Technology
How Advaxis uses Listeria monocytogenes
The details of Listeria intracellular activity are important for understanding Advaxis technology. 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 specific antigen that we wish to use as the focus for an immune attack 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.
Listeria has a unique life cycle. It infects Antigen Presenting Cells (APC), which are the cells that activate immune cells and tell them what to attack. Because of this, Listeria becomes perfectly positioned to have the maximum effect on the immune system in terms of directing it against specific targets. Even more unusual is Listeria�s ability to stimulate both helper T cells (CD4+) and killer T cells (CD8+), since both are necessary for an antitumor response and it is unusual for a single pathogen to stimulate both in the way Listeria does.
The gene encoding for the fusion protein is engineered into plasmids which are inserted into an attenuated Listeria. Attenuation makes the Listeria less virulent, and the plasmids enable it to secrete the antigen fusion protein as if it were a Listeria antigen.
In this way we can use the strong stimulation of a cell mediated response that we have evolved, which enables us to co-exist with Listeria, and redirect it against a specific tumor type. And we can improve upon the normal response to Listeria by increasing the efficiency of generating specific antigen fragments for the immune system to use in activating cancer killing T cells to attack the specific cancer to which the Listeria has been engineered to mimic.
But there is even more to this story. Not only are we able to stimulate innate immunity and both endogenous and exogenous adaptive immune pathways to stimulate the activation of tumor specific CD4+ and CD8+ T cells, but LLO fusion proteins have other effects as well.
It has recently been shown that LLO has a variety of other effects that have utility in the treatment of cancer. These include the stimulation of a variety of chemical messengers called cytokines, chemokines and co-stimulatory molecules that facilitate a therapeutic immune response. Although activated T cells are necessary to treat cancer immunologically, they need an environment conductive to the promotion of a therapeutic effect. The mediators released in LLO (and the LLO fraction of the antigen fusion protein) create such an environment, not the least of which is a local tissue environment within the tumor that enables activated T cells to kill tumor cells and clear them from the body.
One singularly interesting finding that we have published is the effect of our antigen LLO fusion protein on the generation of regulatory T cells. These Tregs are activated by the same stimuli that activate helper and killer T cells, however they serve to inhibit the immune response, thus lessening the attack against cancer. It is believed that these cells serve to inhibit the immune response so as to control potentially fatal autoimmune responses. When we compared Listeria that have been modified to secrete just an antigen with Listeria that have been modified to secrete an LLO-antigen fusion protein, we have found that Listeria secreting the fusion protein does not result in the activation of Tregs, thus increasing the strength of the cancer directed immune attack.
It is in this way that our engineered Listeria stimulate multiple reinforcing immune mechanisms to attack existing cancers.
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