Clinical experience with HLA-B7 plasmid DNA/lipid complex in advanced squamous cell carcinoma of the head and neck.

OBJECTIVE: To investigate the safety and efficacy of alloantigen plasmid DNA therapy in patients with advanced head and neck squamous cell carcinoma using Allovectin-7 (Vical Inc, San Diego, Calif), a DNA/lipid complex designed to express the class I major histocompatibility complex antigen HLA-B7. DESIGN: Multi-institutional prospective trial. SETTING: Academic medical setting. PATIENTS: A total of 69 patients were enrolled in 3 sequential clinical trials: a single-center phase 1 trial and 2 multicenter phase 2 trials. Eligibility criteria included unresectable squamous cell carcinoma that failed conventional therapy, Karnofsky performance status score of 70 or greater, and no concurrent anticancer or immunosuppressive therapies. INTERVENTION: Patients received 2 biweekly intratumoral injections of 10 microg (phase 1 and first phase 2 trials) or 100 microg (second phase 2 trial) of Allovectin-7 followed by 4 weeks of observation. Patients with stable or responding disease after the observation period were given a second treatment cycle identical to the first. MAIN OUTCOME MEASURES: Patients were assessed for toxic effects, and tumor size was measured after cycles 1 (at 6 weeks) and 2 (at 16 weeks). RESULTS: Allovectin-7 treatment was well tolerated, with no grade 3 or 4 drug-related toxic effects. Of 69 patients treated, 23 (33%) had stable disease or a partial response after the first cycle of treatment and proceeded to the second cycle. After the second cycle, 6 patients had stable disease, 4 had a partial response, and 1 had a complete response. Responses persisted for 21 to 106 weeks. CONCLUSIONS: Intratumoral plasmid DNA immunotherapy for head and neck cancer with Allovectin-7 is safe, and further investigations are planned in patients with less advanced disease, where it could potentially improve patient survival and reduce the need for radical high-morbidity treatments.

Phase I study of direct intralesional gene transfer of HLA-B7 into metastatic renal carcinoma lesions.

Renal cancer cell lines exhibit deficient expression of MHC class I antigens required for appropriate CTL stimulation. Nabel et al. (Proc. Natl. Acad. Sci. USA, 90: 11307-11311, 1993) demonstrated that direct gene transfer of the deficient class I MHC molecule into melanoma cells would stimulate their immune destruction. Stopeck et al. (J. Clin. Oncol., 15: 341-349, 1997) demonstrated the clinical use of this approach in melanoma patients. We investigated the safety and ability to bestow gene expression via intratumoral transfer of escalating amounts of lipid-formulated plasmid DNA encoding for the MHC HLA-B7 gene product Allovectin-7 into metastatic renal cancer lesions. Fifteen patients with histologically confirmed, HLA-B7-negative metastatic renal cancer received intratumoral injection of Allovectin-7 on an escalating dose schedule. Tumors were evaluated serially by computed tomography scan, ultrasound, and physical examination. Presence of the HLA-B7 gene and protein was determined via PCR, flow cytometry, and immunohistochemical staining in serial biopsy specimens. HLA-B7 gene, mRNA, or protein expression could be conclusively demonstrated in 8 of 14 patients (57%). Three patients had tumor biopsy to assess the presence of tumor-infiltrating lymphocytes, and all three had higher posttreatment levels of tumor-infiltrating lymphocytes. There were no significant clinical responses or toxicity at the site of injection or at other, noninjected tumor sites. This study demonstrated that intratumoral injection of Allovectin-7 is safe, feasible, and associated with minimal toxicity. This approach was capable of bestowing gene expression, possible resulting in antitumor CTL response. Despite lack of tumor regression in this series of renal cancer patients, the simplicity and low toxicity of this approach commend it for Phase II studies in renal and other cancers, as well as for transfection with other genes.

Immunotherapy of advanced malignancy by direct gene transfer of an interleukin-2 DNA/DMRIE/DOPE lipid complex: phase I/II experience.

PURPOSE: We have completed a phase I study, followed by three phase I/II studies, in patients with metastatic melanoma, renal cell carcinoma (RCC), and sarcoma in order to evaluate the safety, toxicity, and antitumor activity of Leuvectin (Vical Inc, San Diego, CA), a gene transfer product containing a plasmid encoding human interleukin (IL)-2 formulated with the cationic lipid 1, 2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide/dioleyl-phosphatidyl-ethanolamine (DMRIE/DOPE) and administered intratumorally. PATIENTS AND METHODS: Twenty-four patients were treated in the phase I study. Leuvectin doses were 10 microg, 30 microg, or 300 microg weekly for 6 weeks. In three subsequent phase I/II studies, a total of 52 patients (18 with melanoma, 17 with RCC, and 17 with sarcoma) were treated with further escalating doses of Leuvectin: 300 microg twice a week for 3 weeks, 750 microg weekly for 6 weeks, and 1,500 microg weekly for 6 weeks. RESULTS: There were no drug-related grade 4 toxicities and only one grade 3 toxicity, but the majority of patients experienced mild constitutional symptoms after treatment. In the phase I/II studies, 45 patients were assessable for response (14 with RCC, 16 with melanoma, and 15 with sarcoma). Two patients with RCC and one with melanoma have achieved partial responses lasting from 16 to 19 months and continuing. In addition, two RCC, three melanoma, and six sarcoma patients had stable disease lasting from 3 to 18 months and continuing. The plasmid was detected by polymerase chain reaction assay in the posttreatment samples of 29 of 46 evaluated patients. Immunohistochemistry studies on serial biopsy specimens showed increased IL-2 expression and CD8(+) infiltration after treatment in the tumor samples of several patients (12 and 16, respectively). CONCLUSION: Direct intratumoral injection of Leuvectin is a safe and possibly effective immunotherapeutic approach in the treatment of certain tumor types.

Phospholipid prodrug inhibitors of the HIV protease. Antiviral activity and pharmacokinetics in rats.

The aspartyl protease of the human immunodeficiency virus (HIV) is an important target for chemotherapeutic intervention because of its key role in cleaving the HIV gag-pol polyprotein during viral assembly and budding. Short peptides and peptidomimetics, which bind to the active site of the HIV aspartyl protease and inhibit processing of the polyprotein, have been synthesized. These compounds are active against HIV in vitro, but many face substantial development problems because of their rapid elimination from the body in bile and urine. Refinement of these agents appears to be necessary if they are to become useful clinically. Recently, we developed a novel chemical strategy for increasing plasma levels of HIV protease inhibitory peptides, which involves the attachment of a biodegradable phospholipid group to the C-terminus of a pentapeptide, iBOC-[L-Phe]-[D-beta-Nal]-Pip-[alpha-(OH)-Leu]-Val (7194). We coupled phosphatidylethanolamine to the C-terminal valine of 7194 to make a phospholipid prodrug (7196). In vitro assays in HT4-6C cells infected with HIV-1 showed that the antiviral activity of the C-terminal phospholipid prodrug, 7196, was equal to that of the free peptide, 7194. Similar results were obtained in vitro when a related pentapeptide (7140) was derivatized at the N-terminal with dipalmitoylphosphatidylethanolamine-succinic acid (7172). Tritium-labeled 7194 and 7196 were prepared and injected intravenously into rats at 3 mumol/kg; then the plasma was assayed for native compound and metabolites by HPLC radioactivity flow detection. The peak plasma level of the tritium-labeled lipid prodrug (7196) was 36 microM versus 1.6 microM for the free protease inhibitor pentapeptide (7194). The area under the curve of the phospholipid prodrug (7196) was 48-fold greater and its mean residence time was increased 43-fold versus the free peptide (7194). Phospholipid prodrugs appear to offer an alternative approach to optimizing in vivo performance of HIV protease inhibitors and other small peptides.

Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution.

The structure of a complex between a peptide inhibitor with the sequence N-acetyl-Thr-Ile-Nle-psi[CH2-NH]-Nle-Gln-Arg.amide (Nle, norleucine) with chemically synthesized HIV-1 (human immunodeficiency virus 1) protease was determined at 2.3 A resolution (R factor of 0.176). Despite the symmetric nature of the unliganded enzyme, the asymmetric inhibitor lies in a single orientation and makes extensive interactions at the interface between the two subunits of the homodimeric protein. Compared with the unliganded enzyme, the protein molecule underwent substantial changes, particularly in an extended region corresponding to the "flaps" (residues 35 to 57 in each chain), where backbone movements as large as 7 A are observed.

Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease.

The rational design of drugs that can inhibit the action of viral proteases depends on obtaining accurate structures of these enzymes. The crystal structure of chemically synthesized HIV-1 protease has been determined at 2.8 angstrom resolution (R factor of 0.184) with the use of a model based on the Rous sarcoma virus protease structure. In this enzymatically active protein, the cysteines were replaced by alpha-amino-n-butyric acid, a nongenetically coded amino acid. This structure, in which all 99 amino acids were located, differs in several important details from that reported previously by others. The interface between the identical subunits forming the active protease dimer is composed of four well-ordered beta strands from both the amino and carboxyl termini and residues 86 to 94 have a helical conformation. The observed arrangement of the dimer interface suggests possible designs for dimerization inhibitors.