Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization

The gene coding for cholera toxin subunit B (CT-B) was fused to a modified ompA signal sequence and subsequently cloned into a high expression vector based on the regulatory signals of the arabinose operon of Salmonella typhimurium. Upon induction of gene expression in Escherichia coli, a product of the expected size for CT-B monomer was detected at a level of approximately 60% of total periplasmic protein. At pilot scale, batch cultivation in a 20-liter bioreactor allowed a production level of 1 g/liter of recombinant CT-B (rCT-B), the majority of which was released into the culture medium. The latter phenomenon was dependent on the medium selected for cultivation. A simple and inexpensive purification scheme was developed which enabled the recovery of 81% of rCT-B from the culture supernatant. Comparing amino acid composition, amino-terminal sequence, mass spectrum, pentamerisation, and GM1-binding, rCT-B is indistinguishable from natural CT-B produced by Vibrio cholerae. This rCT-B overproducing E. coli strain represents an interesting alternative to overexpressing systems developed in V. cholera

A phase I trial of immunotherapy with intratumoral adenovirus-interferon-gamma (TG1041) in patients with malignant melanoma

Interferon-gamma (IFN-gamma) has been shown to upregulate MHC class I and II expression, and to promote generation of specific antitumor immune responses. We hypothesized that intratumoral administration of an IFN-gamma gene transfer vector facilitates its enhanced local production and may activate effector cells locally. We conducted a phase I dose-escalation study of a replication-deficient adenovirus-interferon-gamma construct (TG1041) to determine safety and tolerability of intratumoral administration, in advanced or locally recurrent melanoma. Patients were enrolled at four successive dose levels: 10(7) infectious units (iu) (n=3), 10(8) iu (n=3), 10(9) iu (n=3), and 10(10) iu (n=2) per injection per week for 3 weeks. TG1041 was injected in the same tumor nodule weekly in each patient. Safety, toxicity, local and distant tumor responses and biologic correlates were evaluated. A total of 11 patients were enrolled and received the planned three injections per cycle. One patient with stable disease received a second cycle of treatment. A maximum tolerated dose was not reached in this study. No grade 4 toxicities were observed. Two grade 3 toxicities, fever and deep venous thrombosis were observed in one patient. The most frequently reported toxicities were grade 1 pain and redness at the injected site (n=8), and grade 1 fatigue (n=5) patients. Clinical changes observed at the local injected tumor site included erythema (n=5), a minor decrease in size of the injected lesion (n=5) and significant central necrosis by histopathology (n=1). Systemic effects included stable disease in one patient. Correlative studies did not reveal evidence of immunologic activity. Weekly intratumoral administration of TG1041 appears to be safe and well tolerated in patients with advanced melanoma