Cell Checkpoint Blockade Research Articles

Abstract C103: Targeting IL-1β pathway for cancer immunotherapy

Abstract Introduction: Inflammation is a hallmark of cancer wherein diverse immune cells exert either pro- or antitumor properties and affect therapeutic resistance (Ritter B et al, JEM 2019). High concentrations of tumor-promoting inflammatory cytokines such as IL-1β (Litmanovich A et al, Oncol Ther 2018) and IL-6 (Altundag O et al, J Clin Oncol 2005) correlate with advanced malignancies and are associated with reduced survival. A recent phase III clinical trial (CANTOS) demonstrated that canakinumab, a selective IL-1β inhibitor, could significantly reduce lung cancer incidences and lung cancer related mortalities as a secondary endpoint in atherosclerosis patients, providing the first clinical evidence for the role of IL-1β inhibition in lung cancer. Canakinumab and other selective IL-1β inhibitors such as gevokizumab aim to target tumor promoting inflammation (TPI) within the tumor microenvironment (TME) by reducing tumor-associated immune suppression. In an effort to gain further insights into the clinical mechanistic role of canakinumab and gevokizumab in cancer development and progression, we have utilized preclinical models to understand the immunological events following IL-1β blockade. Methods: Preclinical efficacy of anti-human IL-1β antibodies, canakinumab and gevokizumab, was assessed in humanized mouse models of NSCLC (H358) and CRC (SW480) respectively. Anti-mouse IL-1β blocking antibody was used to evaluate efficacy in murine syngeneic models of breast (4T1) and lung (LL-2) cancer either as a single agent or in combination with PD-1 inhibitor and docetaxel. Tumor infiltrating lymphocytes (TIL) characterization was carried out either by flow cytometry or by IHC in syngeneic and humanized models. Results: Using both murine syngeneic and humanized mouse models, we demonstrate that IL-1β inhibition remodels the TME and slows tumor growth either alone or in combination with other agents. In the humanized H358 NSCLC mouse model, the combination of canakinumab and the anti-PD-1 monoclonal antibody, pembrolizumab, significantly slowed tumor growth by more than 50% vs control antibodies. Furthermore, canakinumab alone and in combination resulted in increased CD8 and CD3 positive TIL infiltration within the tumor. Decreased tumor growth was also observed with another IL-1β inhibitor, gevokizumab, in a humanized SW480 CRC mouse model, both alone and in combination with anti-VEGF therapy. In syngeneic 4T1 and LL-2 models, anti-IL-1β alone and in combination with anti-PD-1 resulted in increased infiltration of CD8+ cells along with the reduction of immunosuppressive myeloid derived suppressor cells (MDSCs), neutrophils and regulatory T cells (Tregs) into the tumor, suggesting that the TME may be shifting to a less suppressive phenotype after IL-1β blockade. Similar changes to the tumor microenvironment were observed with the combination of anti-IL-1β with docetaxel. Additional combinations are being explored in other tumor models reflecting different indications. Conclusions: Our results highlight the role for IL-1β in tumor immunomodulation and that the pathophysiological role of the IL-1β pathway in innate immunity might have important consequences on T cell function and checkpoint blockade in cancer. These results support the ongoing clinical evaluation of IL-1β inhibitors such as canakinumab and gevokizumab and in combination with other therapeutic agents across several cancers. Citation Format: Pushpa Jayaraman, John Millholland, Neil O’Brien, Connie Wong, Rohan Diwanji, Muchun Wang, Elizabeth Choi, Ronald Linnartz, Kristine Rose, Vanessa Rodrik-Outmezguine, Marc Pelletier, Alexander Savchenko, Tong Luo, Valerie Rezek, Scott Kitchen, Jason Baum, Catherine Sabatos-Peyton, Anne-Marie Martin, Glenn Dranoff . Targeting IL-1β pathway for cancer immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C103. doi:10.1158/1535-7163.TARG-19-C103

TLR9 activation cooperates with T cell checkpoint blockade to regress poorly immunogenic melanoma

Tumors that lack pre-existing immune infiltration respond poorly to T cell checkpoint blockade immunotherapy. These cancers often surround themselves with high densities of suppressive myeloid stroma while excluding immunostimulatory dendritic cells. Tumor-resident myeloid cells and selected lymphocyte populations retain expression of Toll-like Receptors (TLR) that sense common features of pathogens and activate innate immunity in response. We explored whether agonists of TLR9 could augment innate immunity to promote tumor regression alone or in combination with T cell checkpoint blockade. In the setting of the immunogenic B16-Ova (Ovalbumin) expressing melanoma model, local injection of the CpG oligonucleotide TLR9 agonist ODN1826 combined with systemic CTLA-4 blockade cured 45% of mice of both their treated and an untreated tumor on the opposite flank demonstrating the synergistic potential of this combination. Next, in the non-immunogenic B16-F10 melanoma model, we showed that only intra-tumoral, but not systemic TLR9 activation augments the therapeutic potential of checkpoint blockade. In this setting, intra-tumoral TLR9 activation cooperated equally with either CTLA-4 or PD-1 blockade co-administered locally or given systemically; however, the uninjected tumor rarely regressed. Anti-CTLA-4 combinations were associated with improved intra-tumoral CD8 to regulatory T cell ratios, while anti-PD-1 combinations elicited improved ratios of CD8 T cells relative to suppressive myeloid stroma. Using both a TLR9 agonist (MGN1703) and a CTLA-4 antibody (9D9-IgG2a) of increased potency cured 50% of bi-lateral B16-F10 melanoma. These findings suggest that intra-tumoral TLR9 agonists can improve sensitivity of poorly immunogenic tumors to T cell checkpoint blockade, and that newer, higher potency TLR agonists and checkpoint antibodies can raise the therapeutic ceiling for this combination therapy.

Killers 2.0: NK cell therapies at the forefront of cancer control.

Natural killer (NK) cells are innate cytotoxic lymphocytes involved in the surveillance and elimination of cancer. As we have learned more and more about the mechanisms NK cells employ to recognize and eliminate tumor cells, and how, in turn, cancer evades NK cell responses, we have gained a clear appreciation that NK cells can be harnessed in cancer immunotherapy. Here, we review the evidence for NK cells' critical role in combating transformed and malignant cells, and how cancer immunotherapies potentiate NK cell responses for therapeutic purposes. We highlight cutting-edge immunotherapeutic strategies in preclinical and clinical development such as adoptive NK cell transfer, chimeric antigen receptor-expressing NK cells (CAR-NKs), bispecific and trispecific killer cell engagers (BiKEs and TriKEs), checkpoint blockade, and oncolytic virotherapy. Further, we describe the challenges that NK cells face (e.g., postsurgical dysfunction) that must be overcome by these therapeutic modalities to achieve cancer clearance.

Cancer immunotherapy and flow cytometry in immunotherapy monitoring

Immunotherapy for cancer treatment continues to be developed and various strategies have been carried out including bioengineering. This endeavour requires development of technology, and efforts to find specific and sensitive tools to monitor immune responses during and after therapy. The purpose of this mini-review was to discuss cancer immunotherapy using T cell and immune checkpoint blockade therapy, as well as immunotherapy monitoring methods using flow cytometry (FCM). Bioengineering of T lymphocytes for immunotherapy and immune checkpoint blockades can be combined with nanoparticles as drug delivery carrier against cancer to increase drug distribution to tumor cells, as well as T cell stimulation regulation to reduce autoimmune effects. In addition, T cell engineering can also prevent Host versus Graft alloreactivity in chimeric antigen receptor (CAR) T cell administration. FCM is a monitoring method that is widely used in pre-clinical and clinical cancer immunotherapy studies.

Cancer Neoantigens.

Malignant transformation of cells depends on accumulation of DNA damage. Over the past years we have learned that the T cell-based immune system frequently responds to the neoantigens that arise as a consequence of this DNA damage. Furthermore, recognition of neoantigens appears an important driver of the clinical activity of both T cell checkpoint blockade and adoptive T cell therapy as cancer immunotherapies. Here we review the evidence for the relevance of cancer neoantigens in tumor control and the biological properties of these antigens. We discuss recent technological advances utilized to identify neoantigens, and the T cells that recognize them, in individual patients. Finally, we discuss strategies that can be employed to exploit cancer neoantigens in clinical interventions.

Abstract PD5-03: Characterization of high TIL breast cancers reveals a prognostic and functionally distinct tissue-resident memory subpopulation

Abstract Background: Tumor infiltrating lymphocytes (TILs) assessed via light microscopy are prognostic and predictive in the early stage and advanced triple negative and HER2-amplified breast cancer (BC). Higher TILs can also identify patients more likely to benefit from anti-PD-1 therapy. In this study we interrogated T cell subsets that comprise high TILs to determine if distinct subpopulations are key mediators of anti-tumor immunity. Methods: We characterised TILs with a focus on CD3+ T cells in 129 primary and metastatic BC samples using flow cytometry, bulk RNASeq on flow sorted T cell populations, multiplex immunohistochemistry and microdroplet based single cell 3' mRNA sequencing on the 10X Genomics Chromium platform. Cell type specific gene expression signatures were determined from differential expression between putative T cell subpopulations. These signatures were investigated in clinical cohorts, including trial cohorts treated with pembrolizumab. Results: High TIL Infiltrates consisted primarily of CD3+ T cells, with both CD8 and CD4 populations. Unsupervised clustering of single cell sequencing identified 9 CD8 and CD4 subpopulations with distinct gene expression profiles. In addition to Tregs and CD8 effector memory (TEM) T cells, we found a CD8+ tissue resident memory (TRM) population expressing greater levels of T-cell checkpoints and cytotoxic markers compared to effector memory cells. In 2 primary tumours and 1 liver metastasis, bulk RNASeq of flow sorted TEM and TRM corroborated the single cell mRNASeq results. T cell receptor profiling (TCR) in the 3 samples found non-overlapping repertoires in the 2 primary tumours, but overlap in one metastatic lesion, suggesting divergent developmental origins in the breast, but the potential for nascent TRM differentiation in a metastatic niche. Clustering of these TCRs suggested differing antigen specificities between TRM and non-TRM CD8 T cells. Using Metabric data, the CD8 TRM gene expression signature was prognostic for disease free survival (DFS) in primary TNBCs (n=329, log-rank p=0.003), and was able to further stratify cases with high and low CD8A expression for DFS (log-rank p = 0.03). The CD8 TRM signature was enriched in baseline tumour samples of responders (n = 9) compared with non-responders (n=36) in 45 patients with metastatic melanoma treated with T cell checkpoint blockade (p < 0.0001). Additional single cell sequencing data with TCR sequencing will be combined with these initial results, and an independent data set of single cell mRNASeq and TCR Seq on CD3+ BC TILs will be used to confirm our findings. Cell type specific signatures will be explored in additional clinical cohorts including KEYNOTE-086, and presented at the meeting. Conclusion: Using single cell profiling of the immune microenvironment in BC we demonstrate that high TIL BCs contain multiple T cell subpopulations with different functional and prognostic significance. Our approach identified a CD8 TRM population with a distinct gene expression profile and strong expression of key immune checkpoints likely representing the presence of true tumor specific immunity. This population may be a key target of immune checkpoint blockade. Citation Format: Savas P, Virassamy B, Ye C, Salim A, Mintoff CP, Caramia F, Salgado R, Teo ZL, Dushyanthen S, Byrne A, Luen SJ, Fox SB, Speed TP, Mackay LK, Neeson PJ, Loi S. Characterization of high TIL breast cancers reveals a prognostic and functionally distinct tissue-resident memory subpopulation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-03.

Targeted hypoxia reduction restores T cell infiltration and sensitizes prostate cancer to immunotherapy.

Despite the success of immune checkpoint blockade against melanoma, many "cold" tumors like prostate cancer remain unresponsive. We found that hypoxic zones were prevalent across preclinical prostate cancer and resisted T cell infiltration even in the context of CTLA-4 and PD-1 blockade. We demonstrated that the hypoxia-activated prodrug TH-302 reduces or eliminates hypoxia in these tumors. Combination therapy with this hypoxia-prodrug and checkpoint blockade cooperated to cure more than 80% of tumors in the transgenic adenocarcinoma of the mouse prostate-derived (TRAMP-derived) TRAMP-C2 model. Immunofluorescence imaging showed that TH-302 drives an influx of T cells into hypoxic zones, which were expanded by checkpoint blockade. Further, combination therapy reduced myeloid-derived suppressor cell density by more than 50%, and durably reduced the capacity of the tumor to replenish the granulocytic subset. Spontaneous prostate tumors in TRAMP transgenic mice, which completely resist checkpoint blockade, showed minimal adenocarcinoma tumor burden at 36 weeks of age and no evidence of neuroendocrine tumors with combination therapy. Survival of Pb-Cre4, Ptenpc-/-Smad4pc-/- mice with aggressive prostate adenocarcinoma was also significantly extended by this combination of hypoxia-prodrug and checkpoint blockade. Hypoxia disruption and T cell checkpoint blockade may sensitize some of the most therapeutically resistant cancers to immunotherapy.

Fatal graft-versus-host disease after allogeneic stem cell transplantation in a patient recently exposed to nivolumab.

Allogeneic hematopoietic stem cell transplantation and checkpoint blockade therapy are immune-based salvage therapies for Hodgkin's lymphoma; however, the use of programmed death 1 blocking agents in the allogeneic stem cell transplantation setting could augment the incidence of steroid refractory graft-versus-host disease. Few studies suggest that that nivolumab is safe in patients previously treated with an allogeneic stem cell transplantation. Likewise, there are very limited data on the use of nivolumab before allogeneic stem cell transplantation. Here, we report a case of fatal graft-versus-host disease in a patient who underwent allogeneic stem cell transplantation 26 days after the last administration of nivolumab. Careful monitoring and close clinical assessment of atypical presentation for graft-versus-host disease in these patients, interval of time from nivolumab administration to allogeneic stem cell transplantation, drug dosage adjustments or more effective allo prophilaxys should been evaluated in prospective clinical trial.

Prior irradiation results in elevated programmed cell death protein 1 (PD-1) in T cells

Purpose: In this study we addressed the question whether radiation-induced adverse effects on T cell activation are associated with alterations of T cell checkpoint receptors.Materials and methods: Expression levels of checkpoint receptors on T cell subpopulations were analyzed at multiple post-radiation time points ranging from one to four weeks in mice receiving a single fraction of 1 or 4 Gy of γ-ray. T cell activation associated metabolic changes were assessed.Results: Our results showed that prior irradiation resulted in significant elevated expression of programmed cell death protein 1 (PD-1) in both CD4+ and CD8+ populations, at all three post-radiation time points. T cells with elevated PD-1 mostly were either central memory or naïve cells. In addition, the feedback induction of PD-1 expression in activated T cells declined after radiation.Conclusion: Taken together, the elevated PD-1 level observed at weeks after radiation exposure is connected to T cell dysfunction. Recent preclinical and clinical studies have showed that a combination of radiotherapy and T cell checkpoint blockade immunotherapy including targeting the programmed death-ligand 1 (PD-L1)/PD-1 axis may potentiate the antitumor response. Understanding the dynamic changes in PD-1 levels in T cells after radiation should help in the development of a more effective therapeutic strategy.

Immunotherapy in Colorectal Cancer: Where Are We Now?

This review examines the current state of colorectal cancer (CRC) immunotherapy across multiple treatment modalities and discusses some of the most promising approaches. CRC immunotherapy involving viral vector and dendritic cell vaccines, checkpoint blockade, and adoptive cell therapy has been explored from preclinical to clinical studies. Despite successes in other malignancies, including melanoma, leukemia, lung, and renal cancers, immunotherapies have been FDA approved for only a small subset of CRCs. Recent studies leveraging greater understanding of cellular and molecular mechanisms underlying colorectal tumorigenesis and immunotherapeutic mechanism of action may be exploited in upcoming trials. While immune infiltration of CRC has been an established indicator of patient outcomes, immunotherapeutic strategies to date have not exploited its potential immunogenicity to benefit patients. New vaccine, checkpoint inhibitor, and CAR-T cell therapy paradigms promise to change that. With continued research, we could see a rapid increase in the number of FDA-approved immunotherapies for CRC in the coming years.

Identification of essential genes for cancer immunotherapy.

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.

Abstract 3083: Prior irradiation results in elevated programmed death 1 (PD-1) in T cells

Abstract It is well known that radiation has long-lasting effects on the immune system. In this study we address the question whether these radiation-induced adverse effects on T cell activation are associated with alterations of T cell inhibitory receptors. Expression levels of inhibitory receptors on T cells were analyzed at multiple post-radiation time points ranging from one to four weeks in mice receiving whole body irradiation. The level of the checkpoint receptor, PD-1, on different T cell subpopulations was determined. Our results showed that prior irradiation resulted in significant elevated expression of PD-1 in both CD4+ and CD8+ populations. T cells with elevated PD-1 mostly were either central memory or naïve cells. In addition, the feedback induction of PD-1 expression in activated T cells declined with time after radiation. Taken together, the elevated PD-1 level observed at weeks after radiation exposure is connected to T cell dysfunction. Similar changes were seen in samples from tumor patients receiving radiotherapy. Recent preclinical and clinical studies have shown that radiation alters the immune-modulatory microenvironment in tumor tissue, and implies that a combination of radiotherapy and T cell checkpoint blockade immunotherapy including targeting the PD-L1/PD-1 axis may potentiate the antitumor response. Therefore, understanding the dynamic changes in PD-1 levels in T cells after radiation should help in the development of a more effective therapeutic strategy. Citation Format: Renxiang Chen, Deguan Li, Albert J. Fornace, Henghong Li. Prior irradiation results in elevated programmed death 1 (PD-1) in T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3083. doi:10.1158/1538-7445.AM2017-3083

Improving immunotherapeutic efficacy against metastatic tumors in a pre-clinical model of murine renal cell carcinoma.

Abstract Renal and pelvic cancers are the ninth most common cancer in the U.S., and although five-year survival rates for localized renal tumors are >90%, those for metastasized tumors are <12%. Metastatic renal cell carcinoma (RCC), the most common type of renal neoplasm, is resistant to radio- and chemotherapies. Immune-based therapies, including checkpoint blockade, are being pursued in an attempt to amplify protective adaptive immunity but overall response rates remain at 20–30%. We hypothesized that a therapeutic strategy employing T cell priming plus checkpoint blockade would result in improved clearance of renal tumors verses checkpoint alone. Utilizing our previously described in vivo T cell priming therapy, adenovirus-encoded tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (AdTRAIL) with class B oligonucleotides (CpG), we evaluated therapeutic effects on primary tumor outgrowth and metastatic burden in a pre-clinical model of orthotopic murine RCC. We report here that administration of intra-renal AdTRAIL/CpG in combination with α-CTLA-4, but not α-PD-1, causes significant decreases in primary tumor burdens. Strikingly, we observed that this therapeutic strategy caused significant reductions in metastatic lung burdens – equating to a 98% reduction over no therapy controls. Flow cytometric analysis revealed increases in CD8+ T cells in AdTRAIL-treated groups over no therapy/checkpoint alone; results were further supported by immunosuppressive CD4+ to CD8+ T cell ratios that were in favor of anti-tumoral responses. Our results suggest AdTRAIL/CpG can be used as an effective upstream therapeutic approach to enhance the efficacy of α-CTLA-4 checkpoint blockade in a pre-clinical model of murine RCC.

FCγ Chimeric Receptor-Engineered T Cells: Methodology, Advantages, Limitations, and Clinical Relevance.

For many years, disappointing results have been generated by many investigations, which have utilized a variety of immunologic strategies to enhance the ability of a patient’s immune system to recognize and eliminate malignant cells. However, in recent years, immunotherapy has been used successfully for the treatment of hematologic and solid malignancies. The impressive clinical responses observed in many types of cancer have convinced even the most skeptical clinical oncologists that a patient’s immune system can recognize and reject his tumor if appropriate strategies are implemented. The success immunotherapy is due to the development of at least three therapeutic strategies. They include tumor-associated antigen (TAA)-specific monoclonal antibodies (mAbs), T cell checkpoint blockade, and TAA-specific chimeric antigen receptors (CARs) T cell-based immunotherapy. However, the full realization of the therapeutic potential of these approaches requires the development of strategies to counteract and overcome some limitations. They include off-target toxicity and mechanisms of cancer immune evasion, which obstacle the successful clinical application of mAbs and CAR T cell-based immunotherapies. Thus, we and others have developed the Fc gamma chimeric receptors (Fcγ-CRs)-based strategy. Like CARs, Fcγ-CRs are composed of an intracellular tail resulting from the fusion of a co-stimulatory molecule with the T cell receptor ζ chain. In contrast, the extracellular CAR single-chain variable fragment (scFv), which recognizes the targeted TAA, has been replaced with the extracellular portion of the FcγRIIIA (CD16). Fcγ-CR T cells have a few intriguing features. First, given in combination with mAbs, Fcγ-CR T cells mediate anticancer activity in vitro and in vivo by an antibody-mediated cellular cytotoxicity mechanism. Second, CD16-CR T cells can target multiple cancer types provided that TAA-specific mAbs with the appropriate specificity are available. Third, the off-target effect of CD16-CR T cells may be controlled by withdrawing the mAb administration. The goal of this manuscript was threefold. First, we review the current state-of-the-art of preclinical CD16-CR T cell technology. Second, we describe its in vitro and in vivo antitumor activity. Finally, we compare the advantages and limitations of the CD16-CR T cell technology with those of CAR T cell methodology.

Short-term CTLA-4 blockade directly followed by PD-1 blockade in advanced melanoma patients: a single-center experience

Combination of T cell checkpoint blockade by CTLA-4- and PD-1-blockade is one of the most promising therapies in patients with advanced melanoma. It induces superior response rates when compared with single-agent therapy, but at the cost of a high percentage of grade 3 and 4 adverse events (AEs). This combination therapy was until July 2016 not available in the Netherlands, which prompted several physicians to treat patients with less than standard numbers of courses of ipilimumab followed directly by nivolumab or pembrolizumab. In this retrospective analysis, patients were included who were treated with two courses (day 0 and 21) anti-CTLA-4 (ipilimumab 3 mg/kg q3wk), directly followed by anti-PD-1 (starting at day 22 with nivolumab 3mg/kg q2wk or pembrolizumab 2 mg/kg q3wk). Data on treatment-related AEs were collected from electronic patient records and scored according to CTCAE 4.03 criteria. Overall response was evaluated using RECIST 1.1 for CT-scans and EORTC criteria for PET-scans. Forty advanced melanoma patients could be included (29/40 pembrolizumab, 11/40 nivolumab). Median follow-up (FU) was 51 weeks (range: 4-63 weeks) with a minimum FU of 26 weeks. Treatment-related AEs of grade 3 and 4 occurred in 38% of the patients. The best overall response rate (BORR) was 55% (95% CI 39-70) and disease control rate was 75% (95% CI 59-87). Ongoing responses were observed in 82% of responding patients. Treatment with short-term CTLA-4 blockade directly followed by PD-1 blockade may have similar efficacy but potentially lower toxicity when compared with concurrent therapy with anti-CTLA-4 and anti-PD-1. These results warrant further investigation in a prospective randomized controlled clinical trial.

Next generation approaches for tumor vaccination.

Tumor vaccines have been an attractive concept in the immunotherapy of cancer based on the central role of tumor-associated antigens in allowing the immune system to recognize cancer cells and the large variety of platforms in which to present such antigens to the immune system. Early clinical studies of vaccines, however, were largely disappointing. Recent evidence that cancer-mediated T cell suppression may prevent T cell activation is leading to renewed interest in vaccine development. The use of T cell checkpoint inhibitors alone has revolutionized the contemporary treatment of human cancer, and has suggested that the emergence of neoantigens may be an important biomarker of therapeutic response. Thus, the possibility of using more personalized vaccines targeting relevant neoantigens alone and in combination with T cell checkpoint blockade is a new area of active clinical investigation. In this review we will discuss the central role of antigens in tumor immunotherapy, describe how vaccines may be developed in the context of modern genomic profiling of tumor cells and provide a forward looking perspective on how tumor vaccines may be incorporated into the current landscape of cancer therapy.

PD-1 blockade induces remissions in relapsed classical Hodgkin lymphoma following allogeneic hematopoietic stem cell transplantation

BackgroundAllogeneic hematopoietic stem cell transplantation and checkpoint blockade therapy are immune-based therapies that have activity in selected refractory hematologic malignancies. Interest has developed in combining these treatments for high-risk hematologic diseases. However, there is concern that checkpoint blockade could augment graft-versus-host disease, and very few studies have evaluated the safety of checkpoint blockade in the post-allogeneic setting. Here, we report the outcomes of three patients with relapsed classical Hodgkin’s lymphoma following allogeneic transplant that were treated with the anti-PD-1 antibody, nivolumab.Case presentationsThree patients with Hodgkin’s lymphoma relapsed following allogeneic transplant received nivolumab therapy at our institution. All patients were free of graft-versus-host disease and were off of all systemic immunosuppressive medications at the time of nivolumab treatment. Nivolumab was well-tolerated in two of the patients. However, nivolumab had to be discontinued in one patient due to development of immune-related polyarthritis requiring treatment with systemic corticosteroids and methotrexate. Objective responses were observed in all three patients.ConclusionsOur case series demonstrates that anti-PD-1 therapy with nivolumab can be highly effective following allogeneic transplant for Hodgkin’s lymphoma, but serious immune-related adverse events can occur, requiring very close monitoring and interruption of therapy.

Synergistic effects of the immune checkpoint inhibitor CTLA-4 combined with the growth inhibitor lycorine in a mouse model of renal cell carcinoma.

Renal cell carcinoma (RCC) management has undergone a major transformation over the past decade; immune checkpoint inhibitors are currently undergoing clinical trials and show promising results. However, the effectiveness of immune checkpoint inhibitors in patients with metastatic RCC (mRCC) is still limited. Lycorine, an alkaloid extracted from plants of the Amaryllidaceae family, is touted as a potential anti-cancer drug because of its demonstrative growth inhibition capacity (induction of cell cycle arrest and inhibition of vasculogenic mimicry formation). Moreover, T cell checkpoint blockade therapy with antibodies targeting cytotoxic T-lymphocyte associated protein 4 (CTLA-4) has improved outcomes in cancer patients. However, the anti-tumor efficacy of combined lycorine and anti-CTLA-4 therapy remains unknown. Thus, we investigated a combination therapy of lycorine hydrochloride and anti-CTLA-4 using a murine RCC model. As a means of in vitro confirmation, we found that lycorine hydrochloride inhibited the viability of various RCC cell lines. Furthermore, luciferase-expressing Renca cells were implanted in the left kidney and the lung of BALB/c mice to develop a RCC metastatic mouse model. Lycorine hydrochloride and anti-CTLA-4 synergistically decreased tumor weight, lung metastasis, and luciferin-staining in tumor images. Importantly, the observed anti-tumor effects of this combination were dependent on significantly suppressing regulatory T cells while upregulating effector T cells; a decrease in regulatory T cells by 31.43% but an increase in effector T cells by 31.59% were observed in the combination group compared with those in the control group). We suggest that a combination of lycorine hydrochloride and anti-CTLA-4 is a viable therapeutic option for RCC patients.

Abstract PR11: Neo-antigen landscape dynamics during human melanoma-T cell interactions

Abstract Recognition of the neo-antigens that are formed as a consequence of DNA damage is likely to be a major driving force behind the clinical activity of T cell checkpoint blockade and adoptive T cell therapy for treatment of non-virally induced solid cancers. Consequently, strategies to selectively enhance T cell reactivity against genetically defined neo-antigens are currently under development. In mouse models, T cell pressure has been shown to sculpt the antigenicity of tumors through selection of tumor cell variants that do not express the relevant neo-antigens. However, it is unclear whether the T cell-recognized neo-antigen repertoire is constant over time in human cancers. To address this issue, we analyzed the stability of neo-antigen specific T cell responses and the antigens they recognize in stage IV melanoma patients treated by adoptive T cell transfer. Fresh tumor tissue resected before and after treatment was used to establish autologous melanoma cell lines, to culture tumor-infiltrating lymphocytes (TIL) and to generate T-cell infusion products by autologous mixed lymphocyte tumor cell cultures using peripheral blood lymphocytes. Material from 2 patients with objective clinical responses after infusion of autologous tumor-specific CD4+ and CD8+ T cells was analyzed by whole exome and RNA sequencing. Subsequently, 31-mer peptides covering all the non-synonymous somatic mutations were used to test the reactivity of both the T-cell infusion products and TIL isolated from subsequent tumor lesions by ELISA and flow cytometry. The expression of genes encoding T cell-recognized neo-antigens was then analyzed within the tumor cell lines and within the corresponding paraffin-embedded tumor tissue, by real-time PCR analysis, a competitive allele-specific PCR assay, and by analysis of heterozygosity and next generation amplicon sequencing. Analysis of the infusion products showed that 50-80% of T cells reacted to autologous tumor cells, with only a very low percentage of these cells recognizing known shared tumor-associated epitopes. Instead, tumor reactivity was predominantly mediated by CD4+ and CD8+ T cells recognizing clonally expressed neo-antigens formed by private mutations. CD8+ T cells within the infusion products and within TIL populations generated from subsequent tumor lesions of these two patients recognized a total of 6 different neo-antigens, and analysis of these neo-antigens in both the index lesion and subsequent lesions revealed profound alterations in the T cell-recognized neo-antigen landscape. In particular, in 2 cases, the mutant allele was lost from a subsequent tumor, in one case, expression of the mutant gene was substantially reduced in a recurring lesion, and in another case expression of the mutant gene was increased approx. 40-fold in a subsequent tumor. Notably, in 4 out of 4 cases these changes in neo-antigen expression were paralleled by loss or gain of the respective neo-antigen specific T cell response. Collectively, our data demonstrate that under conditions in which a high frequency of tumor-specific T cells is present, tumor cell variants with reduced or lost neo-antigen expression can emerge, similar to what has been observed in mice. This, and the observation of concurrent acquisition of novel T cell reactivity, reveal the dynamics of T cell - neo-antigen interaction in human cancer, and indicate that immunotherapies that maximize the capacity to respond to an altering neo-antigen landscape may offer the highest chance to achieve long-term tumor control. Citation Format: Els M.E. Verdegaal, Noel de Miranda, Marten Visser, Tom Harryvan, Marit van Buuren, Rikke Andersen, Sine Hadrup, Caroline van der Minne, Remko Schotte, Hergen Spits, John Haanen, Ellen Kapiteijn, Ton Schumacher, Sjoerd H. Van Der Burg. Neo-antigen landscape dynamics during human melanoma-T cell interactions [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR11.

Abstract IA04: T cell recognition and tumor resistance in human cancer

Abstract The T cell-based immune system has evolved to recognize and destroy cells that differ from “self” on the basis of the antigens they express. Many human tumors contain large numbers of mutations, of which many hundreds can be present within expressed genes. As the resulting altered protein sequences are foreign to the immune system, immune recognition of such “neo-antigens” has been proposed as a major factor in the activity of clinically used immunotherapeutics such as anti-CTLA-4 and anti-PD-1. In support of this hypothesis, using in-house developed technologies we have demonstrated that both CD4 and CD8 T cell recognition of the consequences of DNA damage is a common feature in human melanoma and can be boosted by T cell checkpoint blockade. Furthermore, to extend these data to other tumor types, we have benchmarked the foreign antigenic space in a variety of human cancers against a series of human pathogens. Collectively, these data, and data from other groups, establish “tumor foreignness” as one of the critical determinants in effective cancer immunotherapy. Clearly, the sensitivity of tumor cells to the tumor-specific T cell pool that can be present in intrinsically foreign tumors forms an additional requirement for tumor control. To address factors that control such sensitivity, we a currently determining mechanisms of immune escape upon T cell targeting of human tumors. Results from this analysis are expected to contribute to the further definition of biomarkers that predict the outcome of the interaction between human tumors and the T cell-based immune system. Citation Format: Ton Schumacher. T cell recognition and tumor resistance in human cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA04.