CICON18 Day 4 Update: The Role of Bacteria; Technologies to Advance Cancer Immunotherapy

The final day of CICON18—the 2018 International Cancer Immunotherapy Conference (CICON), co-hosted by the Cancer Research Institute (CRI), Association for Cancer Immunotherapy (CIMT), European Academy of Tumor Immunology (EATI), and the American Association for Cancer Research (AACR)—explored two diverse areas that both have the potential to improve immunotherapy’s benefits for patients. 

SESSION 7: Convergence of Technology and Cancer Immunotherapy

The final day of CICON18 began with Darrell Irvine, PhD, of the Massachusetts Institute of Technology, who discussed a universal vaccine strategy to enhance CAR T cell immunotherapy. In addition to equipping T cells with a chimeric antigen receptor (CAR), this strategy involved the delivery of complexes containing the very molecule that the CAR T cells are designed to target. These complexes were packaged in a way that would allow them to be taken up by the lymph nodes, where they would then “decorate” the surfaces of antigen-presenting dendritic cells (DCs), which would subsequently activate CAR T cells that they came into contact with. Using the FITC antigen to test the idea, Irvine first showed that these amphiphilic-FITC complexes were able to promote the expansion of CAR T cells in mice. Additionally, in a preclinical model of glioblastoma that expresses a mutated version of the epidermal growth factor receptor (EGFR), this vaccine was able to boost the killing activity and polyfunctionality of CAR T cells, and ultimately increased survival in tumor-bearing mice. Interestingly, in this setting, improved outcomes were seen when the mice’s natural immune cells were depleted prior to the administration of the CAR T cells. Irvine also demonstrated that this approach could enhance the activity of tandem CAR T cells, which possess CARs against two different targets in a mouse model of melanoma. Lastly, this strategy was also shown to enhance the production of interferon gamma (IFNγ) in tandem CAR T cells made from human immune cells.

Darrell Irvine, PhD, of the Massachusetts Institute of Technology

Next, D. Dan Huh, PhD, of the University of Pennsylvania, highlighted engineering advances that are enabling us to better mimic organs in the lab so their biology can be better understood. Specifically, Huh showcased several varieties of the ‘organ-on-a-chip’ technology, including those involving the pancreas, eyes, and even reproductive organs such as the placenta and the cervix. With these models, scientists may be able to gain insights into how certain diseases develop as well as to test out strategies that might be able to reverse or minimize their impact. With respect to cancer, Huh created a tumor-on-a-chip construct, complete with blood vessels, to characterize how immune cells interact with cancer cells in the tumor microenvironment. Potentially, this could serve also as a drug screening platform to evaluate the effectiveness of various therapies. Lastly, Huh discussed his lung-on-a-chip model, which has enabled analyses of how cigarette smoke impacts inflammatory responses and tissue remodeling in the lungs as well as the biological effects of asthma-related airway constriction. Most interestingly, this lung-on-a-chip model—along with a bone marrow-on-a-chip model—will soon be sent into space as part of a study looking into the mechanisms responsible for the increased infection rates that have been observed in astronauts.

D. Dan Huh, PhD, of the University of Pennsylvania

The following speaker, Martin G. Klatt, MD, of Memorial Sloan Kettering Cancer Center, focused on a new method of detecting the mutated neoepitopes that are bound by HLA molecules and then presented on tumor cells as well as to determine why some of these mutated protein ligands provide better targets for immune responses. This method—which relied in part on HLA-bound neopeitope selection by Gaussian distribution and considered peptides that “scored highly” via mass spectrometry analysis without regard to their predicted HLA-binding—enabled Klatt to identify four times as many HLA-bound neoepitopes when re-analyzing a previously published dataset. Where things got more interesting though, according to Klatt, was when he compared mutated epitopes to non-mutated epitopes. This analysis led to the discovery of a phenomenon he dubbed “central cross-tolerance” in which the ability of neoepitopes to induce immune responses might be compromised if a related but non-mutated epitope—one with a similar sequence in the middle positions of the molecule—is presented in the context of a different HLA complex in the patient. Even Klatt admitted that this finding might shock the immunologists in the room, because it goes against our current beliefs regarding T cell restricted epitopes, but his data nonetheless presented a strong case for at least re-evaluating what we think we know about this paradigm. If correct, this insight could help improve the design of personalized immunotherapies in the clinic, in that doctors would know not to target neoepitopes that wouldn’t stimulate immune responses in a patient because of central cross-tolerance.

Martin G. Klatt, MD, of Memorial Sloan Kettering Cancer Center

Liang Chen, MD, PhD, of Stanford University, also spoke about efforts to classify and characterize the tumor-targeting TCR repertoires in cancer patients. Chen began by acknowledging that while it’s easy to sequence a patient’s T cell receptor (TCR) repertoire, it’s hard to interpret the results and figure out what antigens these T cells are actually targeting because of the immense diversity of possible TCRs. As a proof of principle, Chen first analyzed six patients and characterized all of their TCRs that targeted three common viruses—the flu virus, the Epstein-Barr virus, and cytomegalovirus—in order to “learn the rules of antigen specificity.” Then, using a methodology dubbed GLIPH—which stands for Grouping of Lymphocyte Interactions by Paratope Hotspots—Chen analyzed the TCRs of Mart1-targeting T cells that were isolated from melanoma patients and then expanded, and observed that each patient possesses a relatively unique set of TCRs specific for the Mart1 tumor antigen. The TCR repertoires of individual patients also varied widely in terms of their clonality; some patients had relatively few (~20) Mart1-targeting TCR subpopulations, while others had hundreds. With GLIPH, which allows patients’ T cell responses to be analyzed directly from their genetic sequences without requiring the need for information regarding the target antigen or major histocompatibility (MHC)-binding molecules, Chen was also able to identify dominant TCR motifs shared by different individuals, thus providing potentially important insights into the effectiveness of natural adaptive immune responses against common cancer antigens.

Liang Chen, MD, PhD, of Stanford University

The final speaker of the session was Nir Hacohen, PhD, of Massachusetts General Hospital and the Broad Institute, who revealed his findings regarding “the states of T cells” associated with successful immunotherapy. In addition finding that the tumors of melanoma patients who responded to PD-1 immunotherapy were characterized by fewer monocoytes and macrophages as well as a lower ratio of exhauted T cells to dividng T cells, Hacohen used single-cell analysis to identify two distinct states of “killer” T cells that were associated with responses or the lack thereof. Prior to treatment, the tumors of patients who responded typically had higher levels of T cells with a memory-like signature compared to those with an exhausted-like signature, whereas the tumors of those who didn’t respond were the opposite. Digging deeper, Hacohen showed that patients whose tumors contained higher levels of T cells expressing the transcription factor TCF7 were much more likely to respond to PD-1 immunotherapy. As a result TCF7 was determined to be one of if not the major factors promoting the survival and activity of these memory-like T cell subsets. In contrast, he found that the CD39 surface marker could be used to identify T cells that were terminally exhausted; those that expressed CD39 were not activated, while those that did were strongly activated. Subsequently, he showed that blocking CD39 along with the TIM3 immune checkpoint could better control tumor growth and increase survival in mice with melanoma. With respect to these exhausted T cells, Hacohen concluded that BATF was the primary transcription factor responsible for their behavior. Ultimately, these two subsets differed dramatically in their ability to eliminate tumors—only those lacking both CD39 and TIM3 expression were capable of killing cancer cells in the presence of PD-1 immunotherapy.

Nir Hacohen, PhD, of Massachusetts General Hospital and the Broad Institute

SESSION 8: Microbiome and Metabolism

Hassane M. Zarour, MD, of the University of Pittsburgh School of Medicine, spoke first during the last session of CICON18. He began with a discussion of three studies—conducted in Paris, Chicago, and Houston—that revealed the impact that our gut bacteria, otherwise known as the gut microbiome, can have on patient responses to PD-1 immunotherapy. Each of these studies identified a bacterial species—Akkermansia muciniphila, Bifidobacterium longum, and Faecaliabacterium prausnitzii, respectively—whose presence was associated with immunotherapy-induced benefits. Additionally, Zarour’s own Pittsburgh-based study recently observed immunotherapy benefits in metastatic melanoma patients that were associated with three entirely different bacterial species: Alistipes senegalensis, Bacteroides nordii, and Bacteroides caccae. The source of this variation wasn’t clear, but Zarour suggested that it might be due to geographical diversity, different cancer types, or the sequencing methods used to identify the bacteria. Bolstering that claim, he noted a study from earlier this year that showed that environmental factors had a bigger impact than a person’s genetics when it came to shaping the bacteria in their gut, and demonstrated how mice fed a fiber-rich diet experienced better tumor growth control after treatment with PD-1 immunotherapy. To take full advantage of the microbiome’s potential to improve immunotherapy, Zarour outlined to several challenges that must be overcome. In particular, it will be important to compile a more comprehensive list of the bacteria that can enhance immunotherapy as well as characterize the mechanisms through which they do so. Discovering and validating microbiome-related biomarkers could help doctors predict which patients are likely to respond to immunotherapy. These could also aid in the development and refinement of strategies—such as defined diets, prebiotics, or even fecal microbiota transfer (FMT)—that could be used to actively induce or maintain a beneficial microbiome composition within patients. To this end, Zarour pointed to an ongoing phase II clinical trial in which melanoma patients who were previously resistant to PD-1 immunotherapy are now being re-treated with PD-1 in combination with FMT. The first patient treated in this manner saw their tumor shrink dramatically.

Hassane M. Zarour, MD, of the University of Pittsburgh School of Medicine

Jennifer A. Wargo, MD, of the University of Texas MD Anderson Cancer Center, also spoke about targeting our microbiome to improve the effectiveness and reduce the toxicity of cancer treatments, including immunotherapy. Wargo’s first foray into this area of research involved a bit of serendipity when she unexpectedly discovered that bacteria within tumors were able to protect cancer cells against chemotherapy. Subsequently, in work referenced by Zarour, Wargo identified a gut microbiome signature in metastatic melanoma patients treated with PD-1 immunotherapy that was associated with enhance immune responses within the tumor microenvironment and could also predict who would respond to the treatment. These effects also manifested in mice that received fecal transplants from either responding or non-responding patients. Like Zarour, Wargo also noted the need to standardize different profiling approaches to ensure that any analytically-induced variation was minimized. Most recently, she demonstrated that patients who responded to combined PD-1 and CTLA-4 immunotherapy had more diverse gut microbiomes. Bacteria associated with these responders were also identified, including some that were associated with patients who responded to PD-1 alone. Additionally, certain types of bacteria were found to be associated with toxicity, while others were associated with the absence of toxicity. In one patient who developed colitis after treatment that persisted after steroids, a fecal microbiota transplant (FMT) from a healthy donor was able to completely resolve all of the symptoms. Finally, Wargo highlighted a clinical trial involving metastatic melanoma patients who are being treated with PD-1 immunotherapy in addition to gut microbiome modulation, in the form of either a live bacterial product or a fecal microbiota transplant. This study also examined the impact of several other factors on patients’ microbiome, and found that patients whose diet contained high amounts of fiber had “better,” more diverse gut bacteria, whereas the microbiomes of those who took either probiotics or antibiotics were less diverse. Wargo suggested that, in contrast to popular opinion, probiotics that supply high amounts of a single type of bacteria, even if it’s a “good” species, can be detrimental if it comes at the expense of overall bacterial diversity.

Jennifer A. Wargo, MD, of the University of Texas MD Anderson Cancer Center

The remaining talks of the eighth session switched the focus from the microbiome to metabolism. First, Yasmine Belkaid, PhD, of the National Institute of Allergy and Infectious Diseases, highlighted how our immune system preserves its “memory” during times of nutritional stress. She began by noting that, after infection, our adipose (fat) tissue serves as a “hub” for memory T cells that possess superior proliferation, effector activity, and long-term protection compared to other types of T cells. However, when nutrients become scarce—in this case, when mice in the lab have the calories in their diets reduced by 30%—it causes these memory T cells to accumulate in the bone marrow, while at the same time their levels in the spleen, blood, and lymph nodes drop. Importantly, these effects were reversible once their normal diets were restored. Belkaid then outlined the mechanisms through which these changes occur during calorie restriction, which was found to increase the levels of in glucocorticoids in the blood and stimulate the production of blood and fat cells in the bone marrow. The production of sphingosine-1-phosphate (S1P) by red blood cells also appeared to promote the migration of memory T cells into the bone marrow, as T cells lacking the S1P receptor didn’t accumulate there. The depletion of fat cells—one place where the T cells reside during normal times—also prevented the accumulation of T cells in the bone marrow. Lastly, Belkaid showed that when caloric restriction occurred after the mice had already been exposed to infectious bacteria, their secondary responses against the bacteria were much stronger once their caloric intake returned to normal, thus providing strong evidence that the bone marrow is capable of preserving immunological memory during times when nutrient availability is low.

Yasmine Belkaid, PhD, of the National Institute of Allergy and Infectious Diseases

Next, Mirco Friedrich, an MD, PhD, student in the lab of Michael Platten, MD, at the German Cancer Research Center (DKFZ), spoke about the metabolically induced immunosuppression that can occur in brain cancer—specifically gliomas that have mutated forms of the isocitrate dehydrogenase (IDH) enzyme. When IDH is mutated in these tumor cells, it causes them to produce abnormally high levels of R-2-Hydroxyglutarate (R-2-HG), which is then taken up by T cells, where it can inhibit T cell receptor (TCR) signaling. Friedrich found that other immune cells can also incorporate R-2-HG, and that it induces changes in the gene expression of tumor-associated macrophages and microglial cells, and in general makes these myeloid cells more immunosuppressive. For the last portion of his talk, Friedrich demonstrated that R-2-HG’s effects appear to be mediated through the aryl hydrocarbon receptor (AHR) signaling pathway, and that blocking AHR’s activity improved the effectiveness of PD-L1 immunotherapy, but only against gliomas that possess mutated versions of the IDH enzyme.

Mirco Friedrich, an MD, PhD, student in the lab of Michael Platten, MD, at the German Cancer Research Center (DKFZ)

The final speaker of this year’s conference was Michael G. Constantinides, PhD, of the National Institute of Allergy and Infectious Diseases, who revealed insights into mucosal-associated invariant T (MAIT) cells. While MAIT cells are relatively rare in the body and their potential roles in cancer remain unclear, they are highly enriched in “barrier” tissues such as the skin, lungs, and intestines. Constantinides work specifically focused on the interactions between these MAIT cells and commensal (beneficial) bacteria on our skin. After finding that these MAIT cells were major producers of interleukin 17 (IL-17) in mice, he determined that interleukin-23 (IL-23) is required for their development. Due to bacteria’s ability to stimulate the production of IL-23, he next looked at whether bacteria were also necessary for MAIT cell development and found that in the absence of commensal bacteria, the levels of MAIT cells were significantly lower in all organs. These MAIT cells were most dramatically decreased on the skin, indicating that this population relies especially on bacteria to develop. Constantinides also found that the frequency of MAIT cells in adult mice was influenced by exposure to certain bacteria early in life, whereas the proliferation of MAIT cells on the skin could be promoted by topical application of the commensal Staph epidermidis bacteria. These bacteria-induced MAIT cell responses also required antigen presentation through the MR1 pathway: generation of mice that lacked MR1 exhibited severely diminished MAIT cell activity. Finally, in an acute wound model, he showed that these MAIT cells promoted wound healing.

Michael G. Constantinides, PhD, of the National Institute of Allergy and Infectious Diseases

That’s a wrap for CICON18, but be sure to check back on our blog soon for other updates on advances in the field of cancer immunotherapy. And remember to join us in Paris for CICON19!