March 1, 2007 (Vol. 27, No. 5)
Bacterium-based Stimulation of the Immune System Could Hold the Key
Animals have evolved immune systems to protect themselves from foreign invaders like bacteria and viruses, not from endogenous diseases like cancer. Recently, the discovery that disease immunity might be recruited to fight cancer cells as they mutate from self to nonself has led to interest in recruiting the immune system through vaccines to fight cancer.
Advaxis (www.advaxis.com) has been working on building a cancer vaccine by exploiting the powerful immune response and antigen-carrying capabilities of the bacterium Listeria monocytogenes. By engineering cancer-specific antigens into Listeria, the company co-opts the immune mechanisms normally enlisted against this pathogen and redirects them against various cancers.
“Listeria induces a strong, cell-mediated immune response that results from its unusual life cycle and is particularly suited to killing cancer cells,” says John Rothman, Ph.D., vp of clinical development. The bacterium preferentially infects antigen-presenting cells (APC), which activate the immune system and present specific antigens for the immune system to attack.
A fraction of ingested Listeria avoids digestion in APCs by escaping to the cytosol, where it reproduces and can infect adjacent cells by a cell-to-cell transmission. This capability enables the microbe to activate both helper and killer T cells for specific antigens, which is one of the reasons Listeria vaccines are so effective.
Since Listeria is a bacterium, it also stimulates innate immunity, which viruses do not. Innate immunity involves non-specific activation of the entire immune system to support the activated or adaptive immune response. Listeria also stimulates the Th-1-like cytokine cascade, which liberates chemical messengers that support a therapeutic immune response. Taken together, these immune-stimulating mechanisms provide a directed, powerful, concerted, and integrated cellular immune response capable of killing cancer cells, says Advaxis.
Advaxis has improved upon this panel of immune responses through the use of an antigen fusion protein based on the Listeria enzyme listeriolysin O (LLO). When the APC ingests a foreign Listeria and tries to kill it, ingested Listeria secretes this enzyme, which eats a hole in the wall of the digestive vacuole and allows the bacterium to escape into the cytoplasm.
LLO is potentially dangerous to the host cell because it can compromise the outer cell membrane and may kill the host cell. The body protects itself from this consequence by recognizing an amino acid sequence in the enzyme and rapidly degrading and inactivating it.
Advaxis saw this mechanism as useful because activation of killer T cells requires that the antigen be cleared through a similar mechanism to produce peptide fragments such as antigenic epitopes.
Fusing the antigen to an LLO fragment recruits recognition of LLO to accelerate the breakdown and delivery of antigen. A fragment of LLO that does not puncture membranes is fused to the cancer antigen. This increases the rate of delivery of antigen fragments through the class-1 adaptive pathway, which creates activated killer T cells and generates a robust population of the specific immune cells that destroy the target cancer.
Right Type of Immunity
The use of antigen-specific tumor-killing T cells does not always induce the right type of immunity at the proper levels; many vaccines that stimulate specific antigen-based tumor-killing responses do not work in the clinic. Listeria-based vaccines possess two additional, therapeutically beneficial attributes emanating from the LLO fusion protein.
Part of the immune system’s complexity results from the need to protect normal healthy cells from immune attack. It is believed that protection from autoimmunity comes from a specialized population of T cells that are often activated as part of an immune response called regulatory T cells (Tregs). The activity of these cells can often be stimulated by the same processes that activate killer T cells, but Tregs inhibit the immune system from killing cancer cells.
Advaxis says it has shown that Listeria vaccines that deliver an antigen that isn’t the LLO antigen fusion protein stimulate Tregs (as do other types of tumor-specific vaccines). When this occurs, therapeutic efficacy is diminished.
However, when an LLO antigen fusion protein is delivered by the Listeria vector, there is significantly less activation of Tregs in response to the antigen fusion protein. Thus, Listeria appears to activate a strong anticancer immune response in part because it does not activate Tregs along with the tumor-killing T cells.
Finally, LLO itself has been shown to contribute to therapeutic anticancer activity through non-specific adjuvant effects. These effects create a chemical environment within the tumor that supports other elements of the immune system that increase the efficiency of the cancer-killing immune response. The protein achieves this by providing chemical mediators that support the migration of T cells to the tumor sites, enhance the ability of cytotoxic T cells to recognize and attack tumor cells, and facilitate the release of tumorcidal agents from T cells.
Thus, incorporating LLO within the fusion protein simultaneously activates the immune system, minimizes suppression by inhibitory T cell populations, and stimulates the local tumor environment to support a therapeutic response.
This powerful combination of therapeutic mechanisms explains the observation, in animal models of 100% tumor response rates with 50–100% of animals showing complete tumor disappearance. Similar results were obtained in transgenic animals genetically engineered to express certain cancers. There is also a body of evidence suggesting that the live Listeria vector can overcome tolerance to cancer therapy, a common occurrence in humans.
Advaxis’ has an ongoing, Phase I trial of a Listeria vaccine for treating advanced, recurrent, or progressive cervical cancer, which uses a conservative model consisting of a priming dose followed by a single booster vaccination.
Listeria, as with any new therapeutic paradigm, is likely to encounter resistance from regulators and physicians. The main drawbacks of using a live Listeria antigen-delivery vehicle are related to perceptions of the Listeria organism, a known pathogen, according to Dr. Rothman.
Regulators are rightfully concerned that Listeria administered as part of a vaccine formulation might become virulent, “but numerous studies have demonstrated that the therapeutic organism does not revert to, or mutate into, a more pathogenic strain.” Another regulatory hurdle for an injected drug is the requirement that it be sterile. “Of course sterility is impossible with a live bacterium,” Dr. Rothman adds.
Also, Listeria is not appropriate for some types of antigens. Large macromolecules, for example, are difficult to fit functionally inside the Listeria organism. In some situations, it may be possible to fragment the antigen into manageable pieces and create a composite vaccine from the individual fusion components.
Researchers at the University of Pennsylvania, collaborating with Advaxis, demonstrated the effectiveness of this approach in a murine breast cancer model. In this experiment, Listeria fused with the breast cancer antigen Her2/Neu was delivered. The antigen was delivered not as a single fusion protein, but in five separate Listeria constructs, each with a different overlapping fragment of the Her2 molecule. Each fragment was effective in both normal and transgenic models of breast cancer, as well as in protecting against the eventual tumors that spontaneously occur in transgenic animals.