Attenuated Salmonella Targets Prodrug Activating Enzyme Carboxypeptidase G2 to Mouse Melanoma and Human Breast and Colon Carcinomas for Effective Suicide Gene Therapy

Abstract

Purpose: We engineered the oncolytic Salmonella typhimurium^derived bacterium VNP20009 as a vector to target delivery to tumors of the prodrug-activating enzyme carboxypeptidase G2 (CPG2) and to show enhanced antitumor efficacy on administration of different prodrugs. Experimental Design: We characterized CPG2 expression in vectors by immunoblotting, immunofluorescence, and enzyme activity.We assessed prodrug activation by high-performance liquid chromatography.Target human tumor cell and bacterial vector cell cytotoxicity wasmeasured by flow cytometry and colony-forming assays.Therapy was shownin two human tumor xenografts and one mouse allograft with postmortem analysis of bacterial and CPG2 concentration in the tumors. Results: CPG2 is expressed within the bacterial periplasm. It activates prodrugs and induces cytotoxicity in human tumor cells but not in host bacteria. Following systemic administration, bacteria multiply within xenografts reaching 2 Â 10 7

Gene therapy for cancer: bacteria-mediated anti-angiogenesis therapy

“…10,11 However, the most frequently used anti-cancer approach using bacteria is enzyme-prodrug therapy where systemically administered bacteria are used to deliver a gene/enzyme able to convert systemically administered prodrug into its active compound specifically in tumor tissue. [12][13][14] The utilization of bacterial systems for therapeutic purposes is further enhanced by genetic modifications, which make them a very promising tool for targeted delivery of genes and their products. Specific advantages of using bacteria for anti-cancer gene therapy include the natural oncolytic potential of some strains/species, direct targeting of tumor tissue and the ease of positive regulation/eradication.…”

On Advances in Cancer Suicide-genes Therapy

“…CPG2 has been used as antitumor therapy in breast, colon, hepatocellular and colon carcinomas. [26][27][28][29][30] The ZD2767P prodrug is a substrate of GPG2, which activates this prodrug as a bifunctional DNA interstrand cross-linking alkylating agent. This system has been used in hepatocellular carcinoma cells and xenografts exhibiting inhibition of tumour growth and extending the lifespan of the mice.…”

“…[30] According to this reaction, other prodrugs have been studied. [26][27][28] Oncolytic Salmonella typhimurium-derived bacterium VNP20009 was designed to deliver the CPG2. Human breast carcinoma, human colon carcinoma cells and B16-F10 mouse melanoma xenografts were injected with VNP2009/CPG2 and treated with several derivatives of L-glutamic prodrugs.…”

“…CPG2 activated the prodrug and a significant decrease in the tumor growth of xenografts was observed. [28] Two novel benzoic acid L-glutamate mustards, di-and trifluorinated prodrugs, are activated by CPG2, being cytotoxic for human breast carcinoma cells, while the tetra fluorinated prodrug is a competitive inhibitor of CPG2. [27] Nitroreductase: The nitroreductase gene (NTR) from E. Coli, encodes an enzyme which is frequently used for transforming prodrugs into active drugs.…”

Hyperpolarized 13C magnetic resonance detection of carboxypeptidase G2 activity

“…The significant enhancement of the MR signal by more than a factor of 10,000 has transformed insensitive techniques such as 13 C and 15 N MRS into versatile strategies that afford a window on the dynamics of endogenous enzymatic processes by generating high spatial resolution and real-time maps of the metabolism of hyperpolarized substrates noninvasively (1,2). These techniques have primarily focused on hyperpolarized 13 C pyruvate as a substrate due to its favorable relaxation characteristics and central role in cellular energy metabolism (3,4). Other hyperpolarized imaging reporters have emerged in recent years to probe a range of key endogenous metabolic reactions (5)(6)(7)(8), and further applications such as the measurement of pH in vivo (9).…”