How Advaxis uses Listeria monocytogenes
The first thing Advaxis does is to attenuate the bacterium. We reduce it’s virulence (ability to cause disease) between 10,000 – 100,000 times; thus making the agent much safer to use than wild Listeria.
Then we bioengineer Listeria to become a therapeutic aging based on the details of Listeria’s intracellular activity. Inside the phagolysosome, Listeria produces and secretes the virulence factor listeriolysin O ("LLO"), a protein that generates a hole in the membrane of the phagolysosome and allows the bacteria to escape into the relatively safe cytoplasm. Once in the cytoplasm, however, LLO still retains some activity and is also capable of creating a hole in the cell membrane. This would destroy the host cell, and spill the bacteria back out into the intercellular space where it would be exposed to more immune cell attacks and destruction. To prevent this, a sequence of approximately 30 amino acids is present in the LLO protein, called the PEST sequence (for the predominant amino acids it contains). This PEST sequence is recognized by the host cells and targets the LLO protein for rapid digestion, thus giving LLO a very short life span. The benefit for the Listeria is that the LLO is neutralized and the bacteria can continue to prosper inside the cell, which also remains alive.
Since the activation of the immune system is also dependent upon the digestion of antigens and the creation of small fragments that can be used as recognition sites for an immune attack, Advaxis reasoned that we might use this mechanism that rapidly breaks down LLO to also break down antigens, which are released in both the phagolysosome and in the cytoplasm of the APC. To do so, we created a proprietary method in which we engineer Listeria to secrete a "fusion protein" which is comprised of a tumor specific antigen that we wish to use as the focus for an immune attack that is fused to a segment of LLO. In this way, we can accelerate the breakdown of antigens into immuno-active fragments because the PEST sequence of the fusion protein is recognized and the secreted antigen fusion protein is routed for rapid degradation, thus accelerating both the rate at which antigen fragments are created and the speed with which they are delivered to the immune system for use in the creation of recognition molecules that activate T cells. Furthermore, the fusion of the antigen to LLO is essential to promote the secretion of the antigen, because the signal sequence needed for LLO secretion is kept intact in the fusion protein.
Another reason we use LLO is because it is a strong immune adjuvant. Since Listeria that are killed in the phagolysosome are not virulent, but the Listeria that escape from the digestive phagolysosome via the actions of LLO are virulent, LLO is considered to be a virulence factor for Listeria. As a virulence factor, we have evolved very strong innate immune responses to LLO, and so by engineering Listeria to secrete a tumor specific antigen fused to an immunogenic fragment of LLO Advaxis vaccines secrete both a tumor antigen (tumor target) and an adjuvant in one molecule.
