Bacterial Vectors Show Promise in Cancer Gene Therapy

SAN DIEGO--Genetically engineered bacteria have the potential to deliveranticancer genes directly to a tumor site, according to four presentationsof preclinical data at the American Association for Cancer Research (AACR)annual meeting.

The new technology, known as TAPET (tumor amplified protein expressiontherapy), uses genetically engineered Salmonella bacteria that havebeen attenuated for virulence and infectivity, while retaining sensitivityto antibiotics. Salmonella bacteria were chosen because they multiplyrapidly, can be easily modified genetically, and have been found to growunder both aerobic and anaerobic conditions, such as occur within solidtumors.

These engineered bacterial strains are highly selective for tumor tissueand expand within the tumor to levels 1,000 to 10,000 times greater thanfound in normal tissue. For example, in one of the scientific presentationsat the AACR meeting, a genetically engineered nonpathogenic strain of Salmonellatyphimurium injected into tumor-bearing mice rapidly migrated to thetumor site and achieved tumor-to-liver ratios of approximately 10,000:1.

Furthermore, delivery of the modified bacteria, in this and other mousemodels, led to significant tumor reduction and prolonged survival.

Tumor localization has been shown using TAPET strains in six tumor models,including melanoma, breast, lung, colon, renal, and liver cancer.

The TAPET research program was initiated at Yale University in 1993by scientists David Bermudes, PhD, Brooks Low, PhD, and John Pawelek, PhD,and is currently being developed by Vion Pharmaceuticals, Inc. of New Haven,Conn.

"These modified bacteria have lost their ability to cause diseaseelsewhere in the body but seem to retain it in the tumor and are able togrow to very high numbers in the tumor," Dr. Bermudes said in an interview.

Prodrug-Converting Enzymes

Although the genetically engineered bacteria by themselves have theability to slow tumor growth, he said, they cannot eliminate the cancer,so Vion is developing a portfolio of TAPET organisms to deliver prodrug-convertingenzymes and/or cytokines to tumors.

Dr. Bermudes described research presented at AACR involving a Salmonellastrain engineered to express herpes simplex virus thymidine kinase(HSV TK), an enzyme that converts the prodrug ganciclovir (Cytovene) toits toxic phosphorylated form. When introduced into melanoma-bearing mice,HSV TK-expressing Salmonella showed ganciclovir-mediated, dose-dependentsuppression of tumor growth and prolonged survival.

In another AACR presentation, Ellen Carmichael, PhD, associate directorof biology at Vion, said that TAPET bacteria have been engineered to deliverE coli cytosine deaminase (CD), an enzyme that converts the prodrug5-fluorocytosine (5-FC) to the toxic 5-fluorouracil (5-FU).

Tumor cells from mice injected with the CD-expressing bacteria showedsubstantial levels of CD activity, whereas cells from other organs, suchas liver, had no detectable CD activity.

Given alone, the modified bacteria led to tumor reduction. Studies arenow underway, Dr. Bermudes said, to determine whether co-injection of 5-FCfurther increases the antitumor effect of the CD-expressing bacteria.

"By converting 5-FC to 5-FU directly in the tumor, rather thanadministering 5-FU systemically, hopefully, we will be able to reduce thetoxicity of 5-FU to other parts of the body," he said.

Addresses Vector Problems

Dr. Bermudes pointed out that the TAPET technology addresses severalproblems that may be associated with gene therapy of cancer involving viralor liposomal vectors.

First, whereas most viral vectors are delivered locally and thus canonly treat tumors locally, TAPET vectorswould be delivered systemically and would have the potential to find anddestroy clinically undetectable areas of metastatic disease disseminatedthroughout the body.

At the AACR meeting, Ivan King, PhD, senior director of biology at Vion,showed that attenuated Salmonella typhimurium inhibited the growthof subcutaneously implanted melanoma and lung tumors, as well as lung metastasesin mice.

A second problem with gene therapy is the development of a host immuneresponse, which reduces the effectiveness of subsequent treatments. Toavoid this problem, Vion is developing TAPET vectors in multiple serotypes.

Third, TAPET has the potential to overcome a major hurdle in the genetherapy of cancer, ie, the inability to deliver adequate numbers of genecopies to cancer cells. This is because TAPET bacterial vectors have beenshown to distribute and amplify uniformly within the tumor.

At the AACR meeting, Li Mou Zheng, PhD, associate director of biologyat Vion, reported that attenuated strains of Salmonella targetedand amplified within tumors in mice. When examined by light microscopy,Dr. Zheng found that the Salmonella distributed homogeneously withinthe tumor tissues from the periphery to the center, suggesting that TAPETvectors could potentially deliver the therapeutic proteins throughout theentire tumor.

Fourth, Vion is testing TAPET vectors that may express more than onegene, while viral vectors are generally limited to the expression of asingle gene.

Finally, since TAPET vectors remain fully sensitive to antibiotics,they could be cleared from the body in the presence of an antibiotic atany time, allowing greater therapeutic control.

Phase I human clinical trials of the TAPET technology are expected tobegin in 1998, according to Vion. "In our preclinical data, we havebeen able to show efficacy in melanoma, lung cancer, and colon cancer models,and it seems reasonable that the first clinical efforts in humans willbe in these cancers," Dr. Bermudes said.