Rienk Offringa

Prevailing Role of Contact Guidance in Intrastromal T-cell Trapping in Human Pancreatic Cancer

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive collagen-rich stroma. T cells that infiltrate pancreatic cancers frequently become trapped in the stroma and do not contact tumor cells. Here, we aimed to analyze how chemokines and extracellular matrix (ECM) collagen interact in mediating T-cell infiltration in PDAC. Experimental Design: T-cell distribution and ECM structure within tumors were analyzed. Chemokine concentrations in human PDAC were compared with the levels of immune cell infiltration. We assessed the influences of selected chemokines and collagen on directed and random T-cell movement using in vitro migration systems. Results: PDAC overproduced several T-cell-active chemokines, but their levels were not correlated with intratumoral T-cell infiltration. In the absence of collagen, directed migration of activated T cells was induced by chemokines. Interestingly, collagen itself promoted high migratory activity of T cells, but completely abolished chemokine-guided movement. This effect was not altered by a β1-integrin blocking antibody. Activated T cells actively migrated in low-density collagen matrices, but migration was inhibited in dense collagen. Accordingly, T cells were heterogeneously distributed in the pancreatic cancer stroma, with the majority residing in areas of low-density collagen far from tumor clusters. Conclusion: The excessive desmoplasia in PDAC promotes T-cell migration by contact guidance, which abrogates tumor cell–directed movement. Furthermore, dense collagen networks represent a physical barrier, additionally rearranging T-cell distribution to favor tumor stroma. These mechanisms are mainly responsible for intrastromal T-cell trapping in pancreatic cancer and may hinder the development of T-cell–based immunotherapies. Clin Cancer Res; 20(13); 3422–33. ©2014 AACR.

Development of Next-Generation Immunomodulatory Antibodies for Cancer Therapy through Optimization of the IgG Framework

In this issue of Cancer Cell, Dahan and colleagues demonstrate that the Fc region has a significant impact on the therapeutic capacity of checkpoint inhibitor antibodies targeting the PD-1/PD-L1 axis in pre-clinical tumor models. This work provides important insights with respect to the further clinical development of checkpoint inhibitors.

Association of genetic polymorphisms with survival of pancreatic ductal adenocarcinoma patients

Germline genetic variability might contribute, at least partially, to the survival of pancreatic ductal adenocarcinoma (PDAC) patients. Two recently performed genome-wide association studies (GWAS) on PDAC overall survival (OS) suggested (P < 10(-5)) the association between 30 genomic regions and PDAC OS. With the aim to highlight the true associations within these regions, we analyzed 44 single-nucleotide polymorphisms (SNPs) in the 30 candidate regions in 1722 PDAC patients within the PANcreatic Disease ReseArch (PANDoRA) consortium. We observed statistically significant associations for five of the selected regions. One association in the CTNNA2 gene on chromosome 2p12 [rs1567532, hazard ratio (HR) = 1.75, 95% confidence interval (CI) 1.19-2.58, P = 0.005 for homozygotes for the minor allele] and one in the last intron of the RUNX2 gene on chromosome 6p21 (rs12209785, HR = 0.88, 95% CI 0.80-0.98, P = 0.014 for heterozygotes) are of particular relevance. These loci do not coincide with those that showed the strongest associations in the previous GWAS. In silico analysis strongly suggested a possible mechanistic link between these two SNPs and pancreatic cancer survival. Functional studies are warranted to confirm the link between these genes (or other genes mapping in those regions) and PDAC prognosis in order to understand whether these variants may have the potential to impact treatment decisions and design of clinical trials.

Optimized dendritic cell vaccination induces potent CD8 T cell responses and anti-tumor effects in transgenic mouse melanoma models

ABSTRACT Despite melanoma immunogenicity and remarkable therapeutic effects of negative immune checkpoint inhibitors, a significant fraction of patients does not respond to current treatments. This could be due to limitations in tumor immunogenicity and profound immunosuppression in the melanoma microenvironment. Moreover, insufficient tumor antigen processing and presentation by dendritic cells (DC) may hamper the development of tumor-specific T cells. Using two genetically engineered mouse melanoma models (RET and BRAFV600E transgenic mice), in which checkpoint inhibitor therapy alone is not efficacious, we performed proof-of-concept studies with an improved, multivalent DC vaccination strategy based on our recently developed genetic mRNA cancer vaccines. The in vivo expression of multiple chimeric MHC class I receptors allows a simultaneous presentation of several melanoma-associated shared antigens tyrosinase related protein (TRP)-1, tyrosinase, human glycoprotein 100 and TRP-2. The DC vaccine induced a significantly improved survival in both transgenic mouse models. Vaccinated melanoma-bearing mice displayed an increased CD8 T cell reactivity indicated by a higher IFN-γ production and an upregulation of activation marker expression along with an attenuated immunosuppressive pattern of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg). The combination of DC vaccination with ultra-low doses of paclitaxel or anti-PD-1 antibodies resulted in further prolongation of mouse survival associated with a stronger reduction of MDSC and Treg immunosuppressive phenotype. Our data suggest that an improved multivalent DC vaccine based on shared tumor antigens induces potent anti-tumor effects and could be combined with checkpoint inhibitors or targeting immunosuppressive cells to further improve their therapeutic efficiency.

Abstract A070: Genetic knockdown screens across tumor types unravel a diverse tumor “immune-modulatome” landscape

Clinical trials with immune-checkpoint blockade antibodies have bolstered the importance of immune therapy as the standard of care for cancer patients, but it has also simultaneously highlighted the heterogeneity in patient responses to such treatment. These heterogeneities can be explained to a certain extent by the lack of tumor-specific expression of the targeted immune checkpoint molecules. But in many cases it can also be conceived that tumors either develop resistance to a targeted immune-checkpoint node by circumventing it or more than one player is involved in a concerted action to subvert the T cell response. In either scenario, we lack a holistic understanding of the putative genes in the tumor genome that could functionally suppress the immune response. To bridge this gap, we employed a high-throughput RNAi-mediated knockdown of upto 2800 genes (~50% associated with surface molecules) in MCF7 (breast), M579-A2 (melanoma) and PANC-1 (pancreatic) tumor cell lines and co-cultured them with either antigen-specific T cell clones or respective patient-derived and tumor-specific infiltrating lymphocytes (TILs) to assess the impact on anti-tumor immunity using a luciferase-based readout. Primary hit-list was further subjected to a secondary screen based on multi-cytokine profiling of the T cells. Our investigation revealed a few salient caveats of tumor-mediated immune suppression. Firstly, we discovered a family of orphan receptors, which were never attributed to the immune system before, to actively suppress the T cells in a manner comparable to the currently defined immune-checkpoint molecules, such as PD-L1. Secondly, the trans-versatility of these novel molecules across the tumor types was highly limited, with only 3-14 common molecules being involved in two or more tumor types. This leads us to our third observation that there exists a complex organ-specific orchestration of peripheral immune tolerance, which needs to be taken into account when devising immune-checkpoint blockade therapies. Amongst the key immunosuppressive candidate genes, CCR9 was validated to directly subvert T cell responses in melanoma, breast and pancreatic cancer in the in vitro and in vivo tumor models. Additionally we have verified TiMi1, an orphan G-protein coupled receptor, to mediate strong immunosuppression in melanoma and pancreatic cancer against the respective TILs. Knockdown of both CCR9 and TiMi1, either via siRNAs or shRNAs, in tumor cells significantly increased Th1 cytokine secretion by TILs along with elevated tumor lysis in vitro and in vivo xenotransplanted mouse models. Moreover, they both induce a highly immunosuppressive genetic signature in the encountering TILs. While TiMi1 appeared to modulate calcium-dependent signaling, CCR9 regulated STAT signaling in T cells leading to an immunosuppressed phenotype. Overall, these candidates represent attractive targets for cancer immunotherapy either through function blocking antibodies or small molecules. In conclusion, we here report an effective genetic screen strategy in multiple tumor types that has the potential to uncover novel modifiers of anti-tumor immunity. Extensively validated candidates from these screens are attractive targets for cancer immunotherapy that will allow us to further expand our limited arsenal of immune-checkpoint inhibitors, with the overall goal of increasing patient responses to such treatments. Citation Format: Nisit Khandelwal, Tillmann Michels, Marco Breinig, Antonio Sorrentino, Isabel Poschke, Rienk Offringa, Michal Lotem, Michael Boutros, Philipp Beckhove. Genetic knockdown screens across tumor types unravel a diverse tumor “immune-modulatome” landscape. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A070.

Phenotype, function and T cell receptor repertoire of tumor-infiltrating lymphocytes in patients with pancreatic ductal adenocarcinoma.

Meeting abstracts Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a median survival of only about two years even in the 20% of patients that present early enough to be eligible for surgical resection and adjuvant chemotherapy. With this urgent medical need in mind, we are

Abstract 254: TiMi1 is a novel immune-checkpoint in solid tumors identified via a tumor-infiltrating lymphocyte (TIL)-based RNAi screening

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Immunotherapeutic treatment of melanoma achieved major progress in recent years leading for the first time to improved survival. However, since melanoma cells employ various suppressive mechanisms in order to evade recognition and destruction by immune effector cells many patients still do not benefit from immunotherapy. These mechanisms are far more diverse than reflected by currently used immune modulatory drugs. In this study, we established and utilized a novel high throughput RNAi screening to identify new immune checkpoint molecules in melanoma using antigen-specific patient-derived tumor infiltrating lymphocytes (TILs) in conjunction with primary HLA-matched melanoma cells. Using this approach, we screened a siRNA library targeting more than 1200 surface receptors and kinases to explore novel targets for immunotherapy. Briefly, HLA-A2 and luciferase positive M579-A2-luc melanoma cells were reversely transfected with the siRNA library and then co-cultured with MART1- and gp100-specific TILs to validate the TIL-mediated tumor lysis. Local regression models (LOESS) were applied to generate a hit list of 48 candidates that negatively regulated CTL cytotoxicity. Interestingly, four candidates of a related breast cancer screen were among the top hits. To streamline the discovery process for large scale molecule libraries, we established a secondary screen assaying multiple T cell activation markers, including effector cytokines. One of the strongest candidates from our primary and secondary screening is TiMi1 (name altered), a cell surface receptor belonging to the class of GPCRs. We found that knock-down of TiMi1 increased TIL-mediated killing of M579-A2-luc without affecting their viability. TiMi1 knock-down increased TIL activity measured by production of type 1-associated cytokines (e.g. IFN γ and TNF-α), reduced TC apoptosis and increased markers associated with raised activity and cytotoxicity (4-1BB and CD107a). We were able to verify the immune checkpoint function of TiMi1 in melanoma patients using an autologous set of melanoma cells and TILs. Phosphoplex analysis in T cells revealed an involvement of the transcription factor CREB in the mode of action of TiMi1. Preliminary experiments suggest that TiMi1 inhibits anti-tumor immune responses in pancreatic (PDAC) and colorectal (CRC) cancers as well. In summary, we established a novel antigen-specific screening approach for immune checkpoints expressed in melanoma and were able to identify TiMi1 as a promising candidate. Moreover, TiMi1 inhibits T cell responses in melanoma, PDAC and CRC and might be an interesting target for immunotherapy. Our novel high-throughput screening offers a systematic platform to uncover the “immune-modulatome” of cancer and subsequently discover novel targets for immunotherapy. Since the presented work is considered for patent protection, some gene targets are masked in the presented study. Citation Format: Tillmann Michels, Christina A. Hartl, Nisit Khandelwal, Marco Breinig, Antonio Sorrentino, Christina Mader, Ludmila Umansky, Isabel Poschke, Rienk Offringa, Michael Boutros, Galit Eisenberg, Michal Lotem, Philipp Beckhove. TiMi1 is a novel immune-checkpoint in solid tumors identified via a tumor-infiltrating lymphocyte (TIL)-based RNAi screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 254. doi:10.1158/1538-7445.AM2015-254

Abstract 245: Identification of novel immune checkpoints as potential therapeutic targets in pancreatic ductal adenocarcinoma (PDAC) using RNAi screening

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) accounts for 95% of pancreatic cancers and constitutes the fourth leading cause of cancer related death worldwide. In contrast to other malignancies, PDAC is highly resistant to chemotherapy and radiotherapy. Additionally, few immunotherapies are currently available because this malignancy was thought to be poorly immunogenic. Recent studies have shown that infiltration of immune cells in biopsies of PDAC patients correlates with improved clinical outcome. Nevertheless, tumor cells can elude the immune system through several inhibitory mechanisms including the expression of immune checkpoints. These are a plethora of molecules that can either boost or dampen the T-cell receptor (TCR) signaling. Some of these molecules, such as PD-L1, have been successfully used as therapeutic targets of novel anticancer drugs. AIM: We hypothesize that many immune checkpoint molecules on tumor cells remain undiscovered and we performed a high-throughput RNAi screen to unravel the whole arsenal of immune modulators. METHODS: We generated a luciferase-expressing PANC-1 cell line and knocked down 2514 genes using a siRNA library. Our library included G-protein coupled receptors, protein kinases and 1117 surface proteins. We co-cultured HLA-A201+ matched tumor infiltrating lymphocytes (TILs) derived from a PDAC patient with the transfected tumor cells. We then measured the remaining luciferase intensity of the tumor cells as an estimation of TIL-mediated cytotoxicity. In order to exclude genes whose knock-down affected cell viability per se, we cultivated tumor cells with the siRNA library in the absence of TILs. Data were analyzed with the cellHTS2 R package. RESULTS: Our screen revealed 155 candidate genes whose knock-down enhances TIL-mediated killing more efficiently than PD-L1 down-regulation. 35% of these genes are surface molecules and are most likely to directly mediate tumor immune evasion. Beside novel undescribed immune checkpoints, our list contains well characterized immune modulators, supporting the reliability of our approach. Of note 13 of our hits were also found in a related melanoma screen and might play a role in the regulation of immune surveillance of many solid tumors. Among our candidates, TONI1 was one of the most prominent hits. So far, we confirmed the role of TONI1 in inhibiting TIL-mediated killing both in chromium release and luciferase based kill assays. Additionally we detected increased T-cell activity upon TONI1 down-regulation, as measured with interferon-γ ELISPOT and TNF-α ELISA. Since the presented work is considered for patent protection, some gene targets are masked. CONCLUSION: We set up a robust and systematic method to identify novel immune checkpoints for pancreatic cancer. Further functional validation of our candidate genes will prove their use as therapeutic targets. Citation Format: Antonio Sorrentino, Tillmann Michels, Ayse Nur Menevse, Nisit Khandelwal, Marco Breinig, Isabel Poschke, Rienk Offringa, Michael Boutros, Philipp Beckhove. Identification of novel immune checkpoints as potential therapeutic targets in pancreatic ductal adenocarcinoma (PDAC) using RNAi screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 245. doi:10.1158/1538-7445.AM2015-245