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Cancersymposia.no

HARNESSING INNATEAND ADAPTIVE IMMUNITY IN

In 1975 Kohler and Milstein solved a technical problem that scientists had been battling for years – how to produce large amounts of antibodies in the laboratory in such a way that all antibody-molecules were identical. This invention earned them the Nobel price in 1984. In the wake of their discovery, hopes were high that antibodies could be used to kill cancer cells in patients. The fact that this has not become a reality for large patient groups until the last decade, shows that patience and persistence are two important ingredients in successful translational research.
The development has, however, accelerated the last few years with CARs and a number of other new technologies that manipulate the immune system into showing its power to cure cancer. Although no single cure will ever exist, immunotherapy will probably play a key role in the majority of therapeutic regimens for cancer in the future.
Immunotherapy was long regarded safe but ineffective. Now we begin to see what the immune system can do to eradicate cancer when unleashed, sometimes at a cost. We want to learn more, taking risks in a safe way, like the ski jumpers throwing themselves down the steep slope and over the edge, but landing on their feet.
We thank you for contributing to making this symposium a successful event and hope that you will enjoy the creative optimism in the field as much as the view from Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Radiumhospitalet and the University of Oslo SESSION 1 - UNLEASHING SPECIFIC CYTOTOXIC T CELLS
Chairs: Per Thor Straten Herlev Hospital, Denmark & Kjetil Taskén University of Oslo, Norway
Ton Schumacher The Netherlands Cancer Institute, The Netherlands
What T cells see on human cancer Cassian Yee MD Anderson Cancer Center, University of Texas, USA
Adoptive T Cell Therapy of Cancer: Combination Strategies Mads Hald Andersen Herlev Hospital, Denmark
Self-reactive effector T cells: Suppressing the suppressors SESSION 2 - FINDING AND DIRECTING THE RIGHT TCRs
Chairs: Erik Thorsby University of Oslo and Oslo University Hospital, Norway & Annika Scheynius Karolinska Institutet, Sweden
Hans Stauss University College London, UK
TCR Gene Therapy of Cancer Johanna Olweus University of Oslo and Oslo University Hospital, Norway
Allo-reactive T cells to characterize and target the self-immunopeptidome Rolf Kiessling Karolinska Institutet, Sweden
Immunotherapy of malignant melanoma Short Talks 10 min each with 5 min discussion
Stalin Chellappa: Dual phenotypic regulatory T cells in human pancreatic carcinoma
Jon Amund Kyte: Spacer design strongly influences the in vivo activity of CD19-specific
Chimeric Antigen Receptor redirected T cells
Rikke Andersen: Adoptive cell therapy with tumor infiltrating lymphocytes for patients with
metastatic melanoma
Petter Woll: Genetic fate-mapping of human cancer stem cells
Social program
Get-together at The Holmenkollen Arena
SESSION 3 - HARNESSING NK CELL DIVERSITY IN CANCER THERAPY
SESSION 5 - ENHANCING T-CELL FUNCTION BY GENETIC ENGINEERING
Chairs: John Torgils Vaage University of Oslo and Oslo University Hospital, Norway & Erik Dissen University of Oslo, Norway
Chairs: Gustav Gaudernack Oslo University Hospital, Norway & Geir Tjønnfjord University of Oslo and Oslo University Hospital, Norway
Jeff Miller University of Minnesota, USA
Cedrik Britten BioNTech, Mainz, Germany
Exploiting NK Cells for Cancer and Transplantation Personalized RNA-based Immunotherapy Approaches Dean Anthony Lee MD Anderson Cancer Center, University of Texas, USA
Martin Pule University College London, London, UK
Initial report of adoptive immunotherapy trials delivering IL-21-expanded NK cells Advanced T-cell engineering for Cancer Applications Karl-Johan Malmberg University of Oslo and Oslo University Hospital, Norway
Magnus Essand Uppsala University, Uppsala, Sweden
Adoptive NK cell therapy against myelodysplastic syndrome T cells that are insensitive to oxidative stress and immunosuppressive cytokines SESSION 4 - IMMUNE REGULATION AND SYSTEMS IMMUNOLOGY
SESSION 6 - CD40, CD4 AND DC; MANY WAYS TO THE SAME GOAL
Chairs: Ton Schumacher The Netherlands Cancer Institute, The Netherlands & Petter Höglund Karolinska Institute, Sweden
Chairs: Cedrik Britten BioNTech, Mainz, Germany & Ludvig Munthe Oslo University Hospital, Norway
Petter Brodin Karolinska Institutet, Sweden
Thomas Tötterman Uppsala University, Uppsala, Sweden
Monitoring immune systems by Mass Cytometry Local CD40 agonist immunotherapy of cancer- from bench to bedside Fridtjof Lund-Johansen Oslo University Hospital, Norway
Bjarne Bogen University of Oslo and Oslo University Hospital, Norway
Array-based proteomics for identification of immune targets in cancer How do CD4+ T cells eliminate tumor cells that lack MHC class II molecules? Andreas Lundqvist Karolinska Institutet, Sweden
Iver Langmoen Oslo University Hospital, Norway
Suppression of NK cell responses in patients with cancer An autologous dendritic cell-based vaccine against glioblastoma multiforme Short Talks 10 min each with 5 min discussion
Short Talks 10 min each with 5 min discussion
Nadir Kadr: Dendritic cells dictate natural killer cell education at steady state
Alexandre Corthay: Adoptive transfer of tumor-specific Th2 cells induces an inflammatory reaction at
the tumor site resulting in cancer eradication
Trevor Clancy: An in silico dissection of immune cell activity in tumors
Sébastien Wälchli: Invariant chain as a vaccination vehicle for colorectal cancer
Poster session all topics
Social program
Barbecue at Holmenkollen Park Hotel
SESSION 7 - GOING CLINICAL BY CAR, TIL OR INI (Intra Nodal Immunotherapy)
Chairs: Gunnar Kvalheim Oslo University Hospital, Norway & Karl-Johan Malmberg Oslo University Hospital, Norway
Keynote address: Carl June University of Pennsylvania, Philadelphia, USA
Engineered T cells for cancer therapy Per Thor Straten Herlev Hospital, Denmark
Melanoma therapy using adoptive transfer of expanded tumor infiltrating T cells; can it get any better? Arne Kolstad Oslo University Hospital, Oslo, Norway
Sequential intra-nodal immunotherapy induces systemic anti-tumor immunity and regression of dis-seminated follicular lymphoma – a phase I/II study Poster no: 1
Poster no: 3
Dual phenotypic regulatory T cells in human pancreatic carcinoma
Adoptive cell therapy with tumor infiltrating lymphocytes for patients with metastatic melanoma
University of Oslo
Center for Cancer Immune Therapy
Co-authors:
Co-authors:
Stalin Chellappa, Harald Hugenschmidt, Gro Wiedswang, Kjetil Tasken, Einar Martin Aandahl
Marco Donia, Troels Holz Borch, Eva Ellebæk, Trine Zeeberg Iversen, Per Kongsted, Mads Hald Andersen, Per thor Straten, Inge Marie
Human Regulatory T cells (Tregs) express the signature transcription factor FOXP3 and represent 5-10% of total peripheral CD4+T cells. Tregs ensure protective immunity by resolving inflammation through suppressing the effector functions of various immune cells. Impaired Background: Adoptive cell therapy with tumor infiltrating lymphocytes (TILs) achieved impressive clinical results in several single institution function of Tregs is linked to autoimmune disorder and severe inflammatory diseases, whereas over-reactive Tregs may contribute to phase I/II clinical trials performed outside Europe, and holds the promise to enter the mainstream of standard melanoma care in the near the suppression of tumour immunity. Tumour infiltrating Tregs usually dampen the anti-tumour immune responses initiated by tumour future. However, although transient, the toxicities associated with high-dose IL-2 classically administered together with TILs are severe and surrounding T cells. Therefore, analysis of FOXP3+ Tregs has proved to be an interesting prognostication tool in various cancers. High levels recent results have questioned its use. To this end, we have initiated a trial using TILs after classical lymphodepletion but followed by an of FOXP3+ Tregs correlate with poor prognosis in various malignancies including pancreatic adenocarcinoma. However, for some cancers attenuated regimen of IL-2 the inverse has also been found. Heterogeneity and phenotype changes of Tregs under inflammatory conditions and also location of the Materials and methods: In an ongoing phase I feasibility study (NCT00937625), we enrolled and treated 27 pts with progressive FOXP3+ Tregs could possibly explain some of these conflicting observations in different cancer types, which warrant more detailed analyses metastatic melanoma, PS ≤1, age <70, at least one resectable metastasis and no CNS involvement. TILs infusion was preceded by standard of tumour infiltrating FOXP3+ Tregs. FOXP3+ can also be induced in effector T cells upon continuous antigen exposure in the tumour lymphodepleting chemotherapy but followed by low-dose subcutaneous IL-2 for 14 days (6 consecutive pts), or by an intravenous microenvironment. Here, we report our preliminary phenotypic analysis of Tregs from pancreatic adenocarcinoma patients with dual intermediate dose IL-2 decrescendo regimen (21 consecutive pts). phenotype of co-expressing both RORγt and FOXP3 transcription factors. Expression of RORγt marks Th17 cell lineage that is implicated Results: The lower doses of IL-2 considerably decreased the toxicity of the treatment, and imaging evaluations showed the achievement in many inflammatory diseases. Co-occupancy of both transcription factors in the same cell is questioned due to FOXP3 mediated of four complete responses and seven partial responses (24 patients evaluated). Clinical responses were associated with high numbers of repression of RORγt locus in regulatory T cells. Thus, co-expression of these transcription factors in Tregs from pancreatic cancer patients tumour reactive T-cells infused. Importantly, in most responding patients we observed induction and durable persistence of anti-melanoma may represent a phenotype switch towards Th17 cells or it may represent separate ontogeny with in Treg cell lineage with inflammatory T-cell responses in the peripheral blood.
phenotype. Thus, further analysis of signature cytokine and chemokine receptors are required to mark this subset of Tregs as cancer Conclusions: A high response rate including durable complete responses can be induced after treatment with TILs followed by an promoting inflammatory Treg subsets.
attenuated regimen of IL-2, which reduced the occurrence of severe side effects. Effective TIL treatment is associated with induction and long-term persistence in the blood of T cells producing in vitro anticancer responses. By showing that TIL-based ACT is logistically feasible and accessible to medium-size academic centers, we open the possibility for testing this treatment in a large randomized setting in Europe. Poster no: 2
Spacer design strongly influences the in vivo activity of CD19-specific Chimeric Antigen Receptor
Poster no: 4
redirected T cells
Dendritic cells dictate natural killer cell education at steady state
Jon Amund Kyte
Oslo University Hospital, Dept. for Cell Therapy
Nadir Kadri
Co-authors:
Hilde Almåsbak, Even Walseng, Aleksandr Kristian, Marit Renée Myhre, Else Marit Suso Inderberg, Ludvig Munthe, Gunnar Kvalheim,
Co-authors:
Nadir Kadri
We are developing T cell therapy against cancer using biodegradable chimeric antigen receptors (CARs), based on transient CAR expression Studies have demonstrated that the anti-tumour effect of natural killer (NK) cells can be employed therapeutically in mice and in patients after mRNA-electroporation. Severe side effects reported in several CAR T cell trials point to a need to develop safer methods for testing with several cancers. NK cells acquire effector functions via a developmental process termed "licensing". This process is dictated by NK cell new receptors. The mRNA approach may be useful for this purpose and may also be preferable in settings where a temporary treatment expression of inhibitory Ly49 receptors, which bind to self-MHC class I molecules on most nucleated cells in the body. When, where and period is desirable, e.g. up front of allogeneic bone marrow transplant. Herein, we investigated therapy with transiently redirected T cells which cell types that exert influence on NK cell licensing is still under vigorous debate. Because Dendritic cells (DCs) are known to support in a xenograft NSG mouse model. We constructed a series of CD19-specific CARs based on the same fmc63 single chain fragment variable, homeostasis of NK cells, and also prime NK cells during responses to viral and bacterial pathogens, we postulated that NK-DC cross-talk but with different spacers and co-stimulatory domains (CD28, OX40, or CD28-OX40). The CAR constructs all conferred T cells with potent plays a role in NK cell education. To test this, we punctually and specifically ablated DCs in the CD11c.DOG mouse model. DCs depletion CD19-specific activity in vitro. Unexpectedly, the constructs incorporating a commonly used IgG1-CH2CH3 spacer showed lack of anti- led to a decrease in effector functions in vitro and in vivo already at 4 days after depletion. Adoptive transfer of DCs, but not B cells, to leukemia activity in vivo and induced severe, partly CD19-independent toxicity. By contrast, identical CAR constructs without the IgG1- DCs-depleted mice restored NK cell functions. Our data suggest that MHC-I and IL-15 on the same DCs was required to maintain NK cell based spacer eradicated leukemia in vivo, without notable toxicity. We further demonstrated that the toxicity and lack of efficiency was function. Furthermore, the absolute number of NK cells decreased after 6-8 days post DC depletion, which was paralleled by a significant related to the CH2-domain in the spacer, which harbours the binding site for Fcγ-receptors (FcγR). There is substantial crosstalk between increase in proliferation of immature NK cells. High resolution flow cytometry analysis of inhibitory and activating receptors at this stage human immunoglobulins and murine FcγR. We speculate that binding of motifs in the spacer to murine FcγRs may bring CAR T cells over showed alterations in the NK cell repertoire, dominated by an increase in the frequency of Ly49 receptor-null NK cells as well as a decrease their threshold for activation induced cell death. Our findings point to a need for tailoring the spacer for each CAR construct and highlight of some activating receptors. Taken together, our data suggest that continuous interactions with DC in vivo is required to maintain the NK the importance of testing new CARs in vivo. Moreover, the results show that transiently modified T cells are capable of controlling leukemia cell repertoire and to secure NK cell function; properties that determine the ability of NK cells to kill tumor cells and MHC-deficient cells.
in vivo and support the rationale for developing an mRNA-based platform for clinical use.
Poster no: 5
Poster no: 7
An in silico dissection of immune cell activity in tumors
Invariant chain as a vaccination vehicle for colorectal cancer
Oslo University Hospital
Co-authors:
Co-authors:
Trevor Clancy, Eivind Hovig
Else Marit Suso Inderberg, Ana Kucera, Tone Fredsvik Gregers, Marit Renée Myhre, Gustav Gaudernack, Gunnar Kvalheim, Oddmund
Numerous studies in recent years have identified key patterns of immune activity associated with patient outcome for several cancer types. Immune cell activity in tumors, linked to tumor destruction, often corresponds with the immune cell infiltration profile of We and others have previously shown that the HLA-class II scaffolding CLIP peptide of invariant chain (Ii) had the faculty to bind HLA-class the tumor. Immunotherapy strategies can be guided by knowledge of such infiltration activity by activated immune cells .We have I. We further replaced it with an HLA-class I antigenic peptides (Ii-pep) and showed that the loading of this peptide on HLA-class I occurred. developed a computational approach to monitor and gauge the immune cell activity in tumors and link these patterns to the immune- When this construct was produced as mRNA and electroporated in dendritic cell (DC), it evoked a specific T-cell response, suggesting that mediated oncogenic signaling networks in the tumor cell. Presently to quantify the immune component of a tumor one must rely on the Ii-pep had the potential to work like a vaccination vector. With the aim of bringing Ii-pep construct closer to the clinic, we focused our study identification of the immune factors by laborious wet-lab efforts. To address this challenge, we grade the immune component of cancer on a therapeutically relevant peptide, namely the peptide derived from a TGFbRII frameshift mutation. Indeed, this mutation is observed in transcriptomes based on a computational score for several different immune cell types (Cytotoxic T cells, Th1, Th2, NK cells, dendritic cells, 76% of colorectal cancer patients with microsatellite instability (MSI). It leads to a change in the coding sequence which, when processed macrophages, granulocytes, etc.). To this end, we created a genome-wide ranked immune subtype relevance score for all human genes, by the proteasome machinery, generates a peptide presented by the common MHC-class I HLA-A*0201 (-A2). Since this peptide is a neo- developed using combined approaches in text mining and network biology. With immune subtype scores for each human gene in hand; antigen, it is able to induce an efficient immune response as demonstrated in 10/11 patients vaccinated with a similar synthetic peptide. we explore signatures from patient expression profiles in melanoma to demonstrate the power of applying this computational strategy to We here show that Ii-TGFbRIIp construct has the ability to load HLA-class I and stimulate specific CD8 T cells. Furthermore, in order to identify prognostic markers linked to the role of immunity in cancer progression. New avenues for exploring the dynamics of immune cell increase the potency of Ii-pep, we (1) mutated the Ii scaffold and selected for improving mutations and (2) tested the possibility to expand infiltration during cancer progression, and to explore the consequence for cancer immunotherapy, may arise through the provision of this the HLA coverage using larger peptides. One of the mutants we tested was efficiently transported directly from TGN to late proteolytic endosomes and had therefore a shorter half life compared to Iiwt, suggesting a different protolytic processing of the antigen. Preliminary experiments testing HLA-Class I antigens presentation demonstrated that this sorting mutant elicited a stronger T-cell response. We also generated Ii constructs with longer peptides derived from the frameshift and present here evidence that their potency was kept for CD8 T cell activation and furthermore that CD4 T cells were also stimulated. This suggests that both HLA-Class I and II were loaded when long peptides was introduced in the Ii molecule. We are now performing the remaining set of experiments before submitting a clinical application of the Ii-TGFbRII construct as a vaccine against MSI positive colorectal cancer. Poster no: 6
Adoptive transfer of tumor-specific Th2 cells induces an inflammatory reaction at the tumor site
resulting in cancer eradication
Poster no: 8
University of Oslo
Regulation of natural killer cell responses by dendritic cells via lymphotoxin-alpha, interleukin-12,
and tumor growth factor-beta
Co-authors:
Kristina Berg Lorvik, Clara Hammarström, Marte Fauskanger, Ole Audun Werner Haabeth, Michael Zangani, Guttorm Haraldsen, Bjarne
Although T helper 2 (Th2) cells are known to be potent effector cells capable of protecting against parasites, few studies have investigated the potential usage of Th2 cells for cancer immunotherapy. Here, we tested the efficacy of tumor-specific Th2 cells for the treatment of Co-authors:
mice with MHC class II-negative myeloma. Transfer of Th2 cells efficiently eradicated myeloma in an antigen-specific fashion. Transferred Andreas Lundqvist, Marzia Palma, Yumeng Mao, Lars Adamson, Anders Österborg, Rolf Kiessling, Dhifaf Sarhan
Th2 cells persisted in vivo and conferred long-lasting immunity. Myeloma eradication did not require B cells, natural killer T cells, CD8+ T cells, or interferon-gamma. Upon transfer, Th2 cells provoked an inflammatory reaction at the tumor site with recruitment of macrophages. Dendritic cells (DC) induce T-cell responses in cancer patients. However, less is known about the role of DC-vaccines in shaping natural killer Th2 cells induced arginase production and M2 phenotype in tumor-infiltrating macrophages. Blocking experiments revealed that arginase (NK) cell responses. To investigate this, NK cells from patients with chronic lymphoid leukemia (CLL) and melanoma following vaccination was required for efficient cancer eradication by Th2 cells. These results suggest that cancer eradication may be achieved by induction of a with antigen-loaded DC were examined for the expression of activating markers and for IFN-gamma production. A significant down- tumor-specific Th2 inflammatory immune response at the tumor site.
regulation of NKp30, NKp46 and CD16 (p<0.01) expression and a reduction of IFN-gamma production was observed in post-vaccination samples (n=7). To investigate the underlying mechanisms of DC-mediated NK cell suppression, monocytes from healthy individuals were differentiated in vitro with IL-4, GM-CSF and LPS and thereafter co-cultured with NK cells for 3-5 days in presence of interleukin-2 (IL-2). Following co-culture, DC significantly suppressed NK cell proliferation (p=0.04), cytolytic activity against K562 cells (p=0.001), IFN-gamma production (p=0.03) and down-regulated the expression of several activation receptors including, NKG2D, NKp30, NKp46, DNAM-1 and CD16. In vitro, NK cell activity was restored when co-cultured with STAT3-inhibited DC. Compared with untreated DC, STAT3-inhibited DC produced higher amounts of lymphotoxin-alpha (LTA) and IL-12 and less tumor growth factor-beta (TGFβ). Neutralization of TGFβ in co- cultures of DC and NK cells restored the lytic activity of NK cells. Similarly, neutralization of LTA or IL-12 in co-cultures of STAT3-inhibited DC and NK cells restored the lytic activity of NK cells. These results show that LTA, IL-12 and TGFβ are involved in the cross-talk between NK-cells and DC. These findings have implications for the development of DC-based vaccination strategies to maintain NK-cell responses in patients with cancer. Poster no: 9
Poster no: 11
Myeloid-derived suppressor cells inhibit NK cell activity through prostaglandin-E2 regulated TGF-ß
T-cells co-transduced with Chimeric Antigen Receptors and Catalase are protected from Reactive
Co-authors:
Tumors can suppress the host immune system by recruiting a variety of cellular immune modulators, such as regulatory T cells, tumor- Maarten Ligtenberg, Kristina Witt, Dimitrios Mougiakakos, Hinrich Abken, Rolf Kiessling
associated macrophages and myeloid-derived suppressor cells (MDSCs). MDSCs accumulate in high frequencies in peripheral blood of cancer patients and mediate potent inhibition of innate and adaptive anti-tumor immune responses. Emerging evidence has emphasized Treatment of cancer patients with adoptive T cell therapy (ATC) has lately become a potentially curative therapy. Re-programing T cells the importance of COX-2/PGE2 pathway in the induction of MDSCs. In this study, we show that PGE2 induced MDSC-like cells similar by retroviral transduction of tumor specific TCRs or chimeric antigen receptors (CAR) allows for the specific targeting of the tumor. T cells to CD14+HLA-DRlow/- monocytic MDSCs isolated from blood of advanced stage melanoma patients inhibited NK cell functions via the transduced with CARs do not depend upon MHC expression on tumors, thus avoiding loss of MHC class 1 as a mechanism of immune production of TGF-β. Binding of PGE2 to EP2 and EP4 receptors on monocytes activated the p38MAPK/ERK pathway and resulted in evasion. Since the tumor stroma is a hostile environment for the transferred T cells, these are functionally impaired by inflammatory elevated secretion of TGF-β. To assess the functional consequences of COX-2 expressing tumors in vivo, we used a murine model, where mediators in the tumor milieu. In this study we have developed a strategy to protect the transduced T cells from high levels of reactive control or COX-2-silenced 4T1 mammary carcinoma cells were s.c inoculated. Significantly higher percentage of CD11b+Gr1+ MDSCs and oxygen species (ROS). By co-expressing catalase along with the tumor specific CAR, H2O2 will be enzymatically reduced to water and lower percentages of NK cell population were observed in mice inoculated with COX-2 silenced 4T1 cells compared with wt 4T1 cells. oxygen, thereby protecting the T cells from functional defects or apoptosis induced by ROS. T cells transduced with a bi-cistronic CAR Furthermore, mice bearing COX-4 silenced 4T1 tumor had better rejected NK cell-sensitive YAC-1 cells compared with mice bearing wt expression vector which expresses catalase produced increased intracellular catalase and had a reduced intracellular oxidative state. 4T1 cells. Taken together, our data demonstrated a novel role of PGE2 in inducing suppressive functions of MDSCs on NK cells. Given the Introduction of excess catalase in the transduced T cells prevented an oxidized environment both in a basal state of the T cells and when prevalence of COX-2 over-expression and the central immune-regulatory role of TGF-β, these mechanisms might be relevant in several activated by α-CD3 (OKT3) α-CD28 (15E8), PMA or DHNQ. Catalase transduced T cells were able to maintain functional anti-tumor activities types of human cancers. Consequently, combining COX-2 inhibitors or EP antagonists with adoptive NK cell therapy may consolidate and even when confronted with high concentrations of H2O2. Importantly, the protection offered by the CAR-CAT transduced T cells was enhance the clinical benefits for cancer patients. extended to bystander cells, allowing for NK cells to functionally eliminate their K562 target cells under H2O2 induced oxidative stress. This novel approach will be able to protect CAR transduced T cells from the tumor microenvironment (TME) as well as providing an antioxidant bystander effect. Poster no: 10
Cripto-1 encoding DNA vaccine elicits tumor protective immune response in vivo
Poster no: 12
Interferon-gamma-inducible-protein-10 stimulates infiltration of human NK cells into solid tumors
resulting in reduced tumor burden
Co-authors:
Maarten Ligtenberg, Helena Tufvesson, Alvaro Lladser, Rolf Kiessling
DNA vaccines have a great potential in the field of tumor immunology. The in vivo expression of plasmid-encoded antigen generates innate and antigen-specific adaptive immune responses. Targeting broadly expressed tumor-associated antigens will increase the applicability of DNA vaccines against cancer. Cripto-1, a glycoprotein that plays a critical role during embryogenesis but is expressed in low amounts Co-authors:
on normal differentiated tissues, has been shown to be overexpressed in more than 50% of human carcinomas. These include melanoma Erik Wennerberg, Andreas Lundqvist, Richard Childs
as well as colon, and breast cancers. Cripto-1 is involved in multiple cellular processes such as cell-proliferation, migration, epithelial- mesenchymal-transition as well as tumor-related angiogenesis. DNA vaccines offer an attractive immunotherapeutic platform from which Adoptive transfer of natural killer (NK) cells is being increasingly used for cancer treatment. With the exception of hematological to study the feasibility of targeting Cripto-1 in mouse models. In this study, the ability of DNA vaccines encoding Cripto-1 to induce a tumor malignancies, however, NK cell-based therapies have largely failed to demonstrate objective clinical responses. This could potentially protective adaptive immune response was tested. We show protection against mouse Cripto-1 (mCripto-1) transduced D2F2 breast cancer be explained by ineffective homing of infused NK cells to the tumor site. A major regulator of lymphocyte chemotaxis is the chemokine model in Balb/c mice after immunization with human Cripto-1 (hCripto-1) or mCripto-1 encoding DNA vaccines. The vaccination with receptor CXCR3. It is expressed on activated NK cells and governs NK cell migration toward gradients of interferon (IFN)-gamma-inducible hCripto-1 as well as with mCripto-1 in Balb/c mice led to the generation of mCripto-1 specific antibodies. Immunization with hCripto-1 chemokines including IFN-gamma-inducible-protein-10 (IP-10, CXCL10). encoding DNA vaccine protected C57Bl/6 mice against endogenously mCripto-1 expressing melanoma B16F10. In C57Bl/6, mCripto-1 and In this study, we aimed to induce IP-10 secretion in melanoma tumors through IFN-gamma treatment in order to improve migration of hCripto-1 15-mer overlapping peptides were screened and elicited IFN-y responses against specific hCripto-1- and mCripto-1-peptides. adoptively transferred expanded NK cells. Upon expansion, NK cells up-regulated CXCR3 resulting in significantly increased IP-10-dependent In summary, our data indicates that DNA vaccination with either hCripto-1 or mCripto-1 results in a protective immune response against migration toward IFN-gamma-treated melanoma cells. Adoptively infused human NK cells accumulated preferentially in IP-10-transfected Cripto-1 expressing tumors, which could lead to the development of therapeutic tumor vaccines to be tested in the clinic.
subcutaneous 526 melanoma tumors in contrast to IP-10-negative tumors in a xenograft mouse model. NK cell adoptive transfer significantly prolonged survival in mice bearing IP-10-positive tumors compared with mice with IP-10-negative tumors. Intratumoral Acknowledgments: administration of IFN-gamma elicited IP-10 production in mice bearing subcutaneous EST112 melanoma tumors. Mice treated with IFN- Research described here have been supported by grants to R.K. from The Swedish Medical Research Council, The Swedish Cancer Society, gamma prior to adoptive NK cell transfer showed an increase in intratumoral NK cell infiltration and a reduction in metabolic activity of the The Cancer Society of Stockholm, Radiumhemmets Forskningsfonder, ALF-Project grant from Stockholm City Council.
tumor compared with PBS-treated mice. In summary, we have shown that adoptively transferred NK cells infiltrate into solid tumors due to the IP-10/CXCR3 interaction, resulting in reduced tumor growth and increased survival. This is a promising strategy to potentiate the success of NK cell-based therapies.
Poster no: 13
Poster no: 15
Effects of Ipilimumab on myeloid derived suppressor cells in advanced melanoma patients
Spontaneous presence of FoxO3-specific T cells in cancer patients
Yago Pico de Coaña
Stine Kiær Larsen
Center for Cancer Immune Therapy
Co-authors:
Co-authors:
Isabel Poschke, Giusy Gentilcore, Yumeng Mao, Maria Nyström, Johan Hanson, Giuseppe Masucci, Rolf Kiessling
Shamaila Munir Ahmad, Manja Idorn, Özcan Met, Inge Marie Svane, Per thor Straten, Mads Hald Andersen
Blocking the immune checkpoint molecule Cytotoxic T-lymphocyte antigen-4 (CTLA-4) with Ipilimumab has proven to induce long-lasting FoxO3 may serve as a target antigen for tumor-reactive T cells as it is frequently over-expressed in cancer cells. In addition, expression of clinical responses in patients with metastatic melanoma. In order to study the early response that takes place after CTLA-4 blockade, FoxO3 plays a critical role in immune suppression mediated by tumor-associated dendritic cells (TADCs). In the present study we describe peripheral blood immune monitoring was performed in five patients undergoing ipilimumab treatment at baseline, three and nine weeks that FoxO3-specific, cytotoxic CD8 T cells exists among peripheral-blood mononuclear cells (PBMCs) of cancer patients. In contrast, we did after administration of the first dose. Along with T cell population analysis, this work was primarily focused on an in-depth study of the not detect reactivity towards FoxO3 in healthy individuals. Indeed, FoxO3-specific cytotoxic T lymphocytes (CTLs) were able to specifically myeloid derived suppressor cell (MDSC) populations. Ipilimumab treatment resulted in lower frequencies of regulatory T cells along with recognize and kill both FoxO3-expressing cancer cells as well as dendritic cells. Thus, FoxO3 was processed and presented by HLA-A2 on the reduced expression levels of PD-1 at the nine-week time point. In addition to this, CD4+ T cells showed an increase in ICOS expression. cell surface of both immune cells and cancer cells. As FoxO3 programs TADCs to become tolerogenic and thereby comprises a significant Three weeks after the initial ipilimumab dose, the frequency of granulocytic MDSC was significantly reduced and was followed by a immune suppressive mechanism, the targeting of FoxO3 by means of specific T cells is an attractive clinical option that ought to boost reduction in the frequency of arginase1 producing CD3- cells, indicating an indirect in trans effect that should be taken into account for additional immunotherapy. In addition, the natural occurrence of FoxO3-specific CTLs in the periphery suggests that these T cells hold a future evaluations of ipilimumab mechanisms of action. function in the complex network of immune regulation in cancer patients. Poster no: 16
Poster no: 14
Immune regulating role of cytotoxic, PD-L1 specific T cells
Genetic engineering of T cells for increased homing to the tumor site
Shamaila Munir Ahmad
Manja Idorn
CCIT, Herlev Hospital
Center for Cancer Immune Therapy (CCIT)
Co-authors:
Co-authors:
Stine Kjær Larsen, Inge Marie Svane, Mads Hald Andersen
Gitte Holmen Andersen, Hjalte Larsen, Joost Huibert van den Berg, Özcan Met, Per thor Straten
Many different mechanisms contribute to the inhibition of cellular immunity, e.g. cell surface-expression of immune checkpoint ligands Adoptive cell transfer (ACT) using in vitro expanded T cells from biopsy material represents a highly promising treatment of disseminated such as programmed cell death protein 1 (PD1) ligand (PD-L1). PD-L1 is widely expressed by tumor cells and, accordingly, cancers use cancer. ACT in its present form is rather crude and improvements seem within reach. Recruitment of transferred lymphocytes to the tumor PD-L1 to evade the host immune system. Of late we identified and characterized spontaneously occurring PD-L1-specific, CTL. However, site is a crucial step in ACT efficacy; however, quite few T cells actually reach the tumor site upon administration. In the present pre-clinical the potential role of such auto-reactive T cells in immune regulation is still an open question. Thus, the induction of PD-L1-specific CTL study we have genetically engineered T cells aiming at increasing the homing of T cells by matching expression of chemokine receptors on should boost immunity by killing immune suppressive tumor cells as well as PD-L1 expressing stroma cells contributing to the permissive T cells to chemokines secreted by the tumor, thus improving anti-tumor efficacy of ACT. By PCR analysis we found that several malignant melanoma (MM) cell lines showed expression of cytokines CXCL8/IL-8, CXCL12/SDF-1 and CCL2, which was confirmed by ELISA analysis We examined the effect of PD-L1-specific CTL on T-cell immunity towards viral antigens from cytomegalovirus (CMV) or Epstein-Barr virus of MM conditioned medium. Taking advantage of mRNA electroporation we successfully transfected T cells with mRNA encoding the (EBV). Interestingly, the effect of the addition of PD-L1-specific T cells seemed to depend on the timing. Hence, virus stimulation in the chemokine receptors CXCR2, chimeric receptor CXCR4-R2 or chimericreceptor CCR2-R2 on the cell surface, of which the chimeric constructs presence of PD-L1-specific CTL resulted in decreased numbers of viral specific T-cells. In contrast, addition of PD-L1 specific T-cells seven contain the intracellular region of CXCR2 allowing expression in T cells. Work is in progress, but so far chemokine receptor CXCR2 and days subsequent to virus stimulation resulted in a vast increase in the number CMV-specific CD8+ T cells. The overall biological function of chimeric receptor CXCR4-R2 transfected T cells are capable of migrating towards their ligands, CXCL8 and CXCL12 respectively, as well as PD-L1-specific CTL may thus vary depending on the microenvironment and the state of the immune response. In both situations, however, MM conditioned medium in in vitro transwell migration assays. In vitro studies on the transfection efficacy and function of the CXCR4-R2 they may be important for the fine-tuning of immune responses. and CCR2-R2 chimeric receptors as well as in vivo migration studies have been initiated, and data will be presented at the meeting. Poster no: 17
Poster no: 19
PGE2 signaling pathways in cancer and immunomodulation
The role of B cells as APCs in Human CD4+ T cell Based Cancer Immunotherapy
Anna Mari Lone
Biotechnology Center of Oslo
Co-authors:
Cancer is one of the leading causes of death with seven million deaths world wide, each year. An attractive alternative to fight cancer Anna Mari Lone, Anne Jorunn Stokka, Kjetil Taskén
is to use cell based immunotherapy. The first tumor draining lymph node, also called the sentinel node, is a source of tumor specific lymphocytes. Previous work by our group has demonstrated that the in vitro expansions of sentinel node-acquired, tumor specific CD4+ Several tumor immune evasion mechanisms converge on an inhibitory cAMP-PKA pathway in effector T cells (Teffs). Both tumor cells and T cells are promising to be used in adoptive immunotherapy. Naive T helper cells recognize foreign and mutated self antigens presented induced regulatory T cells (Tregs) secrete prostaglandin E2 (PGE2), which activates this inhibitory pathway in Teffs and thus impairs Teff to them in MHC class II by professional antigen-presenting cells (APCs) after which they get activated. There are three distinct classes of function and anti-tumor immunity. Here, I use novel small molecules and peptidomimetics to block this inhibitory pathway to restore Teff APCs in a lymph node, namely Macrophages, Dendritic cells and B cells. The comparatively large proportion of B cells compared to the function. The lab has already developed a stable peptidomimetic which targets this pathway at the level of PKA anchoring and I am working other two classes of APCs in sentinel nodes indicates that B lymphocytes may deserve special attention. In this study, we have used FASCIA to develop a second peptidomimetic which interferes at a different point in the pathway. We have also obtained preliminary hits from (Flowcytometric Assay of Specific Cell-mediated Immune response in Activated cells) to study B cell activation in patients with urinary a screen to identify small molecule inhibitors of this pathway and have performed in silico analysis to identify more potent compounds bladder cancer. We isolated lymphocytes from blood, sentinel node and tumor of the patients and stimulated them with autologous tumor targeting this interaction which we are currently testing. By using these tools to target the cAMP-PKA inhibitory pathway in heterotopic extract. After cultivation for 6-7 days, the cells were analyzed by multi-color flow cytometry, using FASCIA. As control, we used either syngeneic animal tumor models, we hope to improve anti-tumor immune function and limit tumor growth. stimulation with non-malignant epithelium of the bladder and/or unstimulated lymphocytes. Our preliminary data display activation and blast formation of B cells after stimulation with autologous tumor extract for 6-7 days. Several patients displayed a higher activation in In the second part of the project, I am using a combined phosphoproteomics and phospho-flow cytometry approach to study, more sentinel node acquired B cells compared to their counter parts in non-sentinel node and blood, indicating tumor antigen specificity of those globally, PGE2 signaling in T cells. In particular, a previous study in the lab examined signaling networks elicited by PGE2 in T cells and I am B cells. B cell blasts in all patients showed increased activation phenotypes. Upregulation of CD86 and HLA-DR by B cells upon activation now building on these studies and investigating the signaling networks elicited by specific agonists of the four different PGE2 receptors with tumor antigen, indicated an increased tumor specific antigen presenting ability to CD4+ T cells. in different T cell subsets including helper, cytotoxic and regulatory T cells. While phosphoproteomics allows a global overview of PGE2 signaling through its different receptors in these different T cell types, phosphoflow cytometry combined with fluorescent cell barcoding Further studies about antigen presenting ability of B cells in the context of solid tumors are needed to optimize their long term cultivation will allow easy multiplexing and investigation of signaling timing and response to dose or combinations of stimuli such as receptor agonists invitro. We plan to address this by investigating sentinel node acquired CD4+ T cell activation and expansion with the assistance of tumor or antagonists. These phosphoflow cytometry and phosphoproteomics studies may lead to new insights into the functional roles of the stimulated sentinel node isolated B cells. We will also investigate cytokine secretion of both B- and CD4+ T cells to reflect on functionalities four distinct PGE2 receptors in T cell subtypes and the interdependencies of the signaling pathways. Together with the studies aimed at on B cell tumor antigen presentation. targeting the inhibitory pathway in T cells, this research will increase the knowledge about PGE2-elicited pathways in T cells that influence anti-tumor immune function. Altogether, our results suggest a CD4+ T cell dependent tumoral B cell response upon stimulation with autologous tumor antigen which may be exploited for immunotherapy. Poster no: 20
Poster no: 18
T helper TCRs isolated from long term survivors after cancer vaccination for use in adoptive cell
Human anaplastic thyroid carcinoma cells are sensitive to NK cell-mediated lysis via ULBP2 and
Jon Amund Kyte
chemoattract CXCR3-positive NK cells
Co-authors:
Sebastien Walchli, Else Marit Suso Inderberg, Paal Brunsvig, Johanna Olweus, Gunnar Kvalheim, Gustav Gaudernack, Martin Pule
Adoptive cell therapy with retargeted T cells has produced remarkable clinical responses in recent trials, but also serious toxicity. For most Co-authors:
cancer forms, there is a lack of safe targets for therapy with cytotoxic T cells. We hypothesize that therapy with redirected T helper cells Erik Wennerberg, Lars Ekblad, Veronika Kremer, Vitaliy Kaminskyy, Christofer Juhlin, Anders Höög, Inger Bodin, Vitalijs Svjatoha,
may transform the inflammatory milieu and induce epitope spreading, while circumventing key toxicity concerns. Human telomerase Catharina Larsson, Jan Zedenius, Johan Wennerberg, Andreas Lundqvist
reverse transcriptase (hTERT) is overexpressed in most human cancers. We have conducted a series of hTERT cancer vaccine trials. From long term survivors after vaccination, we isolated >100 CD4+ T helper cell clones recognizing hTERT epitopes. These T cell clones were Anaplastic thyroid carcinoma (ATC) is one of the most aggressive forms of solid cancer with no curative therapies currently available. To characterized with regard to HLA restriction, affinity, fine specificity, proliferative capacity, cytokine profile and recognition of naturally date, strategies to target ATC by immunotherapy have not been evaluated. In this study we investigated whether ATC would be a suitable processed epitopes. Based on their characteristics, two DP4-restricted T cell clones (A1 and A2) were selected for molecular T cell receptor target for NK-cell-based immunotherapy.
(TCR) cloning. DP4 is among the most prevalent HLA molecules in Caucasians.
We established 10 new cell lines, 7 from ATC tumors and 3 from papillary thyroid carcinoma (PTC) tumors, and analyzed them together with After TCR sequence identification, TCR A1 and A2 were cloned into the retroviral vector MP71, together with the GMP-applicable suicide/ 8 additional ATC cell lines. Cells were analyzed for sensitivity to NK cells lysis and their ability to chemoattract and regulate NK cell activity. marker/sorter gene RQR8. The RQR8 gene comprises an epitope from CD34, facilitating identification and purification of transduced cells, In addition, NK cell infiltration and phenotype was analyzed in fresh tumor samples and peripheral blood from 6 ATC patients. and two minimal epitopes for rituximab, allowing for effective elimination of transduced cells if needed. We investigated the expression ATC cell lines proved to be sensitive to lysis by ex vivo expanded NK cells and the lysis correlated with the surface expression of UL16- and functionality of TCR A1 and A2, after retroviral transduction. The results demonstrated that both TCRs were expressed well and that binding protein 2 on tumor cells. Blocking NKG2D abrogated NK-cell mediated lysis of ATC cell lines. Moreover, we observed high levels that the transduced T cells acquired the desired hTERT specificity. Interestingly, the T helper TCRs were expressed and functional both in of CXCL10 being produced by ATC cell lines leading to stimulated migration of expanded NK cells. ATC tumors were enriched for NK cells CD4+ and CD8+ T cells. Both cell populations specifically produced IFNγ and CD107α upon hTERT peptide stimulation. expressing the cognate chemokine receptor CXCR3. However, ATC-tumor-derived NK cells displayed a suppressed phenotype with a down- Next, we cloned TCR A1 and A2 into the mRNA expression vector pCIpA102, for transient redirection of T cells with biodegradable TCRs. regulated expression of NKG2D compared with NK cells in peripheral blood. In vitro, suppression of NK-cell-mediated lysis and NKG2D This strategy allows for safer clinical testing and dose escalation, based on repeated injections of redirected T cells that express the TCR for expression by ATC cells was restored upon neutralization of prostaglandin-E2. 4-7 days. We transfected T cells with TCR mRNA by use of electroporation. The results showed that both TCRs were expressed in >90% of T ATC cell lines are sensitive to NK-cell-mediated lysis via ULBP2 and chemoattract CXCR3-positive NK-cells. Patients with ATC may benefit cells, and that the transfected T cells specifically recognized APCs pulsed with the relevant hTERT peptide. from NK-cell-based immunotherapy.
The findings demonstrate that the cloned TCRs confer recipient T cells with the desired hTERT-specificity and functionality in vitro. Preclinical experiments may provide limited information on the efficacy or toxicity of these TCRs, as T helper cell therapy depends on mobilizing the host immune system. We therefore plan to use mRNA-based biodegradable TCRs for a phase I trial evaluating these Poster no: 21
Poster no: 23
Human TCR??+ CD4-CD8- double-negative (DN) T-cells in patients after aHSCT
XENO-FREE SERUM REPLACEMENT FOR EX VIVO CULTURE AND EXPANSION OF T CELLS
Thermo Fisher Scientific
Co-authors:
Co-authors:
Rebecca Axelsson-Robertson, Thomas Poiret, Aditya Ambati, Lalit Rane, Olle Ringden, Markus Maeurer
Grethe Økern, Tanja Aarvak, Sandra Kuligowsky, Mark Bonyhadi
Human TCRab+ CD4-CD8- double-negative (DN) T-cells represent a minor subset in peripheral blood, yet are important in infectious Background: The manufacture of a majority of clinical T cell products for immunotherapy applications requires ex vivo T cell culture and diseases and autoimmune responses. We examined the frequency of DN T-cells in 17 patients after allogeneic hematopoietic stem cell expansion. Commercialization of T cell manufacturing processes requires reagents that meet regulatory guidelines and ultimately help transplantation (aHSCT) at 1, 2, 3, 6 and 12 months post aHSCT and show that these cells are increased early after aHSCT and decrease with reduce manufacturing cost of goods. A key component in many T cell culture protocols is human serum, which is expensive and may time after aHSCT. DN T-cells reside in the terminally differentiated effector (CD45RA+CCR7-) T-cell population and are polyclonal, require testing prior to use for the manufacture of a cGMP-compliant T cell product. To this end, we have developed a xeno-free serum determined by T-cell receptor VbCDR3 analysis. Gene expression analysis of ex vivo-sorted DN T-cells showed a distinct set of gene replacement supplement with defined components that can be used in combination with several different cell culture media to support ex expression, including Interleukin-8, as compared to CD4+ or CD8+ T-cells. DN T-cells contributed to MHC class I restricted EBV-directed vivo expansion of T cells.
immune responses, defined by antigen-specific cytokine production and by detection of HLAA*02:01 restricted EBV BMLF-1 (GLCTLVAML), Results: T cells activated ex vivo and expanded with Dynabeads® CD3/CD28 CTSTM and cultured in OpTmizer™ CTS™, X-VivoTM 15, or LMP-2A (CLGGLLTMV) and HLAA* 24:02 restricted EBV BRLF-1 (DYCNVLNKEF) and EBNA-3 (RYSIFFDY) specific Tcells.
AIM-V® CTS™ supplemented with pooled human serum or serum free T cell serum replacement showed similar growth kinetics, total We created retroviral- transfected Jurkat cell lines with a Melan-A/MART-1 specific TCR plus or minus the CD8a chain to study TCR+ DN fold expansion and transduction efficiency after 2 weeks in culture. Numbers of expanded CD4+ and CD8+ T cells were comparable in the T-cells in response to their nominal MHC class I/peptide ligand. We show that DN T-cells exhibit increased TCRz chain phosphorylation as expanded cultures regardless in the presence of human serum or the newly developed SRS-XF. Restimulated T cells expanded in serum free compared to the TCR+CD8+ transgenic T-cell line. DN T-cells contribute to antigen-specific T-cell responses and represent an effector T-cell T cell serum replacement show similar cytokine profile and proliferation as T cells expanded in human serum.
population that can be explored as the recipient cell population for retroviral transfer antigen-specific TCRs. Conclusion: This study shows that human serum may be replaced by a xeno-free formulation in several commonly used cell culture media to support ex vivo expansion and lentiviral transduction of polyclonal T cells. Culturing T cells in serum free T cell serum replacement facilitates a favourable culture profile and immune function. The serum free T cell serum replacement contains only fully tested human- derived or human recombinant proteins which facilitates supply security for clinical large scale and commercial therapies. Poster no: 22
T cells engineered to express two allo-restricted T cell receptors recognizing CD20/HLA-A*02:01
Poster no: 24
specifically and efficiently kill malignant B-lymphoid cells - a preclinical study
T cells engineered to express a TCR targeting a neoantigen in solid tumour
Institute for Cancer Research Oslo University Hospital Radiumhospitalet
Else Marit Inderberg-Suso
Oslo University Hospital- The Norwegian Radium Hospital
Co-authors:
Nadia Mensali, Weiwen Yang, Sebastien Wälchli, Johanna Olweus
Co-authors:
Sebastien Walchli, Marit Renée Myhre, MengYu Wang, Johanna Olweus, Hilde Almåsbak, Gunnar Kvalheim, Gustav Gaudernack
Patient T cells genetically modified to express cancer-targeted T-cell receptors have recently shown great efficacy in the induction of clinical responses in malignant melanoma and synovial sarcoma. The challenge is currently to identify immunogenic and cancer-specific targets T-cell receptor (TCR) transfer is an attractive strategy to increase the number of cancer-specific T cells in adoptive cell therapy. However, that induce the generation of high-affinity TCRs. recent findings both in the clinic and in pre-clinical mouse models indicate that careful consideration of the target antigen is required to Although shared self-proteins represent attractive cancer targets, the autologous T-cell repertoire is normally depleted of T cells with TCRs avoid on-target, off-site toxicity. Rather than targeting tumour-associated self-antigens, it may be safer directing engineered T cells against recognizing peptides from such antigens with high affinity, during negative thymic selection. We have previously generated high-avidity T mutated proteins such as frequently occurring frameshift mutations. Furthermore, frameshift mutations result in novel polypeptides cells that recognize a peptide from CD20 (SLFLGILSV) in the context of allogeneic HLA-A*02:01. The T cells were shown to efficiently and allowing selection of TCRs from the non-tolerant T-cell repertoire avoiding the problem of low affinity TCRs due to central tolerance. The specifically kill patient-derived leukemia and lymphoma cells (Abrahamsen et al, Leukemia 2010 and Int J Cancer 2012, Kumari et al, PNAS transforming growth factor β Receptor II frameshift mutation (TGFβRIImut) is found in hereditary non-polyposis colorectal cancers and in 2013). A number of T cell clones were functionally tested and two were selected for cloning and expression of the TCRs and subsequent around 15% of sporadic colorectal and gastric cancers displaying microsatellite instability. The -1A mutation in an adenine stretch of the pre-clinical evaluation. The TCRs A94 and A23 were cloned into the retroviral vector pMP71 as 2A-bicistronic constructs encoding WT TGFβRII gene gives rise to immunogenic peptides which have been used for vaccination of MSI+ colorectal cancer patients in a Phase I sequence or variants modified with codon optimization, murinization of the constant part and addition of an extra cystein bond in the clinical trial. From a responding patient we isolated a CD8neg/CD4neg CTL clone from which we cloned an HLA-A2-restricted TGFβRIImut- constant part, as described by others. The genetic optimization lead to a doubling in A23-transduced peripheral blood T cells staining specific TCR. We showed that both CD8+ and CD4+ T cells transiently redirected with this TGFβRIImut-TCR recognised colon carcinoma cell positively for the HLA-A2/CD20p multimer, whereas a manifold higher expression of the A94 was observed. The A23opt and A94opt lines harbouring the frameshift mutation, indicating CD8+ co-receptor independency. Moreover, we demonstrated that TCR-redirected T also mediated correspondingly stronger degranulation (>90%) than the WT TCRs in response to target cells induced to express a single- cells could reduce the growth of colorectal cancer in a NOD/SCID xenograft mouse model. chain trimer containing the CD20p, or to peptide-loaded HLA-A2posSupT1 cells. The re-directed T cells showed no cross-reactivity to a wide range of HLA-A2pos CD20neg cell lines of different histologies, including colon carcinoma, keratinocytes, HEK, liver carcinoma, lung adenocarcinoma and mesenchymal stem cells. Responses were rescued when target cell lines were loaded with the CD20p. The peptide affinity for the two TCRs was measured in response to target cells loaded with titered amounts of peptide. The A94opt had an EC50 between 100-10pM and was approx. 10-fold more sensitive than A23opt. In comparison, the MART-1-specific TCR DMF5, which has proven efficient in a clinical trial utilizing TCR re-directed T cells, showed almost 100-fold lower sensitivity for the cognate WT MART-1 peptide. Finally, the re-directed T cells responded to 5 different HLA-A2pos lymphoma cell lines, and to chronic lymphocytic leukemia (CLL) cells from 4 HLA-A2pos patients, all endogenously expressing CD20. In contrast, no responses were seen to two HLA-A2negCD20pos lymphoma cell lines and CLL donors. In conclusion, the results show that T cells re-directed with A2/CD20p specific TCRs efficiently and specifically kill malignant B cells, paving the way for the clinical testing of these TCRs.
Poster no: 25
Poster no: 27
NK cell repertoires and functions in pediatric patients with acute leukemia
A new generation of fast DCs: first experiences in patients with solid tumors
Iris Bigalke
Department of Immunology, Oslo University Hospital
Oslo University Hospital
Co-authors:
Co-authors:
Aina Ulvmoen
Kirsti Hønnåshagen, Marianne Lundby, Julitta Kasten, Else-Marit Suso Inderberg, Lisbeth Skoge, Stein Sæbøe-Larssen, Dolores Schendel,
NK cells play a major role in tumour immune surveillance, and are thought to contribute to protection from cancer development. NK cells are increasingly attractive as targets for immunotherapy of several types of cancer, and in particular against leukemia. Approximately 30-40 A new generation of fast DCs using a maturation cocktail containing IL-1β, TNFα, IFNγ, PGE2 and the TLR7/8 ligand R848 have shown to be children are diagnosed with acute leukemia in Norway every year with the highest incidence rate in the age group of 2-5 years old. Acute more efficient in pre-clinical models than standard 7 day DCs generated with a standard maturation cocktail. HLA-DR and co-stimulatory lymphoblastic leukemia (ALL) is most frequent, while acute myeloid leukemia (AML) accounts for approximately 15% of the cases. With this molecules like CD80, CD83, CD86 and CD40 are highly expressed in those DCs and they show a good migratory capacity towards CCL19. project we aim to broadly analyse the NK cell receptor repertoire (NCRs, KIRs, CD94/NKG2, NKR-P1, NKG2D) at time of diagnosis, at the end Their special characteristic is IL-12p70 production when stimulated with CD40L whilst IL-10 production is low. of treatment, and during follow-up using blood and bone marrow samples drawn from patients. In parallel we utilize flow cytometry-based We investigated if the new generation DCs maintain their properties when produced by GMP standards and if they are able to mount methods to measure NK cell effector functions; cytokine production (intracellular staining for IFN-γ, TNF-α, and MIP-1β) or cytotoxicity (by specific immune responses in patients. Monocytes were enriched using the Elutra cell separator and cultivated either fresh or after staining for CD107a, a marker for granule secretion). Also, changes in the proportions of immature, mature, and activated subsets of NK cryopreservation. Monocytes were cultured in Teflon bags in the presence of IL-4 and GM-CSF and the maturation cocktail was added cell are assessed. With this study, we hope to find certain phenotypical or functional markers can be correlated with risk of relapse versus on day 2 or 3. After 24 hours DCs were harvested and electroporated with mRNA. After 2-4 hours recovery, cells were frozen with either disease-free survival. 2.5E+6 or 5E+6 transfected DCs per vial. Productions from one lung cancer, one prostate cancer and 4 glioblastoma patients showed similar characteristics, variations only seen in the amounts of IL-12p70 released after CD40L stimulation.
The first patient treated with the new generation DCs transfected with hTERT mRNA suffered from stage IV lung cancer with brain metastases. Following diagnosis in June 2011 she was treated with chemotherapy and radiotherapy. Since December 2011 she has been vaccinated and obtained a status of stable disease. DC vaccination was interrupted in 2/2013 when an attempt was made to re-open an occluded bronchus with radiotherapy. During irradiation the patient developed an inflammation of the pleura, which was treated with Poster no: 26
high dose cortisone. During cortisone therapy the patient developed 2 new brain metastases, which were treated with Dexamethasone and local radiotherapy using Cyberknife. DC vaccination was continued in 6/2013 and health conditions gradually improved bringing the Cancer vaccines with hTERT and Survivin mRNA transfected fast DCs – a simplified and effective
patient again into a status of stable disease. The second patient receiving DC vaccination had a hormone resistant prostate cancer in a very advanced stage and dropped out immediately after start of treatment. Treatment of four glioblastoma patients with hTERT and survivin transfected DCs plus either with autologous tumour mRNA or hCMVpp65 mRNA DCs has started. All patients show strong local DTH responses and flu-like symptoms after vaccination at an earlier time point. The first glioblastoma patient has now been treated with Anne Merete Aa. Tryggestad
DC vaccines for 7 months and remained in stable disease, the other three are still too early to evaluate. Our results show that the new Oslo University Hospital
generation DCs can successfully be produced from patients with different kinds of cancer. Strong DTH reactions and flu like symptoms following DC vaccinations indicate immunological reactions but if this is due to specific T cell responses is still under investigation.
Co-authors:
Iris Bigalke, Gjertrud Skorstad, Turid Kirsti Hønnåshagen, Lisbeth Skoge, Karol Axcrona, Wolfgang Lilleby, Gunnar Kvalheim
Prostate cancer (PC) is the most common cancer among men. Since 2005 more than 4000 new cases each year are diagnosed with PC in Norway and the incidence is increasing. In many cases prostate cancer is an indolent disease and patients often will die with the disease Poster no: 28
and not off it. If the patients are diagnosed with high Gleason Score they will develop relapse following primary therapy and when this occur there is no curative treatment available. We have previously reported that about 50% of hormone resistance patients mount Role for DNAM-1 as a Marker and Effector Molecule in Natural Killer Cell Education
specific immune responses following vaccination with Dendritic Cells (DCs) transfected with mRNA from autologous tumour. Immune responses were also related to overall survival. Initially DCs were produced over 7 days. Recently, we have reduced the production time down to 3 days (Fast DCs) and here we report about our clinical experiences with this new type of DCs. Five metastatic prostate cancer Eivind Heggernes Ask
patients have been included in the study. Prior to the DC vaccination, 3 patients had bone metastasis while 2 were diagnosed with lymph Institute for Cancer research, Oslo University Hospital/University of Oslo
node metastasis. The fast dendritic cells (DC) is produced by differentiation of autologous monocytes to mature DCs by adding GM-CSF (2500IU/mL) and IL-4 (1000IU/mL) for 48 hours, and 24 hours of maturation with GM-CSF (2500IU/mL), IL-4 (1000IU/mL), TNF-α (10ng/ mL), IL-1β (10ng/mL) and PgE2 (1ug/mL) in CellGro DC medium. Mature Fast DCs were then transfected with hTERT- and Survivin-mRNA by Co-authors:
electroporation. After over night incubation in medium without any cytokines added the vaccines were frozen and stored until use. Quality Monika Enqvist, Elin Forslund, Eivind Heggernes Ask, Mattias Carlsten, Greger Abrahamsen, Vivien Béziat, Sandra Andersson, Marie
control of the DCs was performed. All mRNA transfected DC (mDCt) showed a mature phenotype with down-regulation of CD14 and up- Schaffer, Anne Spurkland, Yenan Bryceson, Björn Önfelt, Karl-Johan Malmberg
regulation of CD80, CD83, CD86, CCR7, CD274, CD40 and HLA-DR compared to monocytes. All mDCt showed migration capacity towards CCL19. mDCt had no IL-12p70 secretion and except for one patient with high IL-10 secretion all showed low levels of IL-10. When immune The intrinsic functional capacity of natural killer (NK) cells is tuned in a dynamic fashion by the integrated signalling via inhibitory and responses were tested by T-cell proliferation, no CD4 T-cell responses could be detected. Two of the patient was HLA-A2 positive and activating receptors in a process termed education. However, the understanding of the cellular and molecular mechanisms behind dextramers was used to detect antigen specific CD8 positive T-cells in blood at several time points during the vaccination. Progression free such functional tuning is limited. Here, we show that the expression of the adhesion molecule and activation receptor DNAM-1 is a survival (PFS) assessed by PSA measurement and MRI of the 5 patients were 7, 21, 24, 35 and 36 months. The patient vaccinated with DCs marker for education of human NK cells and correlates with the quantity and quality of expressed HLA class I specific inhibitory killer secreting high IL-10 has the shortest PFS. Two of the patients have been continuously vaccinated and one patient has been revaccinated cell immunoglobulin-like receptors (KIRs) and/or CD94/NKG2A as well as with the magnitude of functional responses. Upon target cell following a treatment interval with chemotherapy and local radiotherapy of bone metastasis. Altogether, our fast DCs show objective recognition, the conformational state of LFA-1 rapidly changed in educated NK cells, leading to a functional pairing with DNAM-1 at the clinical benefit in some of the patients. Only 2 patients were HLA-A2 positive and in both T-cell responses could be measured by dextramers immune synapse. Thus, the coordinated expression of LFA-1 and DNAM-1 is a central component of NK cell education and provides a during the vaccination. The reasons for the lack of CD4T-cell responses in our patients are not fully understood and are under investigation.
potential mechanism for controlling cytotoxicity by functionally mature NK cells. Poster no: 29
Poster no: 31
Allo-reactive CD8+ T cell clones specific for the self-protein CD20 show superior avidity compared
Selective Expansion of Educated NK cells for Cancer Therapy
to autologous T cell clones of the same specificity
Vincent Oei
University of Oslo
Department of Immunology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet
Co-authors:
Lisa Liu, Vivien Béziat, Mats Heyman, Hans-Gustaf Ljunggren, Dan Grandér, Karl-Johan Malmberg
Co-authors:
Fan Ying, Shraddha Kumari, Johanna Olweus
New insights into the fine specificity and molecular regulation of NK cell functionality hold promise for the design of more efficient strategies to activate, expand, and deliver NK cells for cancer immunotherapy. We recently reported that education by self-specific As most tumor antigens represent self-proteins, high-avidity T cells are largely deleted from the autologous repertoire. Allogeneic T cells inhibitory KIRs is required for the proliferation of NK cells in response to stimulation with cellular targets expressing HLA-E. Based on from human leukocyte antigen (HLA)-mismatched donors provide repertoires wherein such cells have not been systematically deleted. We this finding, we have developed a protocol for selective expansion of educated, NKG2C+ NK cells. We generated NK cell cultures with have previously reported the successful generation of allo-reactive cytotoxic T cells (CTLs) from HLA-A*02:01- donors, specific for a peptide distinct specificities (2DL1 or 2DL3) and tested these at day 14 against a panel of primary leukemic blasts from 24 children with acute (SLFLGILSV) from the B cell antigen CD20 in complex with HLA-A*02:01 (A2/CD20p). These CTLs efficiently killed HLA-A*02:01+/CD20+ lymphoblastic leukemia (ALL) in a flow-cytometry-based killing assay. The malignant cells were identified based on the minimal residual target cells, including primary cancer cells from patients with chronic lymphocytic leukemia, follicular lymphoma or acute lymphoblastic disease phenotype determined by the NOPHO panel. The expanded NK cells displayed strictly specific recognition patterns based on the leukemia, while a range of other HLA-A*02:01+/CD20- target cells were spared, indicating a high degree of specificity (Abrahamsen et al., HLA genotype of the leukemic blasts (HLA-C1/C2) and significantly more potent cytotoxicity (on average 80% killing at the E:T ratio 5:1) Leukemia 2010; Abrahamsen et al., Int J Cancer 2011; Kumari et al., PNAS 2013). Another study demonstrated that A2/CD20p-specific CTLs than short-term activated NK cells or the NK92 cell line. The possibility to selectively expand educated NK cells represent a promising first could be generated from HLA-A*02:01+ donors, but these CTLs killed HLA-A*02:01+/CD20+ targets only when high effector-to-target ratios step towards exploiting the adaptive-like behaviour of NK cells in cancer therapy. were used, suggesting low avidity. The aim of the present study was to further investigate and directly compare the avidity of autologous and allo-reactive A2/CD20p-specific T cells. Peripheral blood mononuclear cells from HLA-A*02:01+ or HLA-A*02:01- healthy donors were co-cultured with antigen-loaded autologous monocyte-derived dendritic cells (moDCs). HLA-A*02:01- moDCs were transfected with HLA-A*02:01 mRNA prior to antigen loading. A2/CD20p-specific CTL clones were established from the co-cultures following flow cytometric sorting of single CD8+ T cells staining positively with fluorescent multimers of monomeric HLA*A2:01/CD20p. A2/CD20p multimer positive CTL clones were tested for degranulation in response to peptide-loaded HLA-A*02:01-transduced SupT1 cells or target cells naturally expressing HLA-A*02:01 and CD20, as measured by the percentage of CD8+ cells that were CD107a/b+ by flow cytometry. When target cells were pulsed with high peptide concentrations of 10 nM-1 uM, the responses of autologous and allo-reactive CTL clones were comparable. However, at a concentration of 100 pM CD20p, the median degranulation response in the allo-reactive CTL clones was > 7-fold higher than in the auto-reactive CTL clones. Whereas the median degranulation response was only 4.64% (range 1.75-34.0, n=9) among the auto-reactive CTL clones at 100 pM, the median response for allo-reactive CTL clones was 42.2% (range 12.7-72.6, n=20) even at the lowest peptide concentration of 1 pM. Thus, allo-reactive T cell clones specific for a self-peptide in complex with HLA-A*02:01 seem to be Poster no: 32
of higher avidity as compared to autologous clones specific for the same peptide-HLA complex, as indicated by manifold higher responses at low peptide concentrations and responses to 100-fold lower peptide concentrations. Immune checkpoint control molecules in chronic lymphocytic leukemia (CLL)
Karolinska Institutet, Dept. Of Oncology-Pathology Cancer Centrum Karolinska, CCK R8:01
Poster no: 30
Co-authors:
Characterization of T cell suppressive mechanisms in malignant ascites from ovarian carcinoma
Marzia Palma; Kia Heimersson; Lotta Hansson, Jeanette Lundin, Anders Österborg, Håkan Mellstedt
Chronic lymphocytic leukemia (CLL) patients have impaired humoral and cellular immune functions These are characterized by e.g. profound defects in T-cells such as immunologic synapse formation with antigen presenting cells , impaired signaling via the T cell receptor Simer Bains
(TCR) and cytotoxic function. CD28 is an important T-cell co-stimulatory molecule, constitutively expressed on almost all CD4+ T cells and Norsk senter for molekylærmedisin, UiO
on half of the CD8+ T cells. Upon interaction with the ligands B.7-1 (CD80) and/or B.7-2 (CD86), CD28 transduces a signal enhaning T-cell proliferation and cytokine secretion. In the absence of appropriate co-stimulation, TCR occupancy might lead to T-cell unresponsiveness or clonal anergy, in which T cells are unable to proliferate or secrete cytokines in response to a secondary stimulation. CTLA-4 (cytotoxic Co-authors:
T lymphocyte-associated antigen-4) is transiently expressed on activated T cells and has an inhibitory role in regulating T-cell activation Sheraz Yaqub, Line Bjørge, Erik Rokkones, Kjetil Tasken
upon ligand binding. The immune suppressive molecule programmed death-1 (PD-1) is expressed on activated CD4+ and CD8+ T cells, NK T cells, B cells as well as activated monocytes and DCs. The receptor inhibits T-cell function upon binding to its ligands B7-H1 (PD-L1, CD274) Background: Ovarian cancer (OC) is the sixth most common malignant neoplasm among females and the
and B7-DC (PDL2, CD273). Dysregulated expression of CTLA-4 and PD-1 on T cells of patients with CLL may have an impact on the T-cell leading cause of death from gynecological malignancies. Regardless of cytoreductive surgery and chemotherapeutic regimens, the overall responsiveness and could a mechanism of the immune deficiency in this disease. survival rate remains below 40%. This is mainly due to inherent or acquired drug resistance and the fact that most tumors have developed The aim of the present study was to evaluate the expression of immune checkpoint control molecules (CTLA-4, PD-1 and its ligands) and to an advanced stage at the time of diagnosis. Progressing OC spreads from the epithelium of the ovaries into the peritoneal cavity, and of immune activation markers (CD137, CD103, CD69) in CLL patients. Their possible role as prognostic and/or predictive factors of clinical leads to the accumulation of malignant ascites. The malignant ascitic fluid is an exudate and contains tumor, inflammatory and mesothelial response to treatment strategies targeting these molecules directly or indirectly will also be evaluated in further studies. In a pilot study, cells, representing the tumor microenvironment and battle zone for anti-tumor immune activity. The dynamic interaction between the we assessed the expression of CTLA4 and PD-1 and of the T-cell activation markers CD25, CD69, CD103, CD137 markers by flow-cytometry host immunity and the developing tumor is striking in many malignancies. Activation of adaptive immune cells in response to a tumor may on T cells from 10 indolent and 10 progressive CLL patients. CTLA-4 was found to be almost not expressed on CD4+ and CD8+ cells. The contribute to eradication of malignant cells. However, developing tumors employ different means to obstruct the action of the immune mean % of PD-1+ cells was 0.58 and 0.28 of the CD4+ and CD8+ T-cell populations, respectively, with no significant difference between system by hindering immunological surveillance and providing immunologic escape. Previous studies have shown that ascites in OC indolent and progressive patients. CD4+CD69+ were more frequently seen in progressive patients (p=0.017), while CD4+CD103+ cells were patients is immunosuppressive, though the mechanism has remained elusive.
more frequently noted in indolent patients (p=0.05). The number of patients will be extended as a basis for planning trials, evaluating Objective: The aim of this present study is to further characterize the suppressive effect of cell-free ascites fluid from OC patients using
immune-modulating treatment modalities in CLL.
modern techniques. By identifying inhibitors of effector T cell function, our intent is to see if there are any humoral factor(s) in the tumor microenvironment which may inhibit the clinically important anti-tumor immune responses mediated through cytotoxic T cells.
Results and future plans: Analysis of malignant ascites demonstrated a soluble factor(s) that inhibits T cell function and
proliferation which was not secreted by ovarian cancer cells. Future plans include a characterization of signaling pathways in T cells in the presence of malignant cell-free ascites supernatant. We also plan to perform T cell proliferation assays with add-back of inhibitors to different cytokines (IL-8, IL10, IL-6, etc.) known to be present in ascites, to see if this can somehow abolish the inhibitory effect that ascites supernatant exhibits on T cells proliferation.
Poster no: 33
T cells from non-tolerized repertoires provide tools to discover the immunopeptidome for use in
immunotherapy and vaccination
Department of Immunology, Institute for Cancer Research, Oslo University Hospital
Co-authors:
Sebastien Walchli, Lars-Egil Fallang, Weiwen Yang, Fridtjof Lund-Johansen, Ton N. Schumacher, Johanna Olweus
Epitope discovery is a bottleneck for development of immunotherapy and vaccination in cancer and infectious disease. Here, we describe a new technology for the rapid and direct identification of multiple naturally processed and immunogenic cytotoxic T cell (CTL) epitopes, as well as of the T cells recognizing the epitopes. The high-throughput technology was based on the transfection of monocyte-derived dendritic cells (DCs) with mRNA encoding the full-length target protein and subsequent co-culture with T cells. CTLs reactive to processed and presented epitopes were directly detected using a panel of peptide-HLA multimers containing computationally predicted epitopes. The approach was first tested in a setting in which foreign antigens were presented on self-HLA. DCs from Hepatitis C virus (HCV) seronegative HLA-A2pos donors were transfected with mRNA encoding full-length HCV non-structural (NS) protein 3 or core Ag. Collectively, the CTLs that were generated from two donors covered the majority of the tested 20 peptide specificities, including 5 novel epitopes in NS3, indicating that naïve T cell repertoires can be efficiently used for epitope discovery in foreign antigens. Next, we used T cells from HLA-A2neg donors for the detection of HLA-A2-bound peptides from two leukemia-associated differentiation antigens; CD20 and the novel cancer target myeloperoxidase (MPO). Human Leukocyte Antigen (HLA) molecules presenting peptides derived from shared tumor-associated self-antigens (self-TAA) represent attractive targets for T-cell based immunotherapy of cancer. However, self-tolerance and limitations in the sensitivity of mass spectrometry hampers the detection of such epitopes. Here, the DCs from HLA-A2negative donors were co-transfected with full-length transcripts of self-TAA and HLA-A2 to allow presentation of all naturally processed peptides from a pre-defined self-protein on foreign HLA. Remarkably, cytotoxic T cells (CTLs) were generated against 36 novel epitopes out of 50 peptides predicted to bind HLA-A2. These allo-restricted T cells showed exquisite peptide- and HLA-specificity, and responded strongly to HLA-A2 positive leukemic cells endogenously expressing CD20 or MPO. In conclusion, our data demonstrate that the repertoire of self-peptides presented on HLA class I has been underestimated, and that non-tolerized T cell repertoires can be utilized to efficiently and specifically target such self-TAA. Furthermore, naïve, non-tolerized T cell repertoires can be efficiently used to discover epitopes in foreign antigens using the described technology.
The approach was validated by discovery of a large number of novel epitopes in two leukemia-associated differentiation antigens; the novel cancer target myeloperoxidase and CD20, and in Hepatitis C virus.
Poster no: 34
Vector-encoded Helicobacter pylori neutrophil activating protein promotes maturation of dendritic
cells with Th-1 polarization and improved migration
Science for Life Laboratory, Uppsala University
Co-authors:
Mohanraj Ramachandran, Chuan Jin, Fredrik Eriksson, Magnus Essand
Helicobacter pylori (H. pylori) neutrophil activating protein (HP-NAP) is a major virulence factor involved in H. pylori infection. Both HP- NAP protein and oncolytic viruses encoding HP-NAP have been suggested as immunotherapeutic anti-cancer agents and adjuvants for vaccination but with little known about its mode of action to activate adaptive immunity. Dendritic cells (DCs) are key players in bridging innate and adaptive immune responses and here we aim to evaluate the effect of HP-NAP on DC maturation, migration and induction of adaptive immune responses. Maturation markers CD80, CD86, HLA-DR, CD40, CCR7 were upregulated on human DCs after treatment with supernatants from HP-NAP adenovirus-infected cells. HP-NAP-activated DCs had a Th1 cytokine secretion profile, with high IL-12 and low IL- 10 secretion and migrated towards CCL19. Antigen-specific T cells were efficiently expanded by antigen-presenting HP-NAP-activated DCs, which is an important property of functionally mature DCs. Furthermore, intradermal injections of HP-NAP-encoding adenovirus in C57BL/6 mice enhanced resident DC migration to draining lymph nodes, which was verified by imaging lymph nodes by two-photon microscopy and by phenotyping migrating cells by flow-cytometry. In conclusion, therapeutic effects of HP-NAP are mediated by maturation of DC and subsequent activation of antigen-specific T cells in addition to provoking innate immunity. PRODUCTIVE
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Source: http://www.cancersymposia.no/bilder/2014/NCSprogramSISTE1Juni.pdf