoxic to mammalian cells. In tissue culture assays, using fluorescent-tagged or radiolabeled triple helix-forming oligonucleotides, spermine-cholesterol and spermidine-cholesterol significantly enhance cellular uptake of the oligomers in the presence of serum. Spermine-cholesterol is comparable with DOTMA/DOPE (a 1:1 (w/w) formulation of the cationic lipid N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) and the neutral lipid dioleylphosphatidylethanolamine (DOPE)) in increasing cellular uptake of oligonucleotides, while spermidine-cholesterol is more efficient. The internalized oligonucleotides are routed to the nucleus as early as 20 min after treatment, suggesting that the polyaminolipids increase the permeability of cellular membranes to oligonucleotides. At later times, much of the incoming oligonucleotides are sequestered within punctate cytoplasmic granules, presumably compartments of endosomal origin. Coadministration with polyaminolipids markedly improves the cellular stability of the oligonucleotides; more than 80% of the material can be recovered intact up to 24 h after addition to cells. In the absence of the polyaminolipids, nearly all of the material is degraded within 6 h. These data suggest that the new polyaminolipids may be useful for the delivery of nucleic acid-based therapeutics into cells.

Zero-order ultrasensitivity in the regulation of glycogen phosphorylase.

The activity of glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) is controlled by a cyclic phosphorylation-dephosphorylation process through the action of the interconverting enzymes, phosphorylase b kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38) and phosphorylase a phosphatase (phosphorylase a phosphohydrolase, EC 3.1.3.17). In muscle tissue, the combined concentration of the activated (phospho-) form, phosphorylase a, and the nonactivated (dephospho-) form, phosphorylase b, is substantially greater than the Km of either of the interconverting enzymes for its phosphorylase substrate. It has been predicted that, under such a set of conditions, a sensitivity amplification will occur for phosphorylase regulation due to the zero-order ultrasensitivity effect [LaPorte, D. C. & Koshland, D. E., Jr. (1983) Nature (London) 305, 286-290]. The sensitivity amplification will enhance the responsiveness of the phosphorylase interconversion cycle to changes in the ratio of activities of the kinase to phosphatase. We have studied the cyclic interconversion process using purified muscle enzymes in steady-state reactions and found that there is an enhancement in the control sensitivity of the process due to the zero-order ultrasensitivity effect. The potential for the in vivo enhancement of sensitivity in glycogen degradation by this effect is discussed.

Use of fluoride to inactivate phosphorylase a phosphatases from rat liver cytosol. Presence of fluoride-insensitive glycogen synthase-specific phosphatase.

The diverse metal requirements for activity of the phosphoprotein phosphatases (EC 3.1.3.16) concerned with glycogen metabolism in rat liver were postulated to reflect the diverse binding intensities of their essential metal(s). After inactivation by fluoride, three of these phosphatases had similar metal requirements in contrast to a fourth phosphatase. Further similarities led to a grouping of these enzymes into two general types. Phosphatases designated type 1 consisted of three enzymes which had the following properties; (1) preference for glycogen phosphorylase a as a substrate; (2) molecular weights in excess of 100 000; (3) conversion to an active 30 000 dalton 'subunit' form upon selective denaturation by 80% ethanol; (4) diverse degrees of stimulation by metals (Mg2+ and Mn2+); and (5) changes to an absolute dependence upon added Mn2+ (but not Mg2+) for activity of both the holoenzyme and the subunit after a demetallating treatment with fluoride in EDTA. The phosphatase designated type 2 exhibited the following properties; (1) preference for glycogen synthase D as a substrate; (2) molecular weight of 50 000; (3) no conversion to an active 30 000 dalton subunit form upon selective denaturation by 80% ethanol; (4) complete metal-dependence upon either Mg2+ or Mn2+; and (5) no change to an absolute dependence on added Mn2+ for activity after a demetallating treatment with fluoride in EDTA.

An investigation of the Mini-Mult validity scales.

Examined the ability of the Mini-Mult validity scales to detect invalid MMPI profiles. When 34 invalid MMPI profiles were rescored with the Mini-Mult only 17 of the 34 profiles invalidated by the full MMPI were detected with the Mini-Mult. This included 14 of 27 profiles invalidated by an elevated F scale; 2 of 4 profiles invalidated by an elevated L scale and 1 of 3 profiles invalidated by an elevated K scale. Only 14 of 27 profiles invalidated by an F-K ratio of K11 were detected. When new conversion values for the Mini-Mult were utilized, the detection rate improved considerably for the F scale and the F-K ratio.