The Idea of Checkpoint Blockade and the Cancer Immunotherapy Revolution

The immune system is a powerful network of cells in the human body that is essential for elimination of foreign invaders, such as bacteria and viruses. It has long been a dream of immunologists to harness the power of the immune system to attack tumors as well. Although this idea, called “cancer immunotherapy”, is very appealing, and has previously been shown to work in several mouse models of cancer, it has in general been very difficult to translate cancer immunotherapy approaches to humans. Because of this frustration, by the 1990s, many scientists and biotechnology companies had given up on the idea of cancer immunotherapy.

Prof. James (“Jim”) P. Allison was not one of those that had lost hope. In the late 1990s, while serving as the Director of the UC Berkeley Cancer Research Laboratory, Jim became intrigued by a special biological molecule called CTLA-4. This molecule was displayed on the surface of particular cells in the immune system called T cells. Jim and his colleagues — most notably, graduate student Max Krummel, and postdoctoral fellow Cynthia Chambers — were able to provide evidence that CTLA-4 actually served to inhibit the activity of T cells. Since T cells were known to be critical for orchestrating immune attack, Jim and his colleagues hypothesized that perhaps one reason why T cells are not able to attack tumors is because they are being inhibited by CTLA-4. In a landmark Science paper published in 1996, Jim Allison, Max Krummel, and another postdoctoral fellow in the lab Dana Leach, showed that injection of mice with an antibody that blocks CTLA-4 could actually inhibit or eliminate tumor growth in the mice.

CHECKPOINT BLOCKADE

The series of experiments published in the 1996 Science paper represent the birth of a new concept in immunotherapy, the concept of “checkpoint blockade”. The fundamental idea behind checkpoint blockade is that the immune system is normally held in check by inhibitory molecules such as CTLA-4. The inhibitory effects of CTLA-4 are actually very important for the normal function of the immune system, serving as a “checkpoint” that restrains the immune system from inappropriately attacking healthy self tissue. In fact, several groups, including Jim and his colleague Cynthia Chambers, demonstrated the importance of CTLA-4 in restraining the immune system by generating “knockout” mice that completely lack CTLA-4. These knockout animals, which lack CTLA-4 from birth, become very sick at a young age due to hyperactivation of the immune system. However, transient loss of CTLA-4, such as occurs after injection of anti-CTLA-4 antibodies, can lead to a non-fatal and temporary activation of the immune system. The temporary activation of the immune system, though “checkpoint blockade”, provides a window of opportunity during which the immune system is mobilized to attack and eliminate tumors.

THE REST OF THE STORY

Subsequent to his ground-breaking research at UC Berkeley, Jim moved to Memorial Sloan Kettering Cancer Center in New York City to continue his research on CTLA-4 and cancer immunotherapy. He is now at the MD Anderson Cancer Center. Even after publication of the 1996 Science paper, many experts questioned whether anti-CTLA-4 could ever be successfully used in humans. Despite these doubts, and many false starts along the way, the clinical development of anti-CTLA-4 was ultimately taken over by Bristol Myers Squibb, which successfully shepherded anti-CTLA-4 to the clinic under the name Yervoy (Ipilimumab). In 2010, the results of the first phase III clinical trial with Ipilimumab were published in the New England Journal of Medicine. This paper showed that Ipilimumab can significantly prolong the lives of patients with metastatic melanoma that otherwise exhibit a very poor prognosis. Most importantly, perhaps, was the observation that a subset of patients treated with Ipilimumab exhibited long-lasting beneficial effects, in some cases apparently ‘cured’ of their melanoma. Ipilimumab was the first medication to provide durable remissions in a fraction of patients with metastatic melanoma. By no means did everyone benefit from Ipilimumab, but for a field that is hesitant to talk of ‘cures’, these were unexpected and remarkable results. Tumors in patients with metastatic melanoma and many other cancers often acquire resistance to conventional chemotherapy drugs and ultimately re-emerge. In the case of successful immunotherapy, however, it appears that tumors have a much more difficult time acquiring resistance. Another potential advantage of checkpoint therapies is that they may be effective against multiple types of cancer.

It is no exaggeration to claim that the success of Yervoy (Ipilimumab) has now changed the face of cancer therapy. Many drug companies are attempting to develop anti-cancer antibodies with similar modes of action. One particularly promising target for antibodies is a pair of inhibitory molecules, called PD1 and PDL1. Cancer immunotherapy in general has emerged from its exile and is now generating considerable excitement across the pharmaceutical industry.

LESSONS LEARNED

One important lesson to be learned from the story of Ipilimumab is the value of fundamental research. Jim’s original interest in CTLA-4 was driven by his laboratory’s basic curiosity about how T cells are activated and regulated. There was certainly no guarantee at the outset that fundamental research on CTLA-4 would ever lead to a novel therapy for cancer; indeed, it was unlikely that such hope was even at the forefront of the motivation of the researchers that first worked out the role of CTLA-4 in immunity. Instead, these researchers were motivated by a fundamental curiosity about how the immune system works. The winning strategy was to couple curiosity-based research with keeping an eye towards the potential of any findings for the design of new therapies.

In addition, it is critical to understand that Jim’s work was not performed in isolation. Rather it was based on decades of work, performed in hundreds of laboratories, on the basic architecture and function of T cells and the immune system. Without this fundamental research, Jim’s groundbreaking work would never have been possible. Lastly, given recent and widespread skepticism about the value of mouse models of cancer, it is worth remembering that Jim’s fundamental research, as well as the decades of work leading up to it, was based on and would not have been possible without extensive use of these animal models.

In short, it is impossible to predict which fundamental research will lead to novel therapies and which will not, but what is clear is that fundamental research of all kinds is essential to lay the groundwork for the next breakthrough.

Targeting the Heart of the Human Immune System with Listeria-based Vaccines

The Immunotherapeutics and Vaccine Research Initiative is founded on the belief that fundamental research can lead to novel and entirely unexpected approaches to cancer and infectious disease therapies and vaccines. This belief is exemplified by the remarkable story of how research in Molecular and Cell Biology and Public Health 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 and vaccines for infectious diseases.

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 systems are 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 two 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 an immunotherapeutic vaccine for late stage pancreatic cancer. Similarly, Listeria can be engineered to produced antigens associated with viral, bacterial, or parasitic infection, and then used to stimulate an immune response to multiple pathogens including HIV, tularemia, and leishmaniasis.

Further discoveries based on Listeria. 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.

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.

Activation of the Innate Immune System to Overcome Cancers and Infectious Diseases

Basic research at UC Berkeley is leading to exciting new therapeutic approaches for cancer and infectious diseases. An example of this work involves the discovery and advancement of small molecule immunostimulatory agents called cyclic di-nucleotides, or CDNs.

CDNs were originally discovered as small molecules produced by bacteria. For many years, CDNs were thought to function solely in bacteria, and their potential value in cancer immunotherapy was not appreciated. This started to change with a 2007 paper from the Karaolis Group in Maryland, showing that CDNs could activate immune cells. However, it was still unclear how CDNs were working to activate the immune system. In 2009, the laboratory of UC Berkeley Molecular and Cell Biology Professor and Howard Hughes Medical Investigator Russell Vance published a paper in the Journal of Experimental Medicine showing that delivery of CDNs into mouse cells activated the mouse cells to produce a potent immunostimulatory molecule called type I interferon. At the same time, the lab of Professor Dan Portnoy, also at UC Berkeley, was using bacterial genetics and biochemical approaches to identify Listeria monocytogenes mutants that induced diminished or enhanced immune responses. Subsequently, the Portnoy lab showed in a paper published in Science, that the bacterial pathogen Listeria activates the immune system of mice by secretion of CDNs. It remained a mystery how CDNs activated the immune system until 2011, when the Vance and Portnoy Labs collaborated on a paper published in Infection and Immunity to show that the interferon response to CDNs requires a host protein called STING. The Vance Lab then published a paper in Nature that demonstrated CDNs directly bind to a protein called Stimulator of Interferon Genes or STING, providing the key molecular target for these potent immunostimulatory molecules. The Vance Lab went on to show that CDNs bound directly to and activated STING Recently, the Vance Lab, along with UC Berkeley Chemistry Professor Ming Hammond’s Lab, showed in a 2013 paper in Cell Reports that special “non-canonical” CDN variant molecules were particularly potent activators of human STING.

Building off this basic research, researchers at Aduro BioTech, based in Berkeley, were then able to show in preclinical studies in mice, and published in Cell Reports, CDNs injected directly into tumors leads to their elimination. In 2015, Aduro announced a major $750M partnership with Swiss pharmaceutical maker Novartis to develop this class of molecules as a potential immunotherapy for cancer, and Phase I trials will be initiated in 2016. Aduro, in collaboration with UC Berkeley, is also examining the use of CDNs for infectious diseases, particularly as vaccine adjuvants.

The original motivation for the research on CDNs was driven by curiosity about how the immune system is activated in response to bacterial infection. This story provides yet another outstanding example of how basic curiosity-driven research can ultimately lead to promising new approaches for treatments of human cancer and infectious diseases.