A phase I trial of genetically modified Salmonella typhimurium expressing cytosine deaminase (TAPET-CD, VNP20029) administered by intratumoral injection in combination with 5-fluorocytosine for patients with advanced or metastatic cancer. Protocol no: CL-017. Version: April 9, 2001

An attenuated strain of Salmonella typhimurium, designated VNP20009, was generated by deletion of the msbB and purl genes. When VNP20009 was administered intravenously (IV) to mice bearing spontaneous, syngeneic, or human xenograft tumors, the bacteria accumulated preferentially within the extracellular components of tumors, forming tumor-to-normal tissue ratios exceeding 300-1000 to 1. NVP20009 was administered safely at doses up to 2.5 x 10(9) cfu/kg in monkey toxicology studies. Based on the preclinical data, VNP20009 entered Phase I human clinical trials in November 1999, and has now been administered to >45 patients by IV or direct intratumoral injection. By the intratumoral route, a maximum tolerated dose has not been reached, and dose escalation continues past the current dose level of 4 x 10(7)/m2. Furthermore, VNP20009 persisted in injected tumors for at least 2 weeks in 8/11 patients treated to date. By 30-min IV administration, a maximum tolerated dose (MTD) of 3 X 10(8) cfu/m2 has been established. In all patients treated to date, VNP20009 was not shed in urine or stool. VNP20009 has been further modified by chromosomal insertion of an E. coli cytosine deaminase (CD) gene at the deltamsbB locus which, when expressed, converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). The CD containing VNP20009 was designated TAPET-CD or VNP20029. TAPET-CD had similar efficacy and safety in murine tumor models and similar safety profiles in animal toxicology studies, compared to its parent VNP20009. Specifically, TAPET-CD had a reduced virulence of >10,000 fold, when compared to the wild-type Salmonella strain. It was well-tolerated at doses up to 2 x 10(6) cfu/mouse and 1 X 10(10) cfu/monkey. After an IV or direct tumor injection to tumor-bearing mice, TAPET-CD reached tumor levels as high as 10(8)-10(9) cfu/gm. When compared to the accumulation in liver or spleen, the normal tissues with the greatest colonization of TAPET-CD, tumor-to-normal tissue ratios of TAPET-CD were 300-1000 to 1. TAPET-CD also caused tumor growth inhibition of >90% in several murine tumor models. When 5-FC was administered by intraperitoneal (IP) injection once or 3 times daily to tumor-bearing mice that had been pre-treated with TAPET-CD, high levels of 5-FU (reaching 20-40 microM/g) were detected in the tumor, with low or undetectable 5-FU levels in normal tissues (e.g., spleen, liver, etc.). Furthermore, co-administration of 5-FC and TAPET-CD in 4 different murine tumor models enhanced anti-tumor activity compared to the significant anti-tumor activity of TAPET-CD alone, further confirming the benefit of the inserted CD gene. On the basis of the preclinical data, a Phase I clinical protocol is proposed in which advanced cancer patients will receive TAPET-CD by direct intratumoral injection and 5-FC. TAPET-CD will be administered on day 1. 5-FC will be given orally q8h daily beginning day 4 or when all toxicities of TAPET-CD have resolved to < or = grade 1, and continued for 14 days. Tumor tissues will be sampled to verify TAPET-CD colonization and to measure intratumoral 5-FC and 5-FU concentrations on day 8. A second sample of tumor tissue will be obtained between day 15-17 in selected patients to confirm the persistence of high levels of bacteria in tumor and to obtain a second measurement of 5-FC and 5-FU intra-tumoral concentrations. The TAPET-CD/5-FC treatment cycle will be repeated in appropriate patients on day 29.