The Proceedings on Cancer-Immuno-Oncology offers the opportunity to publish emerging technologies, sharing experience, evaluate developments and future necessities in immuno oncology across industry and academia, and influence next-generation immunotherapy programs. The Proceedings aims to bring together and enlighten the different major fields of microbiota, new targets, liquid biopsies, new ideas of players in the immune response, new therapeutic strategies, new combinatorial approaches bringing together some of the many different ways to unravel the immune systems own power in elimination of cancer cells.
Extracellular Vesicles as biomarkers for cancer progression and cancer associated VTE
Microvesicles (MVs) represent a subgroup of extracellular vesicles (EVs) emerging from various cells by blebbing of their outer membrane. Therefore, they share features such as membrane composition and antigenicity with their parental cells. Released by many immune and tumor cells, MVs act as intercellular messengers, account for horizontal gene transfer and can activate the coagulation system. With the aim to investigate their relevance for tumor cell biology, we characterized MVs released by human tumor cell lines of various origins in the absence or presence of TNF-a. After stimulation, we used the combination of low and high-speed centrifugation to enrich MVs from cell culture supernatants. We analyzed the presentation of phosphatidylserine (PS) and tissue factor (TF) activity on the cell surface and investigated their potency to induce tumor cell migration. In all tumor cell lines, TNF-a stimulation enhanced the release of MVs. While the expression of PS was universally increased, an elevated activity of procoagulant TF could be detected on MVs from lung, pancreatic, and colon carcinoma, but not from breast and ovarian cancer cell lines. Functionally, TNF-a stimulation significantly increased the potency of MVs to induce tumor cell migration. In conclusion, inflammatory conditions promote the release of MVs with increased procoagulant activity from tumor cell lines in vitro. PS-containing and TF-expressing MVs may account for systemic activation of the coagulation system as seen in cancer patients and, since they induce tumor cell migration, they may serve as biomarkers for tumor progression. Publication: Cell Biology International 2018.
Copyright (c) 2018 Frank Gieseler, Fanny Ender https://creativecommons.org/licenses/by/4.0
Immune modulation by extracellular vesicles released from mesenchymal cells
Mesenchymal cells were described for their potency to interact with human immune cells and to modulate thereby immune responses. Besides mesenchymal stromal cells (MSC) from diverse tissue sources, like bone marrow and umbilical cord, also mesenchymal adherent cells within the heart tissue were able to attenuate and modulate induced immune cell activation or inflammatory processes. Mechanistic studies with MSC support the hypothesis, that mainly paracrine acting molecules, in particular extracellular vesicles (EVs), are responsible for the observed functional effects. EVs are known as potent intercellular communicators by delivering proteins, lipids, RNA and other small signaling molecules to a recipient cell. They can be discriminated by their size and biogenesis into the subsets of apoptotic bodies (diameter > 1?m), microvesicles (diameter range = 1 - 0.1 ?m) and exosomes (diameter < 0.1 ?m). While microvesicles and apoptotic bodies are shedded from the plasma membrane, exosomes originate from intracellular located multivesicular bodies, which have to fuse with the plasma membrane for their release into the extracellular space. Crosstalk of EVs from MSCs with immune cells is a secured fact, but the way of up-take into the target cells and especially the mechanism of immune modulation are not entirely understood. In our study, we isolated and characterized EVs from a human mesenchymal cardiac cell type regarding their potential to modulate induced immune responses in vitro. The presence of typical EV surface markers like tetraspanins (CD9, CD63, CD81) was confirmed as well as their low expression of HLA-molecules, which indicates a general low immunogenicity. Furthermore, EVs were able to attenuate triggered T cell proliferation accompanied by significantly reduced levels of pro-inflammatory cytokines (IFN?, TNF?), which was highly dependent on the presence of CD14-positive cells. Interestingly, the EVs of cardiac mesenchymal cells induced a changed phenotype on these myeloid cells; most prominently a reduced HLA-DR and CD86 expression, but enhanced levels for CD206 and PD-L1. Future studies have to identify key molecular pathways involved in EVs` crosstalk with immune cells and to estimate the benefits but also the risks of this new therapeutic based on mesenchymal cells.
Copyright (c) 2018 Martina Seifert https://creativecommons.org/licenses/by/4.0
MDSC as a new target for cancer immunotherapy
Tumor microenvironment is characterized by a strong immunosuppression, where myeloidderived suppressor cells (MDSC) induced by chronic inflammation play a major role. Using ret transgenic mouse melanoma model, which mimics clinical situation in human melanoma, we demonstrated MDSC accumulation in melanoma lesions that correlated with reduced anti-tumor T cell reactivity and accelerated tumor progression. The accumulation and activation of MDSC could be mediated not only by soluble inflammatory factors but also by tumor-derived extracellular vesicles, converting immature myeloid cells into immunosuppressive MDSC. Targeting of MDSC migration and functions significantly prolonged survival of tumor-bearing mice associated with the restoration of T cell anti-tumor reactivity. In advanced melanoma patients resistant to immunotherapy with immune checkpoint inhibitors, MDSC targeting induced a beneficial therapeutic effect. We suggest that chronic inflammatory mediators and MDSC are of critical importance for melanoma pathogenesis and their neutralization should be included in combined melanoma immunotherapies to increase their efficiency.
Copyright (c) 2018 Viktor Umansky https://creativecommons.org/licenses/by/4.0
Neo-adjuvant checkpoint inhibition – the pathway towards personalized immunotherapy
Targeted and immunotherapy have revolutionized the systemic and adjuvant therapy in late stage melanoma. Recent pre-clinical and clinical data indicate that neoadjuvant immunotherapy might be superior to adjuvant therapy. Neoadjuvant therapy can bear several advantages, allowing one to 1) determine therapy efficacy within the individual patient for possible additional adjuvant therapy, if needed, 2) reduce tumor burden before surgery, 3) utilize pathological response data as surrogate outcome markers for relapse free and overall survival, and 4) identify biomarkers in a homogenous patient population. For these reasons (except latter), neoadjuvant therapy has become a standard of care in high tumor burden breast cancer. In the case of T cell checkpoint blockade, neoadjuvant therapies could bear a fifth, and potentially significant, advantage. T cell checkpoint blocking antibodies enhance T-cell activation at the moment antigen is encountered, and drug exposure during the time the in-situ malignancy still provides a major source of antigen may therefore potentially induce a stronger and broader tumor-specific T cell response. I will present pre-clinical and clinical data supporting this notion, present early clinical data supporting these ideas and will give an outline for effective biomarker identification, that will lead towards personalized combination immunotherapies.
Copyright (c) 2018 Christian Blank https://creativecommons.org/licenses/by/4.0
Interference between tumor mutational burden, HPV status, immune signatures and outcome in patients with head and neck cancer treated with chemoradiotherapy: Results from a multicenter retrospective study of the German Cancer Consortium Radiation Oncology
Tumor mutational burden as well as extent and quality of tumor immune-cell infiltration have previously been established as biomarkers of efficacy in immune checkpoint inhibitor trials. Their value for predicting outcome after concurrent chemoradiation, a treatment regimen currently evaluated for combination with immune checkpoint inhibitors remains largely unknown. In the talk, the results from a recent multicenter biomarker study of the prognostic role of tumor mutational burden, immune expression profiles and the HPV status in locally advanced head and neck squamous cell carcinoma treated with definitive chemoradiation will be presented. The value of the estimation of tumor mutational burden by targeted versus whole exome sequencing will be discussed. Results of an interference of tumor mutational burden with immune signatures determined by the nanoString PanCancer Immune panel analysis will be presented. Finally, the associations of tumor mutational burden with patient outcome parameters will be presented.
Copyright (c) 2018 T. Eder, Ingeborg Tinhofer, A. K. Hess, V. Fleischer, P. Balermpas, C. Rödel, A. Linge, M. Krause, M. Baumann, M. Stuschke, D. Zips, A. L. Grosu, A. Abdollahi, J. Debus, U. Ganswindt, C. Belka, S. Pigorsch, G. Multhoff, S. E. Combs, V. Budach https://creativecommons.org/licenses/by/4.0
Dynamics of conversation: Immunotherapy meets tumor microenvironment
The recent exciting success of cancer immunotherapy has emphasized the essential role of dynamic interactions between tumor cells and the immune system. Inflammation and massive infiltration of leukocytes is a hallmark of various tumor entities and recruitment of immune cells into the tumor microenvironment has been shown to contribute to patients’s clinical outcome in certain types of cancer. In contrast to conventional cancer therapies, such as radiation and chemotherapy, the immunotherapeutic approach specifically targets the different subtypes of immune cells and thus orchestrates a systemic response against tumors. However, depending on the context of the reciprocal interaction between cancer cells their surrounded stromal composition, the tumor microenvironment can both, dampen or enhance immune responses, directing patient’s survival.
Copyright (c) 2018 Iris Helfrich https://creativecommons.org/licenses/by/4.0
Immunotherapy works in some patients, in most patients it does not. We have tumor entities that show low objective response rates like CRC or PDAC. We identified the immunosuppressive environment being the main mechanisms of resistance. Therefore strategies that target such suppressive mechanisms are being developed clinically. We currently evaluate CCR5 blockade in combination with checkpoint inhibition in MSS-CRC and PDAC.
Copyright (c) 2018 Dirk Jäger https://creativecommons.org/licenses/by/4.0
Effects of EGFR-MEK-ERK pathway on PD-L1 expression in head and neck squamous cell carcinoma
Objectives: Programmed death ligand-1 (PD-L1) is frequently expressed in human cancers, including head and neck squamous cell carcinoma (HNSCC). However, the regulation of PD-L1 in HNSCC remains largely elusive. The purpose of this study was to address the molecular mechanism of PD-L1 regulation by EGFR-MEK-ERK signaling and to suggest potential clinical relevance of blocking PD-1/PD-L1 in combination with therapeutic options targeting EGFR signaling in HNSCC. Methods: Correlations of PD-L1 protein expression and key nodes of EGFR-MEK-ERK signaling in HNSCC were analyzed by The Cancer Proteome Atlas (TCPA, http://tcpaportal.org). The impact of drugs targeting the EGFR-MEK-ERK pathway on tumor cell survival, clonal expansion as well as PD-L1 expression were addressed in cell culture models. PD-L1 expression and ERK1/2 phosphorylation (p-ERK1/2) were assessed on tissue microarrays with specimens from primary oropharyngeal squamous cell carcinoma (OPSCC) (n=123 patients) by immunohistochemistry and were compared with clinic-pathological features. Results: A highly significant association was found between PD-L1 expression and key nodes of EGFR-MEK-ERK signaling analyzing the TCPA-HNSCC dataset. However, no significant association was found between PD-L1 expression and p-ERK1/2 protein level in the TCPA-HNSCC dataset OPSCC cohort. The impact of cetuximab on PD-L1 expression in HNSCC cell lines after short-term or long-term treatment was heterogeneous but independent of ERK1/2 phosphorylation. Treatment with a MEK1/2 inhibitor confirmed that PD-L1 regulation in HNSCC cell lines is largely independent of MEK-ERK signaling. The majority of HNSCC cell lines with long-term cetuximab treatment regained ERK1/2 phosphorylation as compared to short-term treatment. Up-regulation of PD-L1 was observed in most cetuximab-resistant cell lines after long-term treatment. Conclusion: Our findings suggest a complex and context-dependent PD-L1 regulation upon inhibition of the EGFR-MEK-MAPK signaling cascade and further experimental and clinical studies will be essential to unravel underlying molecular principles.
Copyright (c) 2018 Bohai Feng, Chao Rong, Marie Muller, Dana Holzinger, Christa Flechtenmacher, Julia Thierauf, Annette Affolter, Peter Plinkert, Jochen Hess https://creativecommons.org/licenses/by/4.0
The emerging role of extracellular vesicles in clinical research inflammation, cancer progression, metastasis
The role of extracellular vesicles (EVs) in intercellular communication is being increasingly recognized in clinical research. Being shed from cells in an evolutionarily conserved manner, EVs transfer mediators from different biomolecule categories involving them in inflammation, maintenance of homeostasis, regulation of physiological functions, and the process of horizontal gene transfer. Based on their mechanism of cellular formation, size and cargo, EVs can be subdivided into exosomes, microvesicles (MVs), and bigger vesicles, such as oncosomes and apoptotic bodies. In contrast to exosomes, MVs are released by blebbing of the outer cellular membrane, thus sharing antigenicity with their cellular origin. MVs often express tissue factor (TF) and hence, potently activate the extrinsic pathway of the coagulation cascade. Moreover, TF+MVs induce cellular responses via the PAR2/G protein/ERK pathway, involving them in inflammation, cancer progression and metastatic spreading. In collaboration with several clinics of the UKSH, we are investigating the role of MVs in disease processes and their potential use as biomarkers, in particular (I) the characterization of MVs found in ascites of patients with ovarian carcinoma as markers for cancer progression and thrombosis (gynecology), (II) the suitability of urinary MVs from patients with bladder carcinoma as biomarkers for disease recurrence or progression (urology), (III) the importance of platelet-derived MVs from patients with head and neck tumors functioning as shuttle for clinically relevant biomolecules (e.g. PD-1, PD-L1) (otorhinolaryngology), (IV) the therapeutic and prognostic relevance of TF+MVs as PAR2/G protein activators and GPCR antibodies in rheumatic diseases (rheumatology). Purification of MVs from various body fluids is performed using sequential centrifugation steps; we have established an innovative purification method using annexin-V-coated magnetic beads. Detection, size determination and quantification of MVs are done by high-resolution flow cytometry and electron microscopy (SEM). MVs are characterized biochemically including their exposure of PS, expression of active TF, activation of the extrinsic TENase, as well as PAR2/Gprotein/ERK signaling and, downstream, induction of tumor cell migration as surrogate parameter for their metastatic potency. We plan to establish a translational research network at the UKSH on the emerging role of EVs in various diseases including cancer, and their interaction with inflammatory processes.
Copyright (c) 2018 Melanie Klee, Fanny Ender, Mario Kramer, Axel Merseburger, Ralph Pries, Gabriela Riemekasten, Achim Rody, Barbara Wollenberg, Frank Gieseler https://creativecommons.org/licenses/by/4.0
CD8+ T cell response to HPV16 E7 predicts survival outcome in oropharyngeal cancer
Immunological response to human papillomavirus (HPV) in the development and progression of HPV16+ oropharyngeal squamous cell carcinoma (OPSCC) (accounting for the majority of viral associated cases) is largely unknown and may provide important insights for new therapeutic strategies. In this prospective clinical trial, we examined cell-mediated immune responses to HPV16 E2, E6 and E7 in peripheral blood using IFN-? enzyme-linked immunosorbent spot assay. CD56(+), CD4(+), CD8(+) and regulatory T cell frequencies were also discerned by flow cytometry. Fifty-one study participants with oropharyngeal carcinoma were recruited. Control subjects were those undergoing tonsillectomy for benign disease. All patients were treated with curative intent by radiotherapy ± chemotherapy. Disease-specific survival was investigated by multivariate analysis. HPV16 DNA was detected in 41/51 of the OPSCC participants. T cell responses against HPV16 E6 or E7 peptides were detected in 33/51 evaluable patients, respectively and correlated with HPV status. Matched pre- and post-treatment T cell responses were available for 39/51 OPSCC cases. Within the whole cohort, elevated post-treatment CD8(+) response to HPV16 E7 correlated with longer disease free survival (multivariate DFS p < 0.03). Within the HPV + OPSCC cohort, a significant increase in regulatory T cells (p < 0.02) was noted after treatment. This is the first study to provide survival data in OPSCC stratified by cell-mediated immune response to HPV16 peptides. Within the HPV16+ OPSCC cohort, enhanced immunoreactivity to antigen E7 was linked to improved survival. An increase in regulatory T cell frequencies after treatment may suggest that immunosuppression can contribute to a reduced HPV-specific cell-mediated response.
Copyright (c) 2018 Holger Sudhoff, Liam Masterson, Piyush Jani, Jane Carolyn Sterling, Peter Goon https://creativecommons.org/licenses/by/4.0
On the development of personalized cancer vaccines
Cancer immunotherapy has experienced a breakthrough by the introduction of checkpoint inhibition with antibodies. Response appears to be correlated with mutation load; the majority of cancer patients are nonresponders, possibly due to absence of immunogenic neoepitopes. We analyze the entire detectable landscape of HLA ligands on tumor samples, consisting of 1000 through 5000 peptides per sample, and find dozens to hundreds of peptides in germline sequence with apparently tumor specific expression, based on the absence of these peptides on adjacent autologous benign tissue and absence on a large number of normal tissue samples from all organs and tissue types available for analysis. We have evidence that non-mutated HLA ligands with tumor specific expression should be efficient as targets for personalized antigen specific immunotherapy, in particular if used with an efficient adjuvant.
Copyright (c) 2018 Hans-Georg Rammensee https://creativecommons.org/licenses/by/4.0
Platelets regulate innate immunity in autoinflammatory diseases and cancer
The interleukin (IL)-1 family of pro-inflammatory cytokines are the most potent pyrogens in the body, and their excessive production can cause several auto- inflammatory syndromes, or contribute to a range of inflammatory and metabolic disorders. Furthermore, IL-1? is an important driver of tumor development and metastasis. The recent success of the first and largest randomized trial of the therapeutic monoclonal IL-1? antibody Canakinumab in human beings provided evidence that IL-1 is associated with increased incidence of fatal lung cancer. The expression of the key members of the IL-1 family, such as IL-1? a nd I L-18, is regulated at both the transcriptional and post-transcriptional levels. IL-1? a nd I L-18 are produced as inactive precursors, which require activation of caspase-1 by the inflammasomes for their maturation and release by from cells, occasionally at the cost of caspase-1-mediated cell death. We have recently discovered that inflammasomes are released into the extracellular space where they remain active after the demise of activated cells, and that extracellular inflammasomes can amplify inflammation by sustaining extracellular production of IL-1?. However, the sources of extracellular pro-IL-1? are not known. Recent advances in platelet proteomics have revealed that these non-nucleated cells are able to produce their own cytokines, including soluble IL-1? and membrane-bound IL-1?, and are able to significantly magnify IL-1 production by immune cells. As platelets outnumber leukocytes by several folds, they could potentially be the major source of extracellular inflammasomes in the body, or be a major producer of IL-1 precursors that are cleaved by extracellular inflammasomes released from dying immune cells. In this study, we investigated the mechanism(s) by which platelets produce IL-1, and the specific contribution of platelet- derived IL-1 to sterile inflammation, or host resistance to infection. We believe that a deeper understanding of platelet-IL-1 and their interaction with immune cells during sterile inflammation, or infection might help to uncover new targets for immune therapies.
Copyright (c) 2018 Bernardo Simoes Franklin https://creativecommons.org/licenses/by/4.0
A pan-cancer landscape of interactions between tumors and infiltrating immune cells
Throughout their development, tumors are challenged by the immune system and acquire features to evade its surveillance. A systematic view of these traits is still lacking. We recently performed a computational-based study in which we identified genomic and transcriptomic traits associated to the immune-phenotype of 9,403 tumors of 29 solid cancers. In highly cytotoxic immune-phenotypes we found tumors with low clonal heterogeneity enriched by alterations of genes involved in epigenetic regulation, ubiquitin mediated proteolysis, antigen-presentation and cell-cell communication, which may drive resistance. Tumors with immune-phenotypes with mid cytotoxicity present an over-activation of processes involved in invasion and remodeling of neighboring tissues that may foster the recruitment of immune-suppressive cells. Tumors with poor cytotoxic immune-phenotype tend to be of more advanced stages and present frequent alterations in cell cycle, hedgehog, beta-catenin and TGF-beta pathways, which may drive the immune depletion. These results may be exploited to better understand the outcome of immunetherapies and develop novel combinatorial targeting strategies.
Copyright (c) 2018 David Tamborero https://creativecommons.org/licenses/by/4.0
Rediscovering an old story: HLA class I alterations and immune escape of tumors in the context of immunotherapies
Despite the successful implementation of immunotherapies, the response rates of tumor patients are still limited and often resistances develop. This might be a result of the immune suppressive nature of the tumor microenvironment as well as the immune escape mechanisms of tumor cells. While several tumor infiltrating immune cell subpopulations can foster tumor development, exhibit immune suppressive activity and decrease the efficacy of effector immune cells, tumor cells have also developed multiple mechanisms to escape immune recognition and to modulate immune cell function. These include in particular abnormalities in the classical HLA class I antigen, the interferon (IFN) signal transduction pathways and overexpression of non-classical HLA antigens as well as of immune checkpoint molecules. These different strategies have recently been shown to be crucial for the efficacy of immunotherapies against solid tumors and might be associated with the acquired resistances to these treatment options. Thus, a deeper understanding of the key immune players and regulatory pathways of tumor cells involved in the complexity and dynamic interaction between tumor and immune cells might be important for the identification of prognostic factors and the reversion of the immune escape processes. This might lead to increased immune responses against cancer cells and avoidance of immune resistances in order to improve the efficacy of immunotherapies. HLA class I abnormalities frequently occur in tumors, which are associated with a worse prognosis of patients. These defects are mainly due to deregulation rather than structural alterations of HLA class I antigens and antigen processing machinery (APM) components. Reversion of loss or downregulated HLA class I APM component expression could be obtained by cytokine treatment, in particular IFN-?, but also by epigenetic drugs, proteoglycans, and anti-oxidant substances as well as microRNAs and RNA-binding molecules. Thus, the control of HLA class I APM expression is highly complex and this novel knowledge could be used for the design of immunotherapeutic approaches to enhance treatment efficacy.
Copyright (c) 2018 Barbara Seliger https://creativecommons.org/licenses/by/4.0
Biparatopic heavy chain antibodies (bi-hcAbs): fusion of two distinct CD38-specific nanobodies into a human IgG1 bi-hcAb results in potent CDC vs. myeloma cells
Purpose/Objectives: The purpose of this study was to generate CD38-specific heavy chain antibodies (hcAbs) with improved complement dependent cytotoxicity (CDC) against multiple myeloma cells. Daratumumab is an effective therapeutic mAb targeting CD38, an ecto-enzyme overexpressed on multiple myeloma and other hematological malignancies. Patients that become refractory to daratumumab have myeloma cells expressing increased levels of complement inactivating proteins (CD55, CD59), suggesting that CDC is a mechanism of action of this mAb. Materials/Methods: We fused the soluble variable domain (VHH or nanobody) of CD38-specific llama heavy chain antibodies (hcAbs) to the hinge-CH2-CH3 domains of human IgG1 (Fig. 1). In order to compare the CDC potency of daratumumab and nanobody-based hcAbs, we incubated CD38-expressing LP-1 myeloma and CA-46 Burkitt lymphoma cells with individual Abs or combinations of Abs in the presence of human serum as a source of complement and monitored cell death by flow cytometry. Results: Monospecific and bispecific hcAbs were produced at high yield as highly soluble proteins by transiently-transfected HEK-cells. While daratumumab showed a moderate potency to induce CDC toward LP-1 or CA-46 cells, individual hcAbs showed only little if any CDC potency. In contrast, combinations of two hcAbs recognizing distinct epitopes of CD38 potently induced CDC. Similarly, combining daratumumab with a hcAb recognizing a non-overlapping epitope of CD38 markedly increased its CDC potency. Remarkably, bispecific hcAbs containing nanobodies that recognize two non-overlapping epitopes of CD38 displayed even higher CDC inducing potency than daratumumab. Conclusion: Our results illustrate the therapeutic potential of chimeric nanobody-based human IgG1 heavy chain antibodies. The biparatopic CD38-specific hcAbs may represent alternatives to daratumumab for the treatment of hematological malignancies.
Copyright (c) 2018 Kerstin Schütze, Julia Hambach, Katharina Petry, William Fumey, Birte Albrecht, Levin Schriewer, Jana Röckendorf, Stephan Menzel, Friedrich Haag, Catelijne Stortelers, Peter Bannas, Friedrich Koch-Nolte https://creativecommons.org/licenses/by/4.0
Activation of MDA5 improves anti-tumor therapy and reduces suppressive capacity of myeloid-derived suppressor cells
Objective: Pancreatic cancer is infiltrated by stromal cell and suppressive immune cells, including myeloid-derived suppressor cells (MDSC). MDSC are key determinants for maintaining the immunosuppressive tumor microenvironment. Both, monocytic MDSC (M-MDSC) and polymorphonuclear MDSC (PMN-MDSC), are highly T cell suppressive. Stimulation of RIG-I-like helicases (RLH), such as melanoma differentiation antigen 5 (MDA5), induces a type I interferon (IFN)-driven immune response and induces an immunogenic form of tumor cell death in pancreatic cancer. The aim of the study is to assess the effects of RLH treatment, using the synthetic MDA5 ligand polyinosinic:polycytidylic acid (poly(I:C)), on the MDSC phenotype and function. Methods: C57BL/6 and IFNAR-/- mice were orthotopically injected with KPC-derived T110299 pancreatic cancer cells and treated i.v. with poly(I:C), complexed with PEI. After 21 days, mice were sacrificed and immune cells from tumors and spleens were analyzed by flow cytometry. PMN-MDSC (CD11b+ Ly6G+ Ly6Cint) and M-MDSC (CD11b+ Ly6G- Ly6C+) as well as bulk tumor biopsies were isolated for RNA-sequencing as well as for functional assays. RNAseq data were subjected to KEGG pathway analysis, Gene Set Enrichment Analysis. Results: Survival of poly(I:C)-PEI-treated mice with orthotropic pancreatic tumors was significantly prolonged, and a reduction of tumor size was observed after only two injections of poly(I:C)-PEI. The suppressive capacity of both, PMN- and M-MDSC populations was significantly reduced after poly(I:C)-PEI treatment. KEGG pathway analysis of RNA sequencing data revealed an enrichment of type I IFN signaling, virus-associated as well as antigen processing- and presentation-associated gene signature upon poly(I:C)-PEI treatment. In addition, flow cytometry confirmed the upregulation of MHC-I and CD86 surface molecules in MDSC-subpopulations. In IFNAR-/- mice, expression levels were unaltered pointing towards a type I IFN-mediated effect. CIBERSORT analysis of bulk tumor tissue pointed towards a reduction of the M2 phenotype, which was paralleled by increased expression levels of M1-related genes, after therapy. Conclusion: The survival benefit of RLH ligand-treated mice with pancreatic cancer correlated with the reduction in tumor size, reduced T cell suppressive capacity of MDSC and functional reprogramming towards a M1/G1 phenotype. Type I IFN signaling induced by RLHbased immunotherapy has the potential to revert tumor induced immune suppression.
Copyright (c) 2018 Sabrina V. Kirchleitner, Philipp Metzger, Michael Kluge, Lars M. König, Stefan Endres, Caroline C. Friedel, Max Schnurr, Peter Duewell https://creativecommons.org/licenses/by/4.0
Effect of both PD-1/PD-L1 and ERK inhibitor on Cisplatin-sensitive (HEY) and Cisplatin-resistant (HEY-Cis) ovarian cancer cell lines
Objectives: Diagnosis of ovarian cancer is associated with adverse prognosis caused by late diagnosis, a high recurrence rate and a fast emerging chemotherapy resistance. The goal was to identify a novel therapy approach for treatment of especially chemo resistant ovarian cancer by combination of a PD-1/PD-L1 and an ERK inhibitor. Methods: After single and combined inhibitor treatment the concentration of 50 % reduction of cell viability (IC50) was determined in HEY and Hey-Cis by MTS assay. Impact on MAP kinase pathway was checked by immunoblot. QPCR experiments were performed to prove expression ratios of PD-L1, IL8, IL6, cFos, FosL und VEGFC after treatment. IL8 secretion was checked by ELISA. Results: The PD-1/PD-L1 inhibitor provoked 50 % reduction of cell viability with concentrations of 3.92 ?M in HEY and 4.03 ?M in HEY-Cis. For the ERK inhibitor IC50 values were determined of 4.73 ?M in HEY and of 1.96 ?M in HEY-Cis. Combined treatment effected a synergistic inhibition of proliferation. ERK and S6 were dephosphorylated and the expression of c-Myc was decreased by single and especially combined inhibitor treatment. Gene expression of IL8 and IL6 was strongly increased in HEY while diminished HEY-Cis after ERK inhibitor treatment. Additionally, cFos, FosL and VEGFC expression were reduced in both cell lines. The ELISA confirmed a strong secretion of IL8 in HEY cells after ERK inhibitor treatment. Conclusion: Both inhibitors reduced cell viability and acted synergistically when combined. Intracellular changes mainly occurred by the ERK inhibitor and provoked a stronger effect on HEY-Cis. The reduction of genes involved in inflammatory processes in HEY-Cis could suggest the use of a MAP kinase inhibitor combined with an immunotherapeutic therapy approach by existing Cisplatin resistance.
Copyright (c) 2018 Frank Köster, Paulina Vogt, Stephan Polack, Achim Rody, Karen Bräutigam https://creativecommons.org/licenses/by/4.0
Effect of both PD-1/PD-L1 and ERK inhibitor on triple negative breast cancer cell lines and the non-triple negative cell line MCF-7
Objectives: Diagnosis of triple negative breast cancer (TNBC) is associated with adverse prognosis. The goal was to identify a novel therapy approach for treatment of TNBC cell lines by combination of a PD-1/PD-L1 and an ERK inhibitor. Methods: After single and combined inhibitor treatment the concentration of 50 % reduction of cell viability (IC50) was determined in three TNBC cell lines and the non-TNBC cell line MCF-7 by MTS assay. Impact on Jak/Stat and MAP kinase pathway and apoptosis induction by inhibitor was checked by immunoblot. QPCR experiments were performed to prove expression ratios of PD-L1, CDK5, Notch1, IL-8, IL-6, CXCL1 und CXCR2 after treatment. Results: The PD-L1/PD-1 inhibitor provoked 50 % reduction of cell viability with concentrations of 1.26 to 2.25 ?M in TNBC and 3.20 ?M in MCF-7. For the ERK inhibitor IC50 values were determined of 4.42 to 6.03 ?M in TNBC and of 20.44 ?M in MCF-7. Combined treatment effected a synergistic or additive inhibition of proliferation. The MAP kinase ERK and S6 were dephosphorylated and expression of Stat3 was increased by ERK inhibitor. Gene expression of IL6 and CXCR2 were repressed by combined treatment. Conclusion: Though the use of the PD-1/PD-L1 inhibitor and the ERK inhibitor showed contrary effect on phosphorylation of various proteins of the Map kinase signaling pathway, the combined treatment provoked a synergistic or at least additive inhibition of proliferation especially in TNBC and a reduced expression of genes involved in inflammatory processes.
Copyright (c) 2018 Frank Köster, Elodie Kabore, Stephan Polack, Achim Rody, Karen Bräutigam https://creativecommons.org/licenses/by/4.0
Regulatory T cells in endogenous mouse lymphoma are antigen-specific nTregs and provide a target for immune checkpoint-inhibiting therapies
Purpose/Objectives: Foxp3+ regulatory T cells (Tregs) play an important role in maintaining immune homeostasis. In malignant disease, however, Tregs contribute to the generation of an immunosuppressive microenvironment. To establish therapeutic approaches it is necessary to uncover activating and suppressive mechanisms. Immune checkpoint-blocking (ICB) monoclonal antibodies (mAbs) have been shown to elicit cancer regression and long-lasting tumor control. Particular interest was focused to the mutual influence of Treg cells and therapy by ICB. Materials/Methods: We used a c-MYC transgenic mouse model of spontaneously arising B-cell lymphoma, which mirrors key features of human Burkitt lymphoma. To assess treatment effects mice were intraperitoneally injected with mAbs against PD-1 and CTLA-4. Mice splenocytes and lymphocytes were analyzed by flow cytometry. Results: An augmented fraction of CD4+Foxp3+Tregs was detected in spleens and lymph nodes of tumor-bearing c-MYC mice. Tregs were involved in suppressing antitumor response because specific ablation of Tregs significantly delayed tumor development. Compared to wildtype mice, c-MYC Treg cells showed an activated phenotype as evidenced by upregulated CD69 and CD137. The high expression of the costimulatory molecule CD137 indicated TCR-specific activation. As intratumoral Tregs were predominantly Nrp-1+Helios+nTregs, we suggested that Tregs recognized tumor-associated self-antigens. We identified MHC class II-restricted self-epitopes, which were prevalent in lymphoma as compared to normal B cells and could be recognized by Treg cells resulting in enhanced proliferation (Ki-67) in vitro. Interestingly, effector T cells (Teffs) were able to recognize the same epitopes. Antigen contact apparently led to upregulation of IL-10. In vitro suppression assays showed that Tregs from c-MYC and wt mice potently suppress proliferation of Teffs in a dose-dependent manner. When preventing cell contacts, Teff proliferation was increased and almost restored upon additional IL-10 neutralization. Tregs from c-MYC mice treated with anti-CTLA-4/anti-PD-1 mAbs revealed a lower suppressive capacity. Conclusion: In malignant lymphoma, nTregs directed against self epitopes are involved in cancer immune escape. Taken together, the Treg population is a promising target in future immunotherapies and needs to be further investigated.
Copyright (c) 2018 Fatima Ahmetlic, Vera Bauer, Tanja Riedel, Nico Trautwein, Tim Sparwasser, Stefan Stevanovic, Martin Röcken, Ralph Mocikat https://creativecommons.org/licenses/by/4.0
Specific targeting of oncogenic mutations using CRISPR-technology to destroy activated oncogenes and enhance immune response
Purpose/Objectives: In recent decades, molecular biology tools have enabled researchers to modify the genome of human cells. Today, many methods can be employed to inactivate genes, add them or change their expression. However, survival rates for patients affected by highly malignant cancer types have not improved substantially. If methodology like genome editing, which is routinely used in research laboratories, would be available to clinicians, the patients’ prognosis could be strongly improved. Materials/Methods: CRISPR-Cas9 is a method to precisely target and manipulate genomic loci and recent studies have attempted to use this method as a treatment for genetic diseases including cancer. Some of these approaches target mutant genomic sequences specifically and try to avoid affecting the respective normal loci. We combined knowledge from recent advances in CRISPR-technology with data from cancer genome sequencing studies to develop a concept for the CRISPR-mediated targeting of recurrent or individual mutations in activated oncogenes. Results: We present strategies to target mutations in activated oncogenes in a mutation-specific way using CRISPR-technology. The induction of DNA double strand breaks in key locations of oncogenic drivers is likely to abolish or reduce their oncogenic potential. CRISPR-molecules effective against highly recurrent mutations might be usable off-the-shelf for all patients with that mutation. Clinical electroporators could improve the delivery of CRISPR-molecules into tumor cells after local or systemic injection. Moreover, when combined for example with checkpoint inhibitors, CRISPR-targeting might enhance the immune-recognition of tumor cells and/or decrease the expression of ligands reducing cellular immune responses. However, considering obvious genetic risks opposing the objected benefits, this approach has to be evaluated carefully in preclinical studies. Conclusion: We believe that the mutant-specific targeting of oncogenes activated by mutation can be achieved employing the latest CRISPR-technology and that this approach could be combined with immunotherapy. Current publication: Oppel F, Schürmann M, Goon P, Albers AE, Sudhoff H. Specific Targeting of Oncogenes Using CRISPR Technology. Cancer Research. 2018 Sep 7. PubMed PMID: 30194069.
Copyright (c) 2018 Matthias Schürmann, Felix Oppel, Holger Sudhoff https://creativecommons.org/licenses/by/4.0
Integrin ?4 synergizes with E-and P-selectin to regulate tumor growth: Unravelling the molecular mechanisms
Purpose/Objectives: Metastases are responsible for 90% of cancer-related deaths. Scientific evidence has accumulated showing that adhesion molecules including selectins and integrins play a crucial role in the process of cancer metastasis. In particular, upregulation of integrin ?4 (ITGB4) is associated with tumor progression in various tumor entities. Thus, we aimed at detecting a potential crosstalk between selectins and integrins in tumor formation and metastatic spread. Material/Methods: Knockdown of ITGB4 in PC3 (prostate cancer), PaCa5061 (pancreatic cancer) and SKOV3 (ovarian cancer) cells using shRNA, proliferation and colony forming assays, ELISA, WB, 3D chemotaxis assays with human macrophages, tumor initiation assays in vivo, immunohistochemistry Results: Knockdown of ITGB4 affected prostatic PC3, pancreatic PaCa5061 and ovarian SKOV3 cancer xenografts already at the primary tumor site. In particular, tumor growth was strongly inhibited when the ITGB4-knockdown was combined with E-/P-selectin deficiency of mice while selectin-knockout alone had no effect. In this study we wanted to investigate the molecular mechanisms underlying this synergistic growth retardation. Delayed tumor growth was associated with increased apoptosis. Immunostainings further revealed an enhanced immune cell infiltration in ITGB4-knockdown tumors grown in immunodeficient mice. On the contrary, leukocytes were mainly located at the outer tumor periphery of both ITGB4-knockdown and control tumors in E-/P-selectin-knockout mice. The increased leukocyte infiltration in ITGB4-knockdown tumors in control mice seemed to result from a greater chemotactic potential of ITGB4-knockdown cells. CCL2, 5 and 20 were identified as potential chemotactic factors upregulated upon ITGB4-knockdown in PaCa5061 and SKOV3 cells while the chemoattractant playing a role in ITGB4-knockdown PC3 cells remains to be determined. Conclusion: Taken together, our results suggest that ITGB4-knockdown tumor initiation depends on pro-tumoral signals from attracted tumor-associated leukocytes. These leukocytes appear to depend on endothelial selectins for infiltration of the tumor stroma. Investigation of how leukocytes actually stabilize the survival of ITGB4-knockdown xenografts is the subject of ongoing experiments.
Copyright (c) 2018 Vera Labitzky, Sandra Hanika, Florian Gebauer, Daniel Wicklein, Hanna Maar, Tobias Gosau, Jennifer Schröder-Schwarz, Stefan Linder, Kristoffer Riecken, Udo Schumacher, Tobias Lange https://creativecommons.org/licenses/by/4.0
Primary and recurrent head and neck squamous carcinomas are strikingly different regarding their immune microenvironment
Purpose/Objectives: The tumor immune microenvironment (TIME) has a crucial impact on cancer progression and patients´ survival for various cancer entities. In head and neck squamous cell carcinoma (HNSCC) the development of recurrences in up to 60% is one main factor leading to poor prognosis. We aimed to reveal changes in the TIME of primary tumours and their corresponding recurrences, hypothesising that recurrent HNSCC have successfully escaped anti-tumor immune response. Materials/Methods: The TIME of 70 formalin fixed paraffin embedded (FFPE) tissue samples of HNSCC primary tumours and their corresponding local recurrences was characterised via immunohistochemical staining for the markers CD4, CD8, CD20, FOXP3, CD1A, PD1, CD68 and CD56 and semi-automated quantification with Definines software. RNA was extracted using the Maxwell® 16 LEV RNA FFPE Purification Kit for a subgroup of 18 patients, who underwent (chemo-)radiation therapy between the resection of the primary tumour and the development of a local recurrence. RNA expression levels of 770 immune related genes were identified by nanostring nCounter® PanCancer Immune Profiling Panel and assessed with the nSolver™ Analysis Software. Results: The immunohistochemical TIME analysis demonstrated a loss of CD20+ B lymphocytes (p= 0.0006), an increase in CD1A+ dendritic cells (p= 0.017) and a decrease in the CD8/FOXP3 T-cell ratio (p= 0.106) in HNSCC recurrences compared to their corresponding primary tumours. Nanostring analysis confirmed the depletion of B lymphocytes and additionally revealed a strong decrease in the total number of tumor infiltrating lymphocytes (TILs) as well as an increase in dendritic cells, mast cells, neutrophils and macrophages in HNSCC recurrences. In summary, 141 genes of the nanostring panel were identified to be significantly differentially expressed with the majority being downregulated in the HNSCC recurrences. Significantly downregulated chemokines included CXCL13 and CXCR5, which are involved in B-cell chemotaxis; whereas one of seven upregulated genes, Osteopontin, plays a major role in myeloid cell chemotaxis. Conclusion: Our results reveal significant differences in the TIME of HNSCC primary tumours and recurrences, characterised by a loss of B lymphocytes and an overall shift from antitumor immune response to an increase in pro-tumor immune factors in recurrences.
Copyright (c) 2018 Helen Pasternack, Christian Watermann, Christian Idel, Sven Perner, Rosemarie Krupar, Julika Ribbat-Idel, Mark Philipp Kühnel, Danny Jonigk, Johannes Brägelmann https://creativecommons.org/licenses/by/4.0
The role of NFATc1 in T cell-mediated immune responses during the development of lung cancer
Purpose/Objectives: NFATc1 (nuclear factor of activated T cells 1) belongs to the NFAT family of transcription factors and regulates T cell activation as well as effector and cytotoxic T cell functions in many tissues. In most of the established tumors, inhibitory receptors like programmed cell death protein 1 (PD-1) contribute to the functional impairment of T cell activation by persistent tumour antigen challenge, a process that is called T cell exhaustion. Cancer immunotherapies are aimed to reawake exhausted T cells by blocking inhibitory checkpoint receptors or immunosuppressive cells. As NFATc1 is important for T cell activation we asked whether this factor could be important for the reactivation of exhausted T cells in non-small cell lung cancer (NSCLC). Materials/Methods: We analyzed NFATc1 and its interplay with PD-1 in a human NSCLC patient cohort using qPCR, IHC and FACS. Furthermore, in a murine model of lung adenocarcinoma, we determined lung tumour growth in mice with a conditional inactivation of NFATc1 in T cells (NFATc1?CD4) and analyzed the influence of anti-PD-1 antibody treatment on NFATc1 protein levels in T cells of lung-tumour bearing wild-type mice. Results and Conclusion: In this study, we report a progressive decrease of NFATc1 in lung tumor tissue and in tumor-infiltrating lymphocytes (TIL) of patients suffering from advancedstage NSCLC. NFATc1?CD4 mice showed increased lung tumor growth associated with impaired T-cell activation and function. Furthermore, in the absence of NFATc1, reduced IL2 influenced the development of memory CD8+ T cells. Specifically, we discovered a reduction in effector memory and CD103+ tissue-resident memory (TRM) CD8+ T cell numbers in the lung of tumor-bearing NFATc1?CD4, underlining an impaired cytotoxic T-cell response and a reduced TRM tissue-homing capacity. Targeting PD-1 resulted in NFATc1 induction in CD4+ and CD8+ T cells in tumor-bearing mice and was associated with increased antitumor cytotoxic functions. Thus this study reveals a role of NFATc1 in the activation and cytotoxic functions of T cells, in the development of memory CD8+ T-cell subsets, and in the regulation of T-cell exhaustion. These data underline the indispensability of NFATc1 for successful antitumor immune responses in patients with NSCLC.
Copyright (c) 2018 Juliane Friedrich, Lisanne Heim, Marina Engelhardt, Denis I. Trufa, Carol I. Geppert, Ralf J. Rieker, Horia Sirbu, Susetta Finotto https://creativecommons.org/licenses/by/4.0
Role of glycolysis for intestinal inflammation and carcinogenesis
Purpose/Objectives: Hexokinase (HK) catalyzes the first step of glycolysis and thereby determines the total glycolysis rate. HK and glycolysis are important for inflammation and carcinogenesis. Immune and cancer cells have elevated HK levels and an increased glycolysis rate to fuel their energetic demand. Inhibition of HK in cancer cells is protective. We showed previously that the microbiome modulates HK expression [1] and activity in intestinal epithelial cells (IECs). Now, we aim to test whether ablation of HK in the intestine protects from inflammation and colon cancer and whether modulation of HK through the microbiota could be a novel therapeutic option. Materials/Methods: To that end, we generated HK-IEC mice lacking HK specifically in IECs and subjected them to experimental models of intestinal inflammation (dextran sulfate sodium, DSS) or colitis-associated carcinogenesis (azoxymethane-DSS, AOM-DSS). We also analysed the microbiome composition using 16S amplicon sequencing and transcriptional responses using RNA sequencing. We investigated the regulatory function of specific bacteria on HK using gnotobiotic mouse models and in vitro stimulation experiments. Finally, we investigated HK expression in patients with intestinal inflammation and colon cancer. Results: HK-IEC mice showed reduced susceptibility to intestinal inflammation. Susceptibility to colitis-associated carcinogenesis is currently being evaluated. We identified specific bacteria and metabolites, which modulate HK expression. Finally, we found that HK is dysregulated during intestinal inflammation and in tumours of colon cancer patients. Conclusion: Inhibition of epithelial glycolysis protects from acute intestinal inflammation and potentially colitis-associated carcinogenesis. Probiotic administration of HK-regulating bacteria or their modulation via prebiotics might serve as a novel non-invasive approach for inflammation and cancer therapy. Reference [1] Sommer, F., I. Nookaew, N. Sommer, P. Fogelstrand, and F. Backhed, Site-specific programming of the host epithelial transcriptome by the gut microbiota. Genome Biol, 2015. 16(1): p. 62. Funded by Deutsche Forschungsgemeinschaft CRC1182 “Evolution and Function of Metaorganisms” and Excellence Cluster 306 “Inflammation at Interfaces”.
Copyright (c) 2018 Kenneth Klischies, Jacob Hamm, Nina Sommer, Philip Rosenstiel, Felix Sommer https://creativecommons.org/licenses/by/4.0
MALDI-imaging: Identification of protein signatures of genomic instability in colorectal cancers
Background: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has improved over the years and is increasingly being used for biomarker discovery directly from human tissue. Since DNA aneuploidy has been identified as a prognostic factor for epithelial malignancies, we compared diploid and aneuploid colon cancer tissues of the colorectum by IMS. Material and Methods: DNA image cytometry determined the ploidy status of tissue samples that were subsequently subjected to MALDI-IMS. After obtaining protein and peptide profiles through direct analysis of both, fresh frozen and FFPE tissue sections, a discovery and a validation set were used to predict ploidy and disease status by applying proteomic classification algorithms. Identification of m/z values were performed by using nUPLC mass spectrometry from an adjacent tissue section. Clinical target validations were performed by immunohistochemistry using in-house compiled tissue microarrays (TMA). Results: Classification algorithms categorized normal mucosa and colon carcinoma as well as diploid and aneuploid colon cancers correctly. Proteins could be identified to be differentially expressed between diploid and aneuploid samples that showed expression differences also in clinical samples using a tissue microarray of normal mucosa, diploid and aneuploid colorectal carcinomas. Conclusion: Our data underscore the potential of MALDI-IMS proteomic algorithms for individualized medicine to reveal significant molecular details from distinct tumor subtypes such as different ploidy types.
Copyright (c) 2018 Oliver Klein, Timo Gemoll, Elena Miroll, Christoph Thorns, Jens Habermann https://creativecommons.org/licenses/by/4.0
Investigating a potential role of Got1 in T cell exhaustion.
Purpose/Objectives: During cancer and chronic infections, T cells can become exhausted and gradually lose their effector functions. Although clinical studies targeting immune checkpoint molecules (e.g. PD-1) lead to promising results, not all patients can benefit from that kind of treatment, indicating the mechanism of T cell exhaustion is much more complex and has to be understood in more detail in order to develop better therapies. Regulation of T cell exhaustion is not only mediated by immunosuppressive receptors, but also by cell intrinsic metabolic changes. The cytoplasmatic enzyme Glutamic-oxaloacetic transaminase 1 (Got1) plays an important role during amino acid metabolism and energy production in the cell. Since Got1 has been found to be upregulated during T cell exhaustion, our research aims to investigate a possible role of Got1 in this process. Materials/Methods: To study the T cell-intrinsic role of Got1, we use transgenic mouse lines with Cre-loxP recombination. We are using CD4-Cre*Got1flox/flox mice to study Got1 deficiency in CD4+ and CD8+ T cells. Additionally, we use P14*CD4-Cre*Got1flox/flox mice, which bear a transgenic TCR and allow us to perform adoptive transfer experiments to investigate the CD8+ T cell-intrinsic function of Got1. The mice are challenged with Lymphocytic choriomeningitis virus strains Armstrong or cl-13 to induce acute or chronic viral infections, respectively. Results: Got1-deficient T cells develop and proliferate normal in naïve mice. First preliminary results indicate that, after chronic viral infection, Got1-deficiency resulted in a reduction of EOMES/TCF7 high “stem cell-like” T cells. This population has previously been demonstrated to be needed for a sustained long-term chronic immune response [1, 2] Conclusion: Our preliminary studies of Got1 during T cell exhaustion indicate an importance for this enzyme during the long term chronic immune response. By maintaining the pool of “stem cell-like” T cells that give rise to effector T cells, maintaining the long term chronic immune response, Got1 might positively influence T cell function during chronic stimulus. It will be interesting to see in the future, whether the deficiency of Got1 will also impair T cell function during cancer development. References [1] Im, S.J., et al., Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature, 2016. 537(7620): p. 417-421. [2] Utzschneider, D.T., et al., T Cell Factor 1-Expressing Memory-like CD8(+) T Cells Sustain the Immune Response to Chronic Viral Infections. Immunity, 2016. 45(2): p. 415-27.
Copyright (c) 2018 Nina Weisshaar https://creativecommons.org/licenses/by/4.0
Current status of radiotherapy/radiochemotherapy and immunotherapy combinations– a German survey
Purpose/Objetives: Combinations of immunotherapy with radiation or radiochemotherapy are currently under investigation in several cancer types and clinical settings. In this survey, we solicited members of radiation oncology institutions in Germany to review the current status of research in this field, underline critical issues such as oncological benefit, treatment toxicity and obstacles in clinical research. Method: We surveyed radiation oncology institutions in Germany via an anonymous online questionnaire sent by e-mail to members of the German Radation Oncology Society and yDEGRO (working group of DEGRO e.V.). The questions were based on current studies at their institutions, clinical settings (cancer type, cerebral metastasized situation), emphasizing the oncologic benefit and obstacles of clinical research. Results: We received a total of 26 responses (61.90% response rate). Of which 23 were completed and returned, hence eligible for further evaluation. The responders represent 14 different departments of radiation oncology at German university hospitals. 14 (60.90%) responders perform radio-immunotherapy studies at their institutions and participate in multicenter studies. Radio-immunotherapy combinations were investigated mainly in head & neck tumors (95%), lung cancer (57%), malignant melanoma (48%) and tumors of the upper GI tract (8.7%). Combinations of radiochemotherapy with immunotherapy were only tested in head & neck cancers (52%), non-small cell lung cancer (NSCLC) (8.70%) and malignant melanoma (4%).The oncologic benefit of radio/radiochemotherapy with immunotherapy is assumed to be effective or very effective by >85% of all responders. Regarding possible obstacles to clinical research of radio/radiochemo-immunotherapy combinations, 21 (91.3%) responders mentioned lack of financial resources, 18 (78.3%) responders shortage of human resources, 10 (43.5%) responders lack of sufficient evidence and 6 (26.1%) lack of ethics committee approval. Conclusion: Radioimmunotherapy combinations are under investigation at >75% of all participating centers. Head & neck tumors, NSCLC and malignant melanoma are the most frequently tested cancer types. The treatment of intracranial metastatic disease by the combination of radiotherapy and immunotherapy is assumed to be very effective by the majority of responders. Several obstacles for clinical research were collated. The results of ongoing prospective trials are eagerly awaited and our survey shows great acceptance of new combinations of radio/radiochemo-immunotherapy.
Copyright (c) 2018 Lukas Käsmann, Chukwuka Eze, Olarn Roengvoraphoj, Claus Belka, Farkhad Manapov https://creativecommons.org/licenses/by/4.0
Activation of immune response in refractory patients to standard treatment (T.R.A.N.S.L.A.T.E.)
Purpose/Objectives: The TRANSLATE project started in 2016 to test the immune effects of metronomic cyclophosphamide, daily low-dose IL-2 every other week, and a single flash of radiotherapy (RT) in peripheral blood. The rationale is based on self vaccination induced by radiotherapy (RT) 8 Gy single fraction on one metastatic lesion, T cells expansion by IL-2 treatment and selective Tregs down regulation by lowdose cyclophosphamide administration. Materials/Methods: We enrolled patients with end-stage breast, colon, kidney and prostate cancer. Analysis was performed at baseline, the day after RT, after 28 days from treatment start and at disease progression. Assay focused on Tregs, CD8+, NK, MDSC, CD3-PD1, IL-2, IL-4, IL-5, IL- 6, IL-10, IL-12, IL-13, IL-17a, TNF?, IFN?, TGF?. We divided patients into two groups depending on the time of disease progression (A > 3 months; B, < 3 months). We report preliminary data with the aim to show the changes observed post-RT in 20 pts. Results: At baseline, group B had higher rates of CD3-PD1, higher IL-2, IL-13, TNF?, and TGF?; group A had higher IFN ?, IL-4, and IL-12. After RT, we observed a difference between the two groups in the rates of CD3-PD1 (lower in group B) and Tregs (higher in group A). Among cytokines, only TNF? reached a statistical significance (higher in group B). We also observed that TGF? and IL-6 were higher in group B and IFN? was higher in group A. The longitudinal analyses showed that CD3-PD1 remained stable between basal and post-RT in group A but decreased in group B. Tregs marginally increased in group A. TNF?, TGF?, IL-4, IL-6, and IL-12 increased in group B. IL-4 decreased in group A and IL-6 and IL-12 also marginally decreased in the same group. IFN? slightly increases in group A. Discussion: The limited number of patients reduced the interpretation of the study. However, following RT a positive trend of Th2 cytokines is observed in patients with early progressing disease, without the expected surge of IFN?? that was instead observed in patients with better outcome. Additional analyses are in progress and will be presented.
Copyright (c) 2018 Andrea Abbona, Dario Sangiolo, Marco Merlano, Ornella Garrone, Merlotti Anna, Martino Monteverde, Cristiana Lo Nigro, Antonella Falletta, Marcella Occelli, Chiara Varamo, Massimo Aglietta, Loretta Gammaitoni, Alessia Reali https://creativecommons.org/licenses/by/4.0
Which adjuvants for tumor-antigen vaccination induce powerful non-sialylated IgG antibodies?
Tumor-antigen vaccination is a promissing approach to induce intensive T and B cell and IgG antibody responses for eliminating the tumor. The effector functions of IgG antibodies depend on their subclass and Fc glycosylation pattern. Non-fucosylation and non-sialylation of tumor-antigen-specific IgG antibodies enhance their affinity to activating Fcgamma receptors on immune cells and enhance tumor elimination. Accordingly, it has been suggested that tumor-antigen vaccination should induce non-fucosylated and non-sialylated tumor-antigen-specific IgG antibodies. We have analyzed, which adjuvants induce or not non-sialylated IgG antibodies and what are the responsible mechanisms and have identified an IL-17 dependent mechanism for the generation of non-sialylated IgG antibodies. We conclude that adjuvants interesting for tumor-antigen vaccination can be selected by their potential to induce non-sialylated IgG antibodies.
Copyright (c) 2018 Yannic Bartsch, Marc Ehlers https://creativecommons.org/licenses/by/4.0
Killing melanoma cells with ROS provides immuno-protection against tumor growth in vivo
Purpose/Objectives: Immunogenic cancer cell death (ICD) delivers inflammatory stimuli to elicit anti-tumor immune responses. Release of adjuvant find-me (HMGB1, ATP) and eat-me (calreticulin; CRT) signals enables DC maturation and cross presentation of tumor antigen to T cells. Effective ICD-inducing therapies are drugs such as anthracyclines, ionizing irradiation, or photodynamic therapies. Intriguingly, these antitumor strategies show concomitant generation of reactive oxygen species (ROS) that can drive cell death and immunogenicity signaling responses through redox relays. In cancer cells, the importance of ROS are underappreciated in the onset of ICD. Materials/methods: A novel technology, cold physical plasma, was used to generate various ROS that can be effectively delivered to B16F10 melanoma cells. The oxidation and ATP levels as well as expression levels of MHC class I and CRT were measured. Plasma-treated tumor cells were injected into syngeneic mice with a subsequent re-challenge with live cells. Mitomycin C (MMC) and mitoxantrone (MTX) served as negative and positive control, respectively. Lymph node cells of sacrificed mice were co-cultured with melanoma cells, and T cell activation was assessed. Results: Cold physical plasma oxidized melanoma cells and led to an increase of ATP release as well as MHC class I and CRT expression. Mice “vaccinated” with plasma-treated melanoma cells showed less tumor growth compared to mice receiving MMC-treated cells but more tumor growth compared to positive control MTX. Activation of lymph node-derived T cells ex vivo with melanoma cells was best in MTX mice, followed by the cells from the plasma group, and was lowest in cells from the MMC group. Conclusion: Not only the event of cell death but also its auxiliary signals are important in the formation of anti-tumor immune responses. ROS play a significant role in this process. Deep phenotyping of ROS-treated melanoma cells will decipher the molecular machinery important in eliciting the responses observed.
Copyright (c) 2018 Eric Freund, Ramona Clemen, Rajesh Kumar Gandhirajan, Sander Bekeschus https://creativecommons.org/licenses/by/4.0
Pre- and intratherapeutic cellular monitoring for dose adjustment of chemotherapy in aggressive lymphoma
Purpose: In eukaryotic cells treated with chemotherapeutic drugs DNA double strand breaks (DSB) are the most severe form of DNA damage which usually leads to apoptotic cell death. Cytotoxic chemotherapy is still a mainstay of cancer therapy, but there are few established methods to individually predict efficacy and tolerability. In most regimens dosage is based on the body surface area, a dimension of little biological precision, and individual dose adaptions are usually performed by clinical guessing or in response to adverse events. This project sets out to examine biological parameters of cell damage. Methods: In particular, we measure DNA double strand breaks by an automated yH2AX assay applying the Aklides NUK® system. In preliminary work, PBMCs were stimulated with bendamustine or with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, the components of the R-CHOP protocol, to induce DSB. Results: There was no inter-individual correlation between drug dose and number of foci, whereas does titrations on the cells of each individual donor demonstrated a clear correlation between dose and yH2AX assay result. Conclusion: The lack of correlation across individuals between yH2AX foci and drug dosage based on standard calculation by body surface area suggests that standardized dosing of chemotherapeutic drugs based on gross physical determinants such as weight or body surface area does not correspond to the individual’s biological response and clinical effect. The future plan of this project is the replacement of standard dosage regimens by biologically based, real-time-adapted individual dosage in lymphoma to reduce toxicity while increasing therapeutic efficacy.
Copyright (c) 2018 Julia Schröder, Stefan Rödiger, Madeleine Ruhe, P. Markus Deckert https://creativecommons.org/licenses/by/4.0
Combining anti-VEGF-A and anti-PD-L1 therapy prolongs overall survival in small cell lung carcinomas
Purpose/Objectives: SCLC accounts for approximately 15% of newly diagnosed lung cancer cases, is driven by RB1 and TP53 lesions and represents the most aggressive pulmonary carcinoma. Current chemotherapies are initially effective in SCLC patients, however tumors rapidly reoccur and patients die within a few months. Results from KEYNOTE-028 revealed that PD-L1 positive late stage SCLC patients who received a human-ized monoclonal anti-PD-1 antibody, showed 25% response rates. This finding is remarkable for SCLC, since targeted therapies for this tumor entity are rare. However, most SCLC patients harbor a primary resistance or acquire resistance during treatment by an immune suppressive microenvironment. Thus, there is a critical need to combine immune checkpoint inhibitors with other therapies to overcome these resistances. Materials/Methods: We implemented a combined therapy concept including anti-VEGF-A and anti-PD-L1 monoclonal antibodies in an autochthonous SCLC mouse model. As a read out, we used X-ray computed tomography, flow cytometry and end-point immunohistochemistry. Results: We found that combined anti-VEGF-A/anti-PD-L1 treatment synergistically improved overall survival, compared to monotherapies, and enhanced T cell infiltration into the tumor. Moreover, we identified in murine SCLC tumors which acquired resistance to anti-PD-L1, a significant increase in PD-1+/TIM-3+ exhausted CD4 and CD8 T-cells accompanied by significantly increased expression of Galectin-9, the major TIM-3 ligand, on TAMs. In addition, we observed a significantly enriched fraction of TAMs with a similar exhausted phenotype indicated by the expression of LAG-3 and CTLA-4 upon acquired resistance to PD-L1 blockade. Importantly, the exhaustion of T-cells and TAMs was reverted upon combined anti-VEGF-A/anti-PD-L1 therapy. In line with the results obtained from the mouse model, we showed mechanistically in PBMCs of SCLC patients, that VEGF-A triggers co-expression of PD-1 and TIM-3 on T-cells, indicating an immunosuppressive function of VEGF-A in SCLC patients during anti-PD-1/PD-L1 therapy. Furthermore, we elucidated using CRISPR-Cas9 technology, that VEGF-A liberated from the tumor microenvironment is the likely mediator of acquired resistance to PD1/PD-L1 blockade in SCLC. Conclusion: Taken together, in SCLC there is evidence for a substantial benefit of implementing combined anti-angiogenic and anti-immune checkpoint therapy approaches in the clinic in order to overcome acquired resistances delivered by T-cells and macrophages in the tumor microenvironment. Remarks: The authors declare no conflicts of interest. This work was published in Cancer Res. 2018 Aug 1;78(15):4270-4281. doi: 10.1158/0008-5472.CAN-17-2176. Epub 2018 May 18 with the title 'Combined VEGF and PD-L1 Blockade Displays Synergistic Treatment Effects in an Autochthonous Mouse Model of Small Cell Lung Cancer'.
Copyright (c) 2018 Lydia Meder, Philipp Schuldt, Martin Thelen, Anna Schmitt, Felix Dietlein, Sebastian Klein, Sven Borchmann, Kerstin Wennhold, Ignacija Vlasic, Sebastian Oberbeck, Richard Riedel, Alexandra Florin, Kristina Golfmann, Hans A. Schlößer, Margarete Odenthal, Reinhard Büttner, Jürgen Wolf, Michael Hallek, Marco Herling, Michael von Bergwelt-Baildon, H. Christian Reinhardt, Roland T. Ullrich https://creativecommons.org/licenses/by/4.0
Robust GMP manufacturing process with IL-2 and a glycoprotein antibody cocktail generates highly active human NK cell batches for cancer immunotherapy
Purpose/Objectives: NK cells are innate immune cells crucial for killing of infected and malignant cells. They are able to fight circulating tumor cells thereby preventing metastases formation which account for approximately 90% of all cancer-deaths. Moreover, infiltration of solid tumors with NK cells has been shown to correlate with a better prognosis for the patient. Thus, NK cells became interesting candidates for cancer immunotherapy and ex vivo manipulation and expansion of highly potent NK cells for adoptive transfer in patients is an aim of paramount importance. Using a novel approach for NK cell expansion, we aimed at providing an extensive characterization of the generated cells to evaluate their potential for cancer immunotherapy. Materials/Methods: Human, CD3+ T cell-depleted PBMCs were expanded in a bioreactor with IL-2 and a glycoprotein antibody cocktail following GMP guidelines. Cells were analyzed for NK cell purity, expression of different chemokine receptors, cell adhesion molecules, activating receptors and death ligands as well as IFN? production using flow cytometry. Further, cytotoxicity towards different tumor cell lines was assessed via LDH assays and flow cytometry-based degranulation assays. Results: Upon expansion NK cell purity reached 85 to 96%. The cells showed expression of the chemokine receptors CXCR3, CXCR4 and CCR7 and the cell adhesion molecules Lselectin, LFA-1 and VLA-4. Further, they expressed the activating receptors NKp30, NKp44, NKp46, NKG2D, DNAM-1 and CD16, the death ligands Fas and TRAIL and produced IFN?. NK cells showed cytotoxicity towards the tumor cell lines K562 (leukemia), PaCa5061 (pancreatic cancer), and SKOV3 (ovarian cancer). Conclusion: We describe a novel approach for ex vivo NK cell expansion generating a set of highly potent NK cells which represent promising candidates for cancer immunotherapy. The GMP manufacturing process allows the use of these cells in clinical trials, i.e. adoptive NK cell transfer in patients.
Copyright (c) 2018 Udo Schumacher, Katharina Bröker, Ralf Pörtner, Hans Hoffmeister, Evgeny Sinelnikov, Stefan Lüth, Werner Dammermann https://creativecommons.org/licenses/by/4.0
Progression of head and neck cancer is associated with elevated neutrophil extracellular traps formation by circulating neutrophils
Neutrophil extracellular traps (NETs) represent web-like structures consisting of externalized nuclear and mitochondrial DNA decorated with granule proteins. NETs were discovered as structures responsible for distant trapping and killing of bacteria. However, there are also undesirable effects of NETosis during carcinogenesis; such as facilitating of metastasis by anchoring circulating tumor cells to the endothelium. Importantly, tumor cells are able to support this process by expressing multiple neutrophil activating factors, such as G-CSF. Since neutrophils are known to negatively influence the course of head-and-neck cancer (HNC), as their increased numbers are associated with adverse patient prognosis, we addressed changes in neutrophil activity during HNC progression. To this end we assessed NETs production by isolated circulating neutrophils of head and neck cancer (HNC) patients. Percentage of NET-positive neutrophils and the mean area of NETs were estimated in HNC patient specimen (n=37) versus healthy donors (n=10). It was correlated with tumor stage, lymph node metastatic load and the expression of tumor derived factors. While there were no significant differences in spontaneous NETosis, bacteria-stimulated NET release was significantly elevated in T1-T2 and N0-N2 stages, compared to healthy controls. Interestingly, NETosis decreased in advanced cancer (T3-4, N3). Of note, we also observed a strong expression of G-CSF by tumor tissue and a positive correlation between G-CSF levels and NETosis. This is in agreement with our observation that G-CSF stimulates NETs formation in vitro. Additionally, we analyzed the expression of CD11b, the component of beta 2 integrin, which mediates human neutrophil adhesion and migration, as well as interactions between neutrophils and tumor cells. CD11b was up-regulated on blood neutrophils in HNC T2-T3 stage and its expression correlated with G-CSF levels released by tumor tissue. Altogether, based on the above results we suggest that NETosis is significantly elevated in patients prone to lymph nodes metastasis and could be used as a biomarker predicting metastatic spread in HNC. Therapeutic disruption of NET formation or neutrophilic adhesion may offer new roads for treatment of HNC patients in order to prevent metastasis.
Copyright (c) 2018 Infinite Science Publishing https://creativecommons.org/licenses/by/4.0
Tumor and host factors regulating anti-tumor immunity and immotherapy efficacy
Two major phenotypes of human melanoma metastases have been observed based on gene expression profiling and confirmatory assays. One subgroup of patients has a T cell-inflamed phenotype that includes expression of chemokines, T cell markers, and a type I IFN signature. In contrast, the other major subset lacks this phenotype and appears to display immune “exclusion”. The mechanisms of immune escape are likely distinct in these two subsets, and therefore the optimal immunotherapeutic interventions necessary to promote clinical responses may be different. The T cell-inflamed tumor microenvironment subset shows the highest expression of negative regulatory factors, including PD-L1, IDO, and FoxP3+ Tregs. Deep analysis of tumor antigen-specific T cells in the tumor microenvironment has identified additional mechanisms of immune dysfunction and new potential therapeutic targets. Treatment strategies targeting several pathways have been translated back into the clinic, with anti-PD-1/PD-L1 agents being FDA approved for 11 different cancer entities. Combinations with new agents based on the biology of T cell dysfunction are ongoing. In contrast to the T cell-inflamed melanomas, non-T cell-inflamed tumors are largely immunotherapy resistant with current approaches. Natural innate immune sensing of tumors appears to occur via the host STING pathway, type I IFN production, and cross-priming of T cells via CD8a+ DCs, and these factors are absent in non-T cell-inflamed tumors. New strategies are being developed to engage or mimic this pathway as a therapeutic endeavor, including STING agonists. The molecular mechanisms that mediate the absence of the T cell-inflamed tumor microenvironment in patients are being elucidated using parallel genomics platforms. The first oncogene pathway identified that mediates immune exclusion is the Wnt/b-catenin pathway, which argues that new pharmacologic strategies to target this pathway should be developed to restore immune access to the tumor microenvironment. Recent evidence has indicated that host factors, including the intestinal microbiota, are also critical. We recently have identified commensal bacteria in mouse models that augment spontaneous anti-tumor immunity and increase efficacy of anti-PD-L1 therapy. Similar analyses in human melanoma patients revealed commensal bacteria associated with anti-PD-1 efficacy. These results have prompted the pursuit of new probiotics that may improve spontaneous immune infiltration and expand immunotherapy efficacy in the clinic.
Copyright (c) 2018 Thomas F. Gajewski https://creativecommons.org/licenses/by/4.0
Human leukocyte antigens and haplotypes allow for prognostic classification of HNSCC patients
Background: Genetic instability generates somatic mutations which are mostly controlled by the immune system but defects in the T-cell system accompany higher cancer incidences. T-cell responses to tumor-associated antigens (TAA) including neoantigens, oncogenes and viral proteins depend on binding and proper presentation of TAA-derived peptides by human leukocyte antigen (HLA) class I (HLA-A, B, Cw) and II proteins (DR, DQ, DP). Particular HLA antigens and their combinations (haplotypes) may facilitate immune escape of cancer caused by their inability to bind and present such peptides to T cells. We recently demonstrated a link between HLA alleles and haplotypes (HLA-traits) and the development of head and neck squamous cell carcinoma (HNSCC) and varying progression-free survival (PFS) of HNSCC patients. Methods: We developed a HLA-score abstracting from complexity of HLA-typing results to facilitate potential use of HLA-associated hazard ratios (HR) for prognostic stratification. The HR for PFS of 8 HLA-traits shown to be independent predictors (Pi) of PFS in a test cohort (TC) of 90 patients[1] were used to build the HLA-score based on the Pi-associated HR.[2] Crude ln-transformed HR of the alleles HLA-B*13 (2), B*35 (1), B*51 (2), DQB1*06 (1), homozygous Cw (1), homozygous DRB4 (2), and haplotypes A*01/B*08 (-6) and B*08/C*07 (4), were summed up to define the individual patient’s HLA-score. Results: Receiver operating characteristic (ROC; P = 0.0000034) and Kaplan-Meier curves for PFS demonstrated the prognostic significance of the HLA-score (AUC=0.750, CI 95% 0.665-0.836; 70/90 patients in TC with HLA-Score >0 had reduced PFS (P=0.001)[2]. Applying the same classifier (HLA-score >0) to an independent validation cohort (iVC) of 32 DeLOS-II patients confirmed these findings by demonstrating reduced PFS of 25/32 patients (P=0.040)[2). Recent research demonstrates that HLA-traits constitute critical Pi in other cancers (e.g. melanoma) and predicts response to immune-checkpoint inhibitors. Conclusion: Considering HLA for prognostic stratification could reduce outcome variance in clinical trials. The HLA-score may facilitate the use of genetic information from HLA-typing. References [1] Wichmann et al., Oral Oncol. 2017; 69: 115-127; http://dx.doi.org/10.1016/j.oraloncology.2017.04.017. [2] Wichmann et al., Front. Oncol. 2018; 8: 168; http://dx.doi.org/10.3389/fonc.2018.00168.
Copyright (c) 2018 Proceedings on Cancer-Immuno-Oncology https://creativecommons.org/licenses/by/4.0
Breaking the brake: targeting the immunosuppressive cells to optimize immunotherapy of cancer
Introduction: Immune suppression is a physiological phenomenon resulting from the cooperation of many actors, both humoral and cellular, which continuously interplay each other leading to the impairment of immune response. Immune suppression is necessary to avoid tissue damages and, ultimately, life-threatening situations which could be induced by the maintenance over time of the immune response. Unfortunately, during neoplastic disease, immune suppression favors tumor progression. Cell mediated immune suppression: Immune response starts with the antigens recognition by APCs, their presentation to naïve T-cells and the reactive T-cells generation. Tumor has profound influence on the DCs maturation, and immature or partially matured DCs are largely represented in most tumors and mediate immune suppression. A second key-point leading to the impairment of the immune response is the capability of effector T cells to cross the vascular endothelium to reach their targets. Thus, endothelial cells may represent powerful mediators of immune suppression, selecting the populations of cells which can cross the barrier and preventing homing, or directly killing those that must be stopped. The third and most important area of immune-suppression is the local tumor microenvironment (TME) where the plasticity of immune cells and stromal cells allows the polarization toward a Th2 response. Indeed, tumor cells may directly secrete immune suppressive cytokines and chemokines prone to impair immune attack. In addition, the persistence of inflammation changes in the TME, such as pH modification and hypoxia, may impair the activity of effector T cells, both directly and indirectly. Targeting of immunosuppressive cells: There are two main opportunities to target immunosuppressive cells. It is possible target specific cell lines directly using classic anticancer drugs. For example cyclophosphamide and gemcitabine, can directly target Treg and MDSC respectively. It is also possible to exploit the plasticity of immune cells to reprogram them towards a Th1 response. For example, inhibition of PD-1/PD-L1 axis prevents Tregs (iTregs) induction leading to the reduction of this population into the TME. Translational data on targeting immunosuppressive cells will be discussed.
Immunotherapy and head and neck cancer: Focus on recurrent and metastatic disease
The treatment for recurrent and metastatic head and neck cancer is limited and prognosis is poor. Survival is limited to months and patients have significant and severe symptoms and poor quality of life. Recent phase II and III studies have shown modest benefit in survival when using checkpoint blockade in this setting with most of the studies focusing on patients heavily pretreated with chemotherapy. Initial biomarker work from these studies support a potential benefit for biomarker selection with PDL-1 and TMB with patients showing an increased and more sustained response when selected for these biomarkers. The overall response rate in an unselected patient population is less than 20 percent and that could potentially be doubled with a more robust biomarker selected patient population. the ideal ”immunologic profile” to select patients remain elusive though and more work is necessary to answer this question. The addition of chemotherapy to immunotherapy is an interesting concept and could play a role in augmenting the response rates to immunotherapy but data in head and neck cancer is lacking at this stage. Recently disclosed data in the first line setting is very encouraging and could lead to immunotherapy replacing chemotherapy in the first line recurrent and metastatic disease. the focus on a “combined immunologic score“ as a strong biomarker for survival is in keeping with what the second line data was hinting at. Going forward we expect more data looking at combination immunotherapy agents and more biomarker driven studies as is currently being done in a number of solid tumors. It has become increasingly clear that the single agent, unselected patient approach, is not necessarily ideal to improve overall outcome for the head and neck cancer patient and more work is needed to identify responders early on so that we can avoid unnecessary and costly treatments for those who are not going to benefit from this approach.
Adaptive immunotherapy using redirected chimeric antigen receptor (CAR) T cells against leukemia has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products leads to a manufacturing challenge regarding automation, process robustness, and cell storage. In this respect our results from a study for relapsed Melanoma regarding manufacturing of CAR T cells in a closed and automated system gives rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies. In contrast to T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease, which makes them a promising source for third-party-donor immunotherapy. However, tumor cells can escape NK cell immunosurveillance by tumor immune escape mechanisms (TIEMs). In order to overcome TIEMs and to make NK cell-based therapies more specific, we engineered primary human NK cells to express a CAR designed to recognize CD19 or CD123, which are highly expressed on the surface of primary acute lymphoblastic or myeloid leukemia, respectively. NK cells were transduced with state-of-the-art alpharetroviral self-inactivating (SIN) vectors encoding EGFP alone as control or a second or third generation CAR engineered with an anti-CD19 or anti-CD123 single chain variable fragment (scFv) and containing the CD28 transmembrane domain, the 4-1BB costimulatory domain, the CD3? signaling domain and an internal ribosomal entry site (IRES) element for EGFP expression. CAR-modified NK cells showed a strongly improved cytotoxicity against leukemic cells compared to activated NK cells with a nearly complete elimination of leukemic cells after 48 h. Moreover, our side-effect studies demonstrated minimal or no cytotoxicity of CAR NK cells against PBMNCs and lung epithelia cells, respectively. Since autologous T cells cannot consistently be expanded ex vivo for all patients, third-party allogeneic CAR NK cells as an “off the shelf product” may serve as an alternative strategy. Since NK cells have a significantly shorter lifespan than T cells, off-tumor toxicity might be reduced. However, for durable anti-tumor effects immature CAR NK cells or repeated cell infusions of mature CAR NK cells might be necessary.
Copyright (c) 2018 Ruth Esser, Ulrike Koehl, Wolfgang Glienke, Michael Morgan, Andrew Kaiser, Hinrich Abken, Michael Heuser, Krasimira Aleksandrova, Jana Leise, Olaf Oberschmidt, Christoph Priesner, Lubomir Arseniev, Axel Schambach, Stephan Kloess https://creativecommons.org/licenses/by/4.0
Immune biological rationale for the design of radioimmunotherapies
Radiotherapy primarily aims to induce tumor cell death. However, it has become evident that it also impacts on the tumor cell phenotype and its micro-environment thereby exerting both immune suppression and activation. Therefore, a deeper understanding of how radiotherapy modulates the immune system is mandatory for the design of radioimmunotherapies. The talk will focus on immune modulations by radiotherapy, taken also into account the impact of radiation dose and timing of combination with immunotherapies on it. Immune biological challenges for combining radiotherapy with additional immune modulators are discussed. Based on the inflammatory status of a tumor, the dynamics and the interconnections of the immune system and the tumor are outlined. Finally, genetic and immune biomarkers for radiation responses are specified for improvement of radioimmunotherapies. It can be concluded that local and systemic antitumor immune responses can be induced by radiotherapy, but predominantly only in combination with additional immune modulations.
Copyright (c) 2018 Udo S. Gaipl https://creativecommons.org/licenses/by/4.0
Role of resident memory T cells in cancer immunotherapy
Recent studies show that tissue resident memory T cells (TRM) are emerging as essential actors involved in tissue immunosurveillance. This role partly relies on their specific expression of adhesion molecules (CD103 or CD49a integrins, chemokine receptors) facilitating their retention into tissues and consequently into tumor. Their well-positioned location in close contact with tumor cells, their enrichment in tumor specific T cells, as well as their higher cytotoxic capacities explain that their tumor infiltration is correlated with good clinical outcome in many cancers. We and other groups have shown that the efficacy of cancer vaccines depends on their ability to elicit TRM. In adoptive cell therapy, the transfer of cells with the ability to establish TRM at the tumor site correlates with the potency of this therapy. Interestingly, TRM express immune checkpoint molecules and preliminary data showed that they could expand early during anti-PD-1 treatment, and thus be considered as a surrogate marker of response to immunotherapy. Overall, TRM cells appear to represent important components in cancer immunology both as a potential biomarker of response to immunotherapy and as critical effector cells required for the success of various immunotherapy approaches.
Copyright (c) 2018 Eric Tartour https://creativecommons.org/licenses/by/4.0
Perspectives of human gamma/delta T-cells in cancer immunotherapy
?? T-cells comprise a numerically small subset of CD3-positive T-cells in peripheral blood but occur at increased frequencies in mucosal tissues. The major subset of human blood ?? T-cells expresses a V?9V?2-encoded T-cell receptor (TCR) which specifically recognizes pyrophosphate intermediates of the cholesterol synthesis (mevalonate) pathway from microbes and tumor cells. Many tumors have a dysregulated mevalonate pathway and produce increased levels of intermediates (e.g., isopentenyl pyrophosphate, IPP) which are then specifically sensed by ?? T-cells. Recognition of such pyrophosphates is not dependent on HLA presentation but requires the butyrophilin member BTN3A1 (also termed CD277). Due to their HLA-independent killing of many different tumor cells, ?? T-cells have recently raised great interest as potential effector cells for cell-based immunotherapy. In some studies, however, ?? T-cells have also been reported to actually exert pro-tumorigenic activities. I will discuss anti- and pro-tumorigenic activities of ?? T-cells with a focus on strategies how to improve the efficacy of tumor cell killing by human ?? T-cells. This may include the development of bispecific antibodies targeting ?? T-cells to tumor-associated cell surface antigens and the design of other strategies to specifically enhance the cytotoxic capacity of ?? T-cells. On the other hand, it will be important to counteract known inhibitory mechanisms of the tumor microenvironment such as e.g. prostaglandin E2 and certain galectins. Furthermore, the sensitivity of some tumor cells towards ?? T-cell mediated cytotoxicity can be improved by epigenetic modifiers such as histone deacetylase inhibitors. The overall goal is to improve cytotoxic effector activity of ?? T-cells (and therefore to pave the way to clinical application) by enhancing cytotoxic mechanisms while simultaneously inhibiting suppressive mechanisms.
Copyright (c) 2018 Dieter Kabelitz https://creativecommons.org/licenses/by/4.0