CICON18 Day 3 Update: Targeting Tumor Mutations with Vaccine-Based Immunotherapies

The third day of CICON18 explored cutting-edge strategies to identify and target the mutated markers that distinguish cancer cells.

SESSION 5: Novel Vaccine Platforms and Combinations

Filling in for Robert Schreiber, PhD, of Washington University in St. Louis, Matthew Gubin, PhD, gave the first talk on day three of 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).

Matthew Gubin, PhD, of Washington University in St. Louis

Highlighting work in a preclinical sarcoma model with well-defined neoantigens, or antigens that are encoded by tumor-specific gene mutations, that responds to several types of immunotherapy, including vaccination, Gubin provided “a glimpse into the complexity” of tumor-infiltrating macrophages. Five different types were identified using high-dimensional analysis, and successful checkpoint immunotherapy significantly impacted two of them, in opposite ways. One population—distinguished by CX3CR1 and CD206—were a large (~40%) fraction of the entire macrophage pool in untreated tumors, but never reached more than 5% frequency in mice treated with the combination of CTLA-4 and PD-1 immunotherapies. Conversely, this approach enabled the expansion of the other cluster, which was defined by iNOS and high levels of CD38 and remained relatively rare in untreated tumors.

Next, Ugur Sahin, MD, of BioNTech and TRON Germany, showcased advances in personalized, RNA-based vaccines—in particular, the IVAC mutanome approach. In the first-in-human trial involving thirteen patients with stage 3/4 melanoma, every single person generated immune responses against three or more of the neoepitopes included in their personalized vaccines. One patient even developed T cells against all ten targets, and overall more than 60% of those chosen proved immunogenic. All nine patients whose tumors were resected, including some that were metastatic, remain in remission at least a year and a half after treatment, with the longest response ongoing at 33 months. Notably, the manufacturing for this RNA vaccine has improved since this trial began—the time required from sample-to-shipment has been halved since 2015 and is now down to under six weeks.

Ugur Sahin, MD, of BioNTech and TRON Germany

Sahin also discussed a more recent approach that involved incorporating RNA-based vaccines into liposomes. In the Lipo-MERIT trial, this was used to target four non-mutated tumor antigens and enabled systemic delivery of the vaccine products, which elicited immune responses against at least one of the antigens in 81% of the 41 patients treated so far. Of the 32 patients for whom prior PD-1 immunotherapy had been ineffective, 20% responded and another 30% saw their disease stabilize, and long-term persistence of vaccine-induced T cells has been observed. Perhaps unsurprisingly, the more of the targets a patient’s tumor expressed, the more likely they were to respond: of the four patients whose tumors expressed all four, two responded and two stabilized. Lastly, Sahin spoke about recent preclinical breakthroughs in which these liposome delivery vehicles were used to directly induce the formation of CLDN6-targeting CAR T cells within mice.

Catherine J. Wu, MD, Dana-Farber Cancer Institute, spoke next about her work with NeoVax, a synthetic long peptide (SLP)-based vaccine approach, which has also been effective in melanoma, including complete responses in two patients in combination with PD-1 immunotherapy. In addition to melanoma, NeoVax has also been used to treat patients with glioblastoma, which doesn’t have many mutations and doesn’t normally respond to immunotherapy. Here, vaccination was able to “warm” these “cold” tumors and induce infiltration of neoantigen-specific T cells in multiple patients. Wu then discussed “building a new predictor” to help improve the ability to identify optimal vaccine targets.

Catherine J. Wu, MD, Dana-Farber Cancer Institute

Another SLP-based vaccine approach, ISA-101, was discussed by Cornelis J.M. Melief, MD, PhD, of the Leiden University Medical Center (The Netherlands) and ISA Pharmaceuticals. This vaccine was designed to target HPV-derived proteins expressed by cervical cancer cells. While vaccine-induced clinical responses were seen in half of the patients with early-stage disease, combinations were necessary against more advanced disease. Thus far, the generation of HPV-specific T cells was associated with improved survival in 18 patients with late-stage cervical cancer who were treated with both vaccination and chemotherapy. This vaccine is also being combined with PD-1 immunotherapy in a phase II trial, where it was associated with a clinical response in one of every three patients, more than double the reported rate of PD-1 immunotherapy alone. The median overall survival associated with the combination was also twice as high in the 24 patients treated.

Cornelis J.M. Melief, MD, PhD, of the Leiden University Medical Center (The Netherlands) and ISA Pharmaceuticals

The next speaker was Alan Melcher, PhD, of the Institute of Cancer Research in London, who focused on the use of oncolytic viruses to stimulate vaccine-like immune responses against tumors. Specifically, Melcher’s work dealt with reoviruses, which most of us are exposed to as children and already possess antibodies against it. While this could potentially lead to complications after systemic infusion of an oncolytic reovirus, Melcher found that in mice, paradoxically, those that had prior immunity actually had higher survival. In a clinical trial involving patients with either primary brain cancer or metastatic brain lesions that were resectable, an injection of reovirus directly into the tumors was able to turn “cold” tumors “hot” (making them more visible to the immune system) by altering the expression of 32 interferon gamma (IFNγ)–responsive genes. Tumors treated with the reovirus had twice the PD-L1 expression of untreated tumors, indicating they were potentially primed for subsequent PD-1 immunotherapy. Indeed, in preclinical work involving mice with glioma, combining PD-1 immunotherapy with intravenously administered reovirus was able to drastically improve survival. Elsewhere, it was demonstrated that exposing immune cells—either monocytes and dendritic cells—to reovirus-infected cancer cells was effective at priming “killer” T cells against a range of tumor antigens.

Alan Melcher, PhD, of the Institute of Cancer Research in London

Following Melcher, James J. Moon, PhD, of University of Michigan, highlighted a “nanodisc” platform for use in cancer vaccines. These nanodiscs, made out of synthetic high-density lipoproteins (sHDL) can have both tumor antigens and immune-stimulating adjuvants incorporated into them for co-delivery, and were found to enhance and prolong the presentation of these antigens on dendritic cells compared to when the neoantigen peptides and adjuvants were delivered in “free” form. When used to treat tumor-bearing mice, these vaccines also induced far greater levels of neoantigen-targeting T cells (> 31-fold) and were associated with decreased tumor growth and increased survival. In combination with PD-1 immunotherapy, nanodisc vaccination provided even greater benefits in mouse models of colorectal cancer and glioblastoma, where 100% were still alive at 60 days after the tumors implantation.

James J. Moon, PhD, of University of Michigan

Moon also explored loading these nanodiscs with chemotherapy drugs to induce immunogenic cell death, and showed that these chemotherapy-loaded nanodiscs accumulated efficiently, and increased the drug concentration (27-fold), within the colorectal tumors of mice; it also led to decreased tumor growth and increased survival without any apparent cardiotoxicity. Lastly, the combination of chemotherapy-loaded nanodiscs and PD-1 immunotherapy was able to eliminate established tumors in mice and conferred to them long-term protection against tumor re-challenge.

Next, Robert Seder, MD, of the National Institutes of Health’s Vaccine Research Center, discussed the development of a self-assembling, nanoparticle-based vaccine incorporating peptide antigens and adjuvants. Using this method, he demonstrated that the solubility of the peptides influences the extent of subsequent T cell stimulation, with particulate forms providing enhanced lymph node persistence, uptake by important antigen-presenting cells, and greater T cell priming. With respect to adjuvants—in this case a Toll-like receptor (TLR)-7/8 stimulator—their inclusion into polymers within the nanoparticles also improved their activity and limited systemic distribution to potentially reduce toxicity. Ultimately, this approach was able to improve the breadth, magnitude, and quality of “killer” T cell responses against tumor neoantigens, in particular in mouse models of melanoma, colorectal cancer, and human papilloma virus (HPV)-associated cancer.

Robert Seder, MD, of the National Institutes of Health’s Vaccine Research Center

The method discussed by Liang Deng, MD, PhD, of Memorial Sloan Kettering Cancer Center—known as in situ, or on site—vaccination doesn’t explicitly tell the immune system which tumor neoantigens to target. Instead, it involves injecting a modified vaccinia virus Ankara (MVA) into tumors to stimulate the immune system’s natural adaptive abilities by inducing the expression of interferon (IFN) and promoting maturation in dendritic cells (DCs). Through heat-inactivation, she showed, it was possible to hamper the virus’ “infectivity” and even enhance its anti-tumor effects, which required “killer” T cells and the STING pathway in addition to DCs. This heat-inactivated MVA also synergized with checkpoint immunotherapy in a mouse mode of large, established melanoma, with PD-L1-targeting therapy providing the greatest survival benefit. Finally, Deng discussed one version of MVA that was engineered to target the FLT3 and OX40 pathways, which promote the activity of DCs and T cells, respectively, and proved effective at inducing immune responses against distant, non-injected tumors. This version too was more effective in combination with checkpoint immunotherapy in mouse models of large and established melanoma and colorectal cancer.

Liang Deng, MD, PhD, of Memorial Sloan Kettering Cancer Center

Elizabeth E. Evans, PhD, Vaccinex, concluded the fifth session with a discussion of a first-in-class antibody designed to reprogram the myeloid cells in the tumor microenvironment by targeting (and blocking) the activity of semaphorin 4D (SEMA4D), a receptor whose activity has been shown to stabilize regulatory T cells and is associated with poor prognosis. In short, this anti-SEMA4D therapy promoted the migration and differentiation of pro-inflammatory macrophages as well as the recruitment of cross-presenting dendritic cells. It also increased the ratio of “good,” M1 macrophages to “bad,” M2 macrophages, blocked the recruitment of myeloid-derived suppressor cells, and enhanced T cell activity and proliferation. Overall, blocking SEMA4D’s activity shifted the balance of the myeloid cells within the tumor to a less immunosuppressive one more supportive of “killer” T cell responses against tumors. Based on preclinical results that revealed synergy between anti-SEMA4D therapy and both PD-1 and CTLA-4 checkpoint immunotherapies, several clinical trials have now been launched to test these combination approaches in the clinic for patients with lung cancer, melanoma, colorectal cancer, and pancreatic cancer.

Elizabeth E. Evans, PhD, of Vaccinex

SESSION 6: Mutational Analysis and Predicting Response to Immunotherapy

The sixth session began with Ton N. Schumacher, PhD, of the Netherlands Cancer Institute, who focused on his efforts to better understand what patients’ T cells are targeting by characterizing their T cell receptor (TCR) repertoires. In an initial study of four patients, Schumacher found that the majority of the T cells within patients’ tumors didn’t even recognize the tumor cells, and he noted that this may limit the value of checkpoint immunotherapies that target these T cells. He also observed that the “dysfunctional” T cells that expressed the highest levels of PD-1 within tumors had distinct gene expression signatures from other T cells in the tumor that expressed lower levels of PD-1. These high PD-1-expressing T cells were found predominantly within tertiary lymphoid structures (TLS), produced significantly higher levels of the B-cell attracting CXCL13, and had an increased capacity to recognize tumors compared to other T cells. Unsurprisingly, the presence of these cells correlated with higher response rates and increased survival in patients treated with PD-1 immunotherapy.

Ton N. Schumacher, PhD, of the Netherlands Cancer Institute

Next, Benjamin D. Greenbaum, PhD, of Icahn School of Medicine at Mount Sinai, took a look at how immune responses can drive the evolution of tumors, similar to how viruses evolve as they’re recognized and rooted out within the body. Whereas tumors that are more heavily-mutated are more likely to respond to immunotherapy, he also noted that tumor heterogeneity can negatively impact treatment success. In these tumors, some tumor cell clones may be eliminated, but others that are able to resist treatment may then expand and become the dominant population. To determine the “fitness” of different tumor cell clones—and neoantigens—Greenbaum used a model that quantifies both the frequency and immunogenic potential of neoantigens based on their ability to bind major histocompatibility complex (MHC) molecules as well as T cell receptors (TCRs). This method was validated in CTLA-4-treated melanoma and PD-1-treated lung cancer patients, with those whose tumors were characterized by “low fitness” surviving significantly longer than those with “high fitness” tumors.

Benjamin D. Greenbaum, PhD, of Icahn School of Medicine at Mount Sinai

Moving on, Greenbaum turned to the repetitive elements in our genome that can become de-repressed and re-activated within tumor cells, and potentially attract the attention of the innate immune system. In particular, he found that increased expression of one such repeat—HERVK—was associated improved responsiveness to PD-L1 immunotherapy. Expression of another, known as HSATII, correlated inversely with the infiltration of “killer” T cells, with HSATII-low tumors characterized by infiltration of significantly more T cells.

Following Greenbaum, Naiyer Rizvi, MD, of Columbia University Medical Center, highlighted the value of the tumor mutational burden (TMB) biomarker. In general, tumors with higher TMB respond better to PD-1/PD-L1 immunotherapy, although there’s still no agreement on what threshold constitutes a “high” TMB, with several different cutoffs being employed in various trials. Even so, TMB’s clinical value has been observed in several tumor types, including lung and bladder cancer, where patients with high TMB tumors have been shown to benefit more from PD-1/PD-L1 immunotherapy, both alone and in combination with CTLA-4, than from chemotherapy. The TMB biomarker has shown even more predictive power when used in combination with the PD-L1 expression biomarker. In a trial with non-small cell lung cancer (NSCLC) patients, for instance, those with tumors characterized by high TMB and high PD-L1 expression experienced drastically higher rates of progression-free survival after PD-1 immunotherapy compared to those with either high TMB and low PD-L1 or low TMB and high PD-L1. To end his talk, Rizvi explored other strategies that could help expand the clinical applications and benefits of TMB, including the ability to measure TMB from blood samples (as opposed to having to acquire and analyze a tumor sample) as well as harmonizing the different TMB-measuring assays to ensure consistency between different samples and trials.

Naiyer Rizvi, MD, of Columbia University Medical Center

Johns Hopkins University School of Medicine’s Drew M. Pardoll, MD, PhD, spoke next and focused on how tumor mutations influence immunotherapy responses. Like Schumacher, Pardoll also sought to characterize the T cell receptor (TCR) repertoires of the T cells within patients’ tumors, and to see what mutated tumor neoantigens these T cells were targeting. In one patient whose heavily-mutated tumor was characterized by high microsatellite instability (MSI-hi), Pardoll found neoantigen-targeting T cells within the tumor and noticed that their levels in the blood expanded greatly after PD-1 immunotherapy. He also revealed that the T cell responses against these neoantigens can change over time in response to treatment, before highlighting the benefits of PD-1 immunotherapy prior to surgery. In both lung cancer and Merkel cell carcinoma (MCC), this neoadjuvant immunotherapy was associated with encouraging rates of pathological complete response prior to resection. In this setting too, T cells targeting dominant tumor neoantigens expanded greatly in the blood after treatment. Furthermore, there was an association between the diversity of the intratumoral TCR repertoire and tumor mutational burden (TMB) as well as the degree of response. On average, tumors with more mutations had more diverse TCR repertoires after neoadjuvant immunotherapy, whereas patients with less diverse TCR repertoires were associated with more extensive responses prior to surgery.

Drew M. Pardoll, MD, PhD, of Johns Hopkins University School of Medicine

Lastly, Pardoll revealed how immunotherapy can also help patients with low TMB tumors that don’t typically respond to immunotherapy. One patient with microsatellite stable (MSS) colorectal cancer had their disease stabilize—for four and a half years—in concert with the expansion of a population of T cell clones targeting the AKT1 driver mutation stabilized after treatment. Another patient, whose lung tumor had relatively few (30) mutations, had a complete response associated with T cells targeting a BRAF driver mutation.

The next speaker was Vinod P. Balachandran, MD, of Memorial Sloan Kettering Cancer Center, who highlighted advances in our understanding of the factors associated with long-term survival in patients with pancreatic cancer, which rarely responds to current approaches. These long-term survivors, Balachandran noted, were characterized by immunogenic tumor microenvironments, enhanced T cell activity, and more polyclonal (more diverse) T cell receptor (TCR) repertoires. While the quantity of tumor neoantigens couldn’t be used to distinguish long-term responders, neoantigen quality could. By quantifying a tumor’s heterogeneity and the immunogenicity of its individual neoantigens, Balachandran was able to assess the effective immunogenicity of tumors, which was able to stratify long-term survivors. Notably, T cells targeting these high quality neoantigens were found to persist up to 12 years in the blood of these exceptional responders. The presence of high quality neoantigens was also able to predict survival in melanoma and lung cancer patients treated with CTLA-4 and PD-1 immunotherapy, respectively.

Vinod P. Balachandran, MD, of Memorial Sloan Kettering Cancer Center

Next, Yiyi Yan, MD, PhD, of the Mayo Clinic College of Medicine, showcased recent breakthroughs in overcoming PD-1 immunotherapy resistance. Most notably, he revealed the importance of CX3CR1-expressing “killer” T cells, which were found to increase in number in the blood of metastatic melanoma patients who responded to PD-1 immunotherapy by itself. Additionally, in PD-1-resistant patients treated with the combination of chemotherapy and immunotherapy, those who responded had higher levels of these T cells compared to non-responders prior to treatment. After successful treatment, the numbers of these CX3CR1-expressing “killer” T cells also increased in responsive patients, and subsequent experiments revealed that they were required for the beneficial effects of combination chemo-immunotherapy.

Yiyi Yan, MD, PhD, of the Mayo Clinic College of Medicine

The last speaker of day three at CICON18 was Stephen P. Schoenberger, PhD, of the La Jolla Institute for Allergy and Immunology, who discussed a strategy to identify, verify, and then target tumor neoantigens. One of the unique features of Schoenberger’s method—with which roughly 35% of mutations were verified as neoantigens—is that it doesn’t take HLA-binding into account, which enabled them to identify HLA class I- and class II-associated epitopes that otherwise would have been missed. Recently, they’ve taken this approach into the clinic via a phase I trial in which patients with advanced cancers are being treated with personalized neoantigen vaccines in combination with PD-1 immunotherapy. This strategy has already shown benefits in preclinical studies, where mice with squamous cell carcinoma (SCC) were first vaccinated against actionable neoantigens, which endowed them with pre-immunity that was then “unleashed” by subsequent PD-1 or CTLA-4 immunotherapy.

Stephen P. Schoenberger, PhD, of the La Jolla Institute for Allergy and Immunology

That’s all for the third day of the conference. Check back soon for our recap of the final day at CICON18!