From food poisoning to cancer immunotherapy

March 9th, 2016

How has research at UC Berkeley on a soil bacterium called Listeria monocytogenes led to new approaches to cancer treatment?

The Cancer Research Lab is founded on the belief that fundamental research can lead to novel and entirely unexpected approaches to cancer therapy. This belief is exemplified by the remarkable story of how research in Professor Dan Portnoy’s lab at UC Berkeley on a soil bacterium called Listeria monocytogenes has helped lead to new therapeutic approaches for human cancer.

Historical studies on Listeria. Listeria monocytogenes is commonly found in the soil, but when it contaminates food, it can infect humans and cause severe – even lethal – disease. Listeria infections mainly affect pregnant women and individuals whose immune system is compromised. Immunologists have studied Listeria for over fifty years as a model to study the immune system. A very important observation was that mice that have recovered from a previous infection with Listeria are highly immune to subsequent re-infection. It was discovered that immunity to Listeria requires special immune cells called cytotoxic T cells. These cells patrol the body and are specialized in the killing and elimination of infected cells.

Another viewpoint: how does Listeria cause disease? Instead of looking at the infection from the standpoint of the immune system, Portnoy decided to focus on understanding Listeria from a different angle. For more than 2 decades, his lab has been at the forefront of understanding the tricks that Listeria uses to cause disease and avoid immune attack. These tricks are now understood in elaborate molecular detail. For example, it had been known for many years that upon infection, the bacteria are ingested by phagocytic immune cells. Normally these immune cells will digest and kill bacteria within a special cellular structure called a phagosome. However, to counteract this defense, it was discovered that Listeria secretes a pore-forming molecule called listeriolysin O (LLO) that leads to rupture of the phagosome and delivery of the bacteria to the interior of cells (often called the “cytosol”) where the bacteria can rapidly replicate. Once in the cytosol, the bacteria produce a special protein called ActA that exploits the host cytoskeletal system and allows the bacteria to move within and between cells without exposure to the extracellular milieu (as reviewed here).

Application of Listeria to protect against disease. As already mentioned, infection with low doses of live Listeria vaccinates mice and protects them from subsequent challenge with a high dose of virulent Listeria. Importantly, it was observed that killed bacteria or mutants lacking LLO that cannot enter the host cell cytosol fail to effectively immunize. Thus, entry of Listeria into the cytosol of immune cells is critical for the ability of Listeria to induce potent immune responses. Building on this insight, Berkeley Professor Dan Portnoy, then at the University of Pennsylvania, decided to explore the use of Listeria as a vaccine against other diseases. In 1994, in collaboration with Yvonne Paterson, Portnoy showed that Listeria bacteria, engineered to express new molecules, could stimulate potent T-cell responses to those molecules. Shortly thereafter, Paterson, using the same strain, collaborated with Drew Pardoll from Johns Hopkins University and showed that it could be used as an agent of cancer immunotherapy in a model mouse model. In 2002, Paterson co-founded Advaxis Inc to develop this technology, while Portnoy, having moved to UC Berkeley in 1997, began to collaborate with Tom Dubensky, then at Cerus Corp and now at Aduro Biotech, on the development of Listeria-based vaccines for cancer immunotherapy. In an important paper, and along with several collaborators, they showed that Listeria deleted for two virulence genes (ActA and InlB) was highly attenuated, yet retained its capacity to stimulate immune responses. Even more importantly, they showed that this strain could be engineered to produce an antigen normally expressed by a tumor cell. Infection of mice with this strain stimulated an immune response against the tumor and led to complete elimination of the tumor. This work was the basis of subsequent clinical studies culminating in a recently completed Phase 2 study conducted by Aduro BioTech, in collaboration with Liz Jaffee at Johns Hopkins University. This study demonstrated the remarkable potential of Listeria as a immunotherapeutic vaccine for late stage pancreatic cancer.

Discovery of an immunostimulatory small molecule. There are likely multiple reasons that Listeria is such a potent inducer of immune responses, including its predilection for and growth in immune cells. Portnoy suspected that the bacteria might also produce specific molecules that stimulate immune responses. In order to identify these molecules, Portnoy and colleagues used bacterial genetics and biochemical approaches to identify Listeria mutants that induced diminished or enhanced immune responses. These studies culminated in the discovery of a new immunostimulatory molecule called cyclic-di-AMP. Cyclic-di-AMP was in a class of molecules called cyclic dinucleotides (CDNs) that had previously been reported to be immunostimulatory. In a collaboration, the Portnoy Lab then worked with the Vance Lab, also at UC Berkeley, to discover that the immunostimulatory activity of CDNs required a immune protein called Stimulator of Interferon Genes or STING. The Vance Lab went on to show that CDNs bound directly to and activated STING. Remarkably, CDNs injected directly into tumors leads to their elimination and Phase I trials will be initiated this year.

Lessons Learned. When Portnoy embarked on his studies of Listeria, he was motivated by his curiosity for how a bacterial pathogen could cause disease. The lesson here is that studies of an obscure and relatively rare cause of food poisoning has led to both a potent bacterial vaccine and a novel small molecule-based therapy for cancer immunotherapy.

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