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The supplementary Figures 1 to 3 demonstrate the gating procedure post flow cytomety analysis.

Purpose:The chimeric antigen receptor (CAR) T-cell therapy has been effective for patients with CD19+ B-cell malignancies. Most studies have investigated the second-generation CARs with either CD28 or 4-1BB costimulatory domains in the CAR receptor. Here, we describe the first clinical phase I/IIa trial using third-generation CAR T cells targeting CD19 to evaluate safety and efficacy.Patients and Methods:Fifteen patients with B-cell lymphoma or leukemia were treated with CAR T cells. The patients with lymphoma received chemotherapy during CAR manufacture and 11 of 15 were given low-dose cyclophosphamide and fludarabine conditioning prior to CAR infusion. Peripheral blood was sampled before and at multiple time points after CAR infusion to evaluate the persistence of CAR T cells and for immune profiling, using quantitative PCR, flow cytometry, and a proteomic array.Results:Treatment with third-generation CAR T cells was generally safe with 4 patients requiring hospitalization due to adverse reactions. Six of the 15 patients had initial complete responses [4/11 lymphoma and 2/4 acute lymphoblastic leukemia (ALL)], and 3 of the patients with lymphoma were in remission at 3 months. Two patients are still alive. Best predictor of response was a good immune status prior to CAR infusion with high IL12, DC-Lamp, Fas ligand, and TRAIL. Responding patients had low monocytic myeloid-derived suppressor cells (MDSCs; CD14+CD33+HLA−DR−) and low levels of IL6, IL8, NAP3, sPDL1, and sPDL2.Conclusions:Third-generation CARs may be efficient in patients with advanced B-cell lymphoproliferative malignancy with only modest toxicity. Immune profiling pre- and posttreatment can be used to find response biomarkers.

Supplementary Figure S2. Oncolysis over time The human PaCa3 cell line was infected with 100MOI LOAd(-), LOAd700 or LOAd703 virus, or left uninfected. The cells were plated in a 96-well plate in quadruplicates and analyzed by MTS assay at three time points. The plates were analyzed by spectrophotometry at 490nm as per manufacturer instructions. Uninfected cells remained viable while infected cells were killed over time independently of type of LOAd virus demonstrating that the viruses have oncolytic capacity and that the transgene cassette does not interfere with oncolysis.

Detailed description of the patients and infused cells

Purpose: Pancreatic cancer is a severe indication with short expected survival despite surgery and/or combination chemotherapeutics. Checkpoint blockade antibodies are approved for several cancer indications, but pancreatic cancer has remained refractory. However, there are clinical data suggesting that stimulation of the CD40 pathway may be of interest for these patients. Oncolytic viruses armed with immunostimulatory genes represent an interesting approach. Herein, we present LOAd703, a designed adenovirus armed with trimerized CD40L and 4-1BBL that activates the CD40 and 4-1BB pathways, respectively. As many cells in the tumor stroma, including stellate cells and the infiltrating immune cells, express CD40 and some 4-1BB, we hypothesize that LOAd703 activates immunity and simultaneously modulates the biology of the tumor stroma.Experimental Design: Tumor, stellate, endothelial, and immune cells were infected by LOAd703 and investigated by flow cytometry, proteomics, and functional analyses.Results: LOAd703-infected pancreatic cell lines were killed by oncolysis, and the virus was more effective than standard-of-care gemcitabine. In in vivo xenograft models, LOAd703 efficiently reduced established tumors and could be combined with gemcitabine for additional effect. Infected stellate and tumor cells reduced factors that promote tumor growth (Spp-1, Gal-3, HGF, TGFβ and collagen type I), while chemokines were increased. Molecules involved in lymphocyte migration were upregulated on infected endothelial cells. Dendritic cells were robustly stimulated by LOAd703 to produce costimulators, cytokines and chemokines, and such DCs potently expanded both antigen-specific T cells and NK cells.Conclusions: LOAd703 is a potent immune activator that modulates the stroma to support antitumor responses. Clin Cancer Res; 23(19); 5846–57. ©2017 AACR.

Manufactured CAR batches were analyzed for the presence of CAR T cells and phenotyped for different markers as indicated in the Figure

There is a need for therapeutics with novel mechanisms-of-action for patients with advanced solid malignancies. In this first completed part of LOKON002, a phase I/II (NCT03225989) clinical study, the safety and clinical response to LOAd703 in combination with standard-of-care (SoC), or conditioning gemcitabine chemotherapy, was investigated. LOAd703 is an oncolytic adenovirus of serotype 5/35 modified to express two immunostimulatory transgenes, trimerized membrane-bound CD40L and 4-1BBL. In the completed part I (phase I and IIa) of the study, eligible patients with advanced pancreatic-, colorectal-, biliary- and ovarian cancer, were treated with increasing doses of LOAd703 (5 × 1010, 1 × 1011 and 5 × 1011 viral particles) using a standard 3+3 design. LOAd703 was administered intratumorally (ultrasound-guided) for up to 8 bi-weekly injections. The maximum tolerated dose tested was further evaluated in an extended dose cohort to confirm safety and assess preliminary clinical activity, and to select the best two indications for the part II of the study. Twenty-eight patients were enrolled in part I presented herein. Fifteen of these received LOAd703 combined with SoC, and thirteen in combination with conditioning gemcitabine. The most frequently reported treatment- or procedure-related adverse events were fever (82% of patients), chills (54%) and fatigue (43%). Four patients developed transient cytokine release syndrome. Twenty-five patients fulfilled the criteria to be evaluable for efficacy, which required receiving at least 3 doses of LOAd703 and results from a CT scan week 9. Partial response (PR) was achieved in two patients with pancreatic cancer treated at the highest LOAd703 dose level in combination with gemcitabine and nab-paclitaxel. One of the PRs was achieved at week 25, and the patient received one additional month of chemotherapy per protocol. This patient remained treatment-free for 17 months before chemotherapy was restarted due to progression, and was still alive at last follow-up 34 months after the study registration date. The other PR was achieved at week 17, two weeks after the last LOAd703 injection; disease progression was evident at week 33 and OS was 21 months. Three patients experienced stable disease for a minimum of 25 weeks. This included a patient with advanced ovarian cancer who showed SD at least until week 40, and had an OS of 40 months. No association between adverse events and response was identified. Anti-adenoviral antibody levels (IgG), which increased in all patients, could not be related to indices of clinical benefit. Based on the safety of LOAd703 at all dose levels studied, as well as evidence of objective clinical activity in patients with advanced pancreatic cancer, further disease-directed studies of intratumoral administration of LOAd703 are warranted. Citation Format: Amanda Hahn, Sandra Irenaeus, Jessica Wenthe, Emma Eriksson, Aglaia Schiza, Hanna Dahlstrand, Ulla Olsson-Strömberg, Johan Krause, Anders Sundin, Linda Sandin, Justyna Leja Jarblad, Maria Gustafsson Liljefors, Eric Rowinsky, Angelica Loskog, Gustav J. Ullenhag. A phase I/II clinical study of an oncolytic adenovirus expressing the immunostimulatory transgenes TMZ-CD40L and 4-1BBL in advanced solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT235.

Although CD19-targeting CAR T cell therapy has shown remarkable success against B cell malignancies there is still room for improvement. To improve long-term responses amongst lymphoma patients, mechanisms underlying initial response and tumor relapses must be addressed. Recent studies have highlighted the importance of the quality of the starting cell material for CAR T cell production and the composition of the CAR T cell infusion product for clinical response. In our study, we have evaluated the clinical response to third generation anti-CD19 CAR T cell therapy in 23 patients with B cell lymphoma. Further, we evaluated the CAR T cell infusion products using targeted single-cell RNA sequencing and multi-color flow cytometry to find potential T cell subsets associating with clinical response. Patients were divided into responders (CR + PR) and non-responders (SD + PD) at the 1-month follow-up after treatment. The CAR T cell products of both responding and non-responding patients were dominated by CD8+ CAR T cells. When comparing cells from the two patient groups we found that the presence of effector-like CD8+ CAR T cells with a high cytotoxic and cytokine secretion and low dysfunction gene expression profile were associated with clinical response. Furthermore, CD8+ CAR T cells of responders displayed a higher polyfunctional gene expression profile, which has previously been shown to associate with clinical response. In contrast, CD8+ CAR T cells from the infusion products of non-responders displayed an elevated dysfunction gene expression profile. Our study reveals that single-cell RNA expression analysis of individual CAR T cell infusion product can identify subsets of effector cells correlating with clinical response. This can be used as a guidance in the CAR T cell production processes, to obtain CAR T cells with the desired characteristics. Citation Format: Tina Sarén, Mohanraj Ramachandran, Gustav Gammelgård, Tanja Lövgren, Kristina Wikström, Jamileh Hashemi, Eva Freyhult, Claudio Mirabello, Åsa K Björklund, Håkan Ahlström, Rose-Marie Amini, Hans Hagberg, Angelica Loskog, Gunilla Enblad, Magnus Essand. Single-cell RNA analysis reveal effector-like CD8+ CAR-T cell subpopulations associated with response in lymphoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1793.

Ovarian cancer is commonly treated with debulking surgery, followed by a combination of a taxane and a platinum-based chemotherapy. However, >70% of patients relapse within 3 years with tumors resistant to platinum-based agents. 5-year survival is ranging from 30-40%. Thus, delaying recurrence of disease is a high unmet need in ovarian cancer. Combining the chemotherapy regime with immunostimulatory gene therapy utilizing oncolytic viruses as gene vehicles is an attractive treatment option targeting alternative pathways to induce tumor cell death, control tumor growth and prevent development of metastasis. LOAd703 is an oncolytic adenovirus derived from the LOAd platform of replication-restricted Ad5/35 adenoviruses. LOAd703 encodes for the human immunostimulatory genes trimerized, membrane-bound CD40L (TMZ-CD40L) and 4-1BBL, expressed under a CMV promotor which enables gene expression in both tumor cells and tumor stroma while replication is restricted to tumor cells. Herein, we investigated the effect of combining LOAd703 with paclitaxel and cisplatin in vitro and in vivo. Four human ovarian cancer cell lines (CAOV3, COV362, OVCAR3 and PEA2) were treated with LOAd703 alone, in combination with paclitaxel and/or cisplatin or were left untreated. LOAd703 efficiently killed the ovarian cancer cell lines by oncolysis, as evaluated by an MTS cell viability assay. This effect was further improved when LOAd703 was given in combination with paclitaxel and/or cisplatin. Flow cytometry revealed that the ability of LOAd703 to induce expression of the immunostimulatory transgenes TMZ-CD40L and 4-1BBL, was not negatively affected by the combination with paclitaxel and/or cisplatin. The combination treatment was further tested in an in vivo model where BALB/c nu/nu mice were xenografted with 5 million OVCAR3 cells. The mice were subsequently treated with LOAd703 (1 × 10e9 FFU/mouse), both alone as well as in combination with paclitaxel (6mg/kg) and cisplatin (2.5mg/mg). The treatments were given over the course of 4 weeks where LOAd703 was administered intratumorally twice per week while chemotherapy was given intraperitoneally once per week. The results showed that the combination of LOAd703 and the chemotherapeutics controlled tumor growth significantly better than either LOAd703 alone or the standard treatment with paclitaxel and cisplatin. In conclusion, LOAd703 efficiently killed ovarian cancer cell lines in vitro, and could potentiate the effect of standard-of-care chemotherapy. This effect was confirmed in vivo, in which the combination therapy had a better tumor control than the other treatment groups. A clinical trial is ongoing to confirm the effect of adding LOAd703 to different treatment regimes in ovarian cancer (NCT03225989). Citation Format: Sedigheh Naseri, Jonas Härdin, Emma Eriksson, Tanja Lövgren, Gustav Ullenhag, Angelica Loskog. Immunostimulatory gene therapy with oncolytic viruses potentiates the effect of paclitaxel and cisplatin in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 686.

In the LOKON002 phase I/II clinical trial (NCT03225989), therapy with LOAd703 (delolimogene mupadenorepvec) is investigated in combination with gemcitabine-based chemotherapy in patients with advanced cancer. LOAd703 is an immunostimulatory gene therapy utilizing an oncolytic adenovirus that is engineered to express two transgenes (trimerized membrane-bound CD40L (TMZ-CD40L) and 4-1BBL), which are key players in the induction of an anti-tumor immune response. Herein, we present the preliminary immunological evaluation of the first 27 patients enrolled in the trial. The primary tumor was pancreatic cancer (n=16), colorectal cancer (n=5), ovarian cancer (n=3) or biliary cancer (n=3). Patients received a maximum of eight intratumoral LOAd703 treatments, given biweekly, and chemotherapy was administered at the same time according to standard protocol. Matched tumor biopsies from the injected tumor lesion were taken at baseline and after six LOAd703 treatments, and analyzed for gene expression with nCounter® PanCancer Immune Profiling Panel from NanoString (n=12; 7 patients analyzed so far). Blood samples were collected for isolation of serum samples and peripheral blood mononuclear cells, and these were analyzed with multiplex assays (Meso Scale Diagnostics) (n=15) and flow cytometry (n=27), respectively. Preliminary results indicate that after treatment, most tumors displayed a strong inflammatory profile with an upregulation of gene profiles, which have been shown to predict responses to immune checkpoint inhibitor therapy (T cell inflamed profile (Ayers et al.) & T effector and IFNγ associated profile (Fehrenbacher et al.)). We also noted an upregulation of genes connected to dendritic cell activation and antigen presentation (MHC class I and II, CD80, CD86, TAP1/2), as well as adenoviral-response genes. Moreover, we observed systemic treatment effects after three LOAd703 treatments, as shown by an increase in serum levels of pro-inflammatory cytokines and chemokines (IFNγ, IL-15, CXCL10, CCL2, IL-8, IL-6), as well as an increased percentage of CD8+ effector memory T cells (CD45RA-CCR7-) and PD-1+ CD8+ T cells in the blood. The fractions of natural killer (NK) cells (CD14-CD3-CD56+CD16+) and M2-like macrophages (CD11b+CD163+) were reduced in the blood at that time point. In conclusion, LOAd703 therapy in combination with chemotherapy generated an inflamed tumor microenvironment in tumors that are normally seen as immunologically “cold”. Hence, LOAd703 may be able to prime tumors for immune checkpoint inhibitors or other immunotherapies, such as adoptive T or NK cell transfer. Citation Format: Jessica Wenthe, Emma Eriksson, Sedigheh Naseri, Linda Sandin, Amanda Hahn, Sandra Irenaeus, Anders Sundin, Justyna Leja-Jarblad, Gustav Ullenhag, Tanja Lövgren, Angelica Loskog. Immunostimulatory gene therapy targeting CD40/4-1BB in combination with chemotherapy induces an inflammatory gene profile in tumors from patients with advanced disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6663.

Immunostimulatory gene therapy utilizing oncolytic viruses (OVs) as gene vehicles is a promising immunotherapy for cancer. Since viruses are immunogenic, systemic delivery can be troublesome due to neutralizing antibodies. Nevertheless, local delivery by intratumoral injection seems to induce systemic immune reactions. In this study, we demonstrate a novel mechanism of action of armed OV therapy suggesting that exosomes released by tumor cells infected with armed OV may participate to activate the immune system and this may also support systemic immunity. Tumor cell-derived exosomes commonly exert immunosuppressive functions. We hypothesized that exosomes derived from OV-infected tumor cells may instead be immunostimulatory. Human melanoma cells were infected by OVs armed with costimulatory molecules CD40 ligand (CD40L) and 4-1BB ligand (4-1BBL). Exosomes were purified and investigated for the presence of CD40L/4-1BBL mRNA and protein, and for their capacity to stimulate immune responses. The results show that the exosomes cargo transgenes. The exosomes from CD40L/4-1BBL-expressing tumor cells, or the viruses themselves, could stimulate robust dendritic cell (DC) activation with an enhanced level of major histocompatibility complex (MHC) and costimulatory molecules. Hence, exosomes after OV infection can locally activate immune responses at the tumor site and encounter immune cells such as DCs., Graphical Abstract, Tumor cells infected with armed oncolytic LOAd703 viruses release exosomes expressing two costimulatory transgenes CD40L and 4-1BBL, and such nanovesicles can activate dendritic cells in vitro. This behavior of tumor-derived exosomes is immunostimulatory rather than their immunosuppressive nature and can elicit antitumor immune responses.

AdCD40L is a replication-deficient virus carrying the gene for CD40 ligand which has previously been evaluated in patients with urothelial cancer and malignant melanoma. Herein, we present the results of repeated intratumoral injections of AdCD40L in seven patients with metastatic solid cancer. One patient who developed urothelial cancer derived from a renal transplant was treated with repeated injections of AdCD40L alone. The remaining patients suffered from cholangiocarcinoma, kidney, breast, rectal, or ovarian cancer and received AdCD40L repeatedly (4x) in combination with cyclophosphamide. The treatment was safe and generally well-tolerated. Two patients had clinical benefit of the treatment and one of them was accepted for re-treatment. Circulating proinflammatory cytokines were commonly increased after treatment, but save for TNFα, significances were not reached which could be due to the low number of patients. Similar to earlier findings in AdCD40L-treated melanoma patients, IL8 plasma levels were high in the present study. In conclusion, gene therapy by repeated intratumoral AdCD40L injections alone, or in combination with cyclophosphamide, is feasible and safe in patients with solid cancers. The potential of intratumoral CD40L gene transfer as treatment of cancer was illustrated by the clinical improvement in two out of seven patients.

Immunotherapy aims at activating or re-activating the immune system to create an anti-tumor response that could lead to eradication of the tumor cells. Central is the activation of dendritic cells (DCs) capable of presenting tumor antigens to T cells that can expand and kill tumor cells. However, the tumor is a harsh environment for the adaptive immune system and many of the conditions needed for the establishment of an immune response are lacking. Oncolytic viral therapy presents an opportunity to overcome the immunosuppression in tumors by destroying tumor cells, releasing antigens and immunostimulatory factors. The effects of the oncolytic virus can be further amplified by the introduction of transgenes expressed by the virus. LOAd oncolytic adenoviruses belong to a platform of Ad5/35 adenoviruses that have replication restricted to tumor cells. The viruses express human immunostimulatory transgenes under a CMV promotor that enables expression in all infected cells. LOAd703, encoding trimerized (TMZ)-CD40L and 4-1BBL, is under clinical investigation in solid tumors. Herein we present the data from the investigation of our novel oncolytic adenovirus LOAd732 that encodes for three human transgenes; TMZ-CD40L, 4-1BBL and IL-2. Infection of the human cell lines Mel526 and Mel624 with LOAd732 led to cell killing as was evaluated by MTS viability assay and to expression of the transgenes, TMZ-CD40L, 4-1BBL and IL-2 evaluated by flow cytometry and ELISA. Moreover the immmunostimulatory function of LOAd732 was investigated by infecting immature dendritic cells, differentiated from CD14+ monocytes using GM-CSF and IL-4. Flow cytometry assay revealed that DCs infected with LOAd732 showed a mature profile with expression of activation markers and co-stimulatory molecules such as CD80, CD83, CD86, CD70, ICAM-1, and MHC molecules. Analysis of supernatants from the cultures with multiplex immunoassay (MDS technology) showed that LOAd732 DCs produce IL-12p70, IL-15, IL-21 and CXCL10. To assess the functionality of the DCs infected by LOAd732, the capacity of the cells to expand antigen-specific T cells were investigated in a CMV model system where DCs from CMV positive donors are pulsed with a CMV peptide and co-cultured with autologous T cells. LOAd732 infected DCs could expand CMV-specific CD8+ T cells. Since the tumor microenvironment often is immunosuppressive, TGF-β and IL-10 was added to the co-cultures to model these conditions. The addition of the immunosuppressing cytokines did not affect LOAd732 capacity to expand the antigen-specific T cells. In conclusion, LOAd732 infected DCs express activation markers and co-stimulatory molecules and produce cytokines necessary for the initiation of an immune response. Moreover the DCs are functional and can expand antigen-specific T cells and protect them from TGF-β and IL-10 suppression. Citation Format: Jessica Wenthe, Emma Eriksson, Tanja Lövgren, Rafael Moreno, Ramon Alemany, Angelica Loskog. LOAd732 - a novel oncolytic adenovirus with enhanced immunostimulatory properties and resistance to immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3562.

Low tumor immunogenicity and a cold tumor microenvironment are two main characteristics occurring in different cancer types responsible for immunotherapy resistance. Immunostimulatory gene therapy using oncolytic viruses (OVs) has been developed as a new promising cancer therapy for its capacity to induce an anti-tumor immune response and sensitize the non-responding patients to checkpoint blockade therapy. Lokon adenoviruses (LOAds) are serotype 5/35 oncolytic adenoviruses that in addition to their oncolytic capacity encode for immunostimulatory molecules. Here we have evaluated the anti-tumor effects of trimerized membrane-bound (TMZ)-CD40L-armed LOAd700 and TMZ-CD40L/4-1BBL-armed LOAd703 in a variety of solid tumors to understand the underlying tumor modulating mechanisms. A panel of eight different human solid cancer cell lines (T24, PEA2, Mel526, H727, Skov3, MiaPaca2, H29 and A549) was infected with LOAd(-), a virus without transgenes, LOAd700 or LOAd703 and assessed for their response in different assays. MTS assay was used to measure the cell viability, flow cytometry for evaluating the cell surface markers and luminescent assay for measuring apoptosis (caspase 3/7 activity). LOAd viruses efficiently killed all tested cell lines. However, the oncolytic capacity of CD40L-armed LOAd700 and LOAd703 was significantly higher than LOAd(-) in two cell lines, T24 and PEA2, which were found to express the CD40 receptor. Death receptor TRAIL-R1 was significantly upregulated in T24 infected with LOAd703 and PEA2 infected with either LOAd700 or LOAd703. TRAIL-R2 was upregulated post LOAd infection in 7/8 cell lines where the upregulation was significant in T24, PEA2 and H29. Interestingly, the immunogenic cell death marker calreticulin, a damage-associated molecular pattern (DAMP) known as an “eat-me” signal to dendritic cells, was upregulated post LOAd infection in all cell lines. The increase in calreticulin level was statistically significant in 4/8 cell lines (T24, PEA2, Mel526 and H29) post LOAd700 and/or LOAd703 infection. Furthermore, LOAd700 and LOAd703 increased the effector caspase 3/7 activity in 5/8 cell lines (T24, PEA2, Mel526, Skov3 and MiaPaCa2) indicating an augmented apoptosis level. In conclusion, CD40L-armed LOAd viruses were, in addition to their high oncolytic capacity, demonstrated to induce apoptosis. Upregulation of cell surface calreticulin as well as the death receptors TRAIL-R1 and -R2 indicated generation of a more immunogenic phenotype of cancer cells. Thus, our findings suggest that CD40L-armed LOAd viruses could be inducers of apoptosis and immunogenic cell death in a variety of cancer types with an augmented effect in cancer cells expressing the CD40 receptor. Citation Format: Sedigheh Naseri, Mariela Mejia Cordova, Jessica Wenthe, Ann-Charlotte Hellström, Tanja Lövgren, Angelica Loskog, Emma Eriksson, Gustav Ullenhag. Induction of tumor cell apoptosis & immunogenic cell death by CD40L-armed oncolytic adenoviruses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3501.

Mebendazole is used extensively for treatment of local gut helminthic and invasive echinococcus infections. Anticancer effects of mebendazole have been shown in experimental cancer models and in case studies in patients with advanced cancer. Given these observations, the aims of this study were to investigate safety and efficacy of individualized dosed mebendazole in the cancer indication. Patients with treatment refractory gastrointestinal cancer were treated with individualized dose adjusted mebendazole up to 4 g/day to target a serum concentration of 300 ng/ml. Efficacy and safety were assessed by CT-scans, clinical surveillance and blood sampling. Eleven patients were included in the study and 10 started the treatment phase. Two patients stopped treatment prior to and the remaining eight after tumour evaluation by CT-scan at 8 weeks, all due to progressive disease. Four patients also fulfilled criteria suggested for hyperprogression. Only five patients reached the target serum-mebendazole concentration. No severe adverse effects were observed. Individualized dose adjusted mebendazole is safe and well tolerated in patients with advanced cancer but all patients experienced rapid progressive disease. New approaches such as prodrug development and combination with other anticancer drugs seem needed for further exploration of mebendazole as an anticancer drug.

We recently showed that the anti-helminthic compound mebendazole (MBZ) has immunomodulating activity in monocyte/macrophage models and induces ERK signalling. In the present study we investigated whether MBZ induced ERK activation is shared by other tubulin binding agents (TBAs) and if it is observable also in other human cell types. Curated gene signatures for a panel of TBAs in the LINCS Connectivity Map (CMap) database showed a unique strong negative correlation of MBZ with MEK/ERK inhibitors indicating ERK activation also in non-haematological cell lines. L1000 gene expression signatures for MBZ treated THP-1 monocytes also connected negatively to MEK inhibitors. MEK/ERK phosphoprotein activity testing of a number of TBAs showed that only MBZ increased the activity in both THP-1 monocytes and PMA differentiated macrophages. Distal effects on ERK phosphorylation of the substrate P90RSK and release of IL1B followed the same pattern. The effect of MBZ on MEK/ERK phosphorylation was inhibited by RAF/MEK/ERK inhibitors in THP-1 models, CD3/IL2 stimulated PBMCs and a MAPK reporter HEK-293 cell line. MBZ was also shown to increase ERK activity in CD4+ T-cells from lupus patients with known defective ERK signalling. Given these mechanistic features MBZ is suggested suitable for treatment of diseases characterized by defective ERK signalling, notably difficult to treat autoimmune diseases.

AdCD40L is a replication-deficient virus carrying the gene for CD40 ligand which has previously been evaluated in patients with urothelial cancer and malignant melanoma. Herein, we present the results of repeated intratumoral injections of AdCD40L in seven patients with metastatic solid cancer. One patient who developed urothelial cancer derived from a renal transplant was treated with repeated injections of AdCD40L alone. The remaining patients suffered from cholangiocarcinoma, kidney, breast, rectal, or ovarian cancer and received AdCD40L repeatedly (4x) in combination with cyclophosphamide. The treatment was safe and generally well-tolerated. Two patients had clinical benefit of the treatment and one of them was accepted for re-treatment. Circulating proinflammatory cytokines were commonly increased after treatment, but save for TNFα, significances were not reached which could be due to the low number of patients. Similar to earlier findings in AdCD40L-treated melanoma patients, IL8 plasma levels were high in the present study. In conclusion, gene therapy by repeated intratumoral AdCD40L injections alone, or in combination with cyclophosphamide, is feasible and safe in patients with solid cancers. The potential of intratumoral CD40L gene transfer as treatment of cancer was illustrated by the clinical improvement in two out of seven patients.

Multiple myeloma (MM) is a plasma cell malignancy that is characterized by immune dysregulation. MM is commonly treated with immunomodulating agents, but still remains incurable. Herein, we proposed and evaluated immunostimulatory Lokon oncolytic adenoviruses (LOAd) for MM treatment. LOAd viruses are serotype 5/35 chimera, which enables infection of hematopoietic cells. Oncolysis is restricted to cells with a dysregulated retinoblastoma protein pathway, which is frequently observed in MM. Further, LOAd viruses are armed with human immunostimulatory transgenes: trimerized membrane-bound CD40L (LOAd700, LOAd703) and 4-1BBL (LOAd703). LOAd viruses were assessed in a panel of MM cell lines (ANBL-6, L363, LP-1, OPM-2, RPMI-8226, and U266-84). All cells were sensitive to infection, leading to viral replication and cell killing as analyzed by quantitative PCR and viability assay. Transgene expression was verified post infection with flow cytometry. Cell phenotypes were further altered with a downregulation of markers connected to MM progression (ICAM-1, CD70, CXCL10, CCL2, and sIL-2Rα) and an upregulation of the death receptor Fas. In a co-culture of immune and MM cells, LOAd viruses promoted activation of cytotoxic T cells as seen by higher CD69, CD107a, and IFNγ expression. This was most prominent with LOAd703. In conclusion, LOAd viruses are of interest for MM therapy.

The success of cancer immunotherapy relies on the knowledge of the tumor microenvironment and the immune evasion mechanisms in which the tumor, stroma, and infiltrating immune cells function in a complex network. The potential barriers that profoundly challenge the overall clinical outcome of promising therapies need to be fully identified and counteracted. Although cancer immunotherapy has increasingly been applied, we are far from understanding how to utilize different strategies in the best way and how to combine therapeutic options to optimize clinical benefit. This review intends to give a contemporary and detailed overview of the different roles of immune cells, exosomes, and molecules acting in the tumor microenvironment and how they relate to immune activation and escape. Further, current and novel immunotherapeutic options will be discussed.

This phase 1 study sought to characterize the safety, tolerability, and pharmacokinetic behavior of VLX1570, a small molecule inhibitor of the deubiquitinases (DUBs) that remove sterically bulky ubiquitin chains from proteins during processing in the19S regulatory subunit of the proteasome, in patients with relapsed and refractory multiple myeloma (MM). Fourteen patients were treated with escalating doses of VLX1570 ranging from 0.05 to 1.2 mg/kg as a brief intravenous (IV) infusion on Days 1, 2, 8, 9, 15, and 16 of a 28-day cycle. Due to its poor aqueous solubility, VLX1570 was formulated in polyethylene glycol, polyoxyethylated castor oil, and polysorbate 80 and administered as a brief intravenous (IV) infusion via a central venous catheter. Anti-myeloma effects were noted at doses at or above 0.6 mg/kg, however, two patients treated at the 1.2 mg/kg dose level experienced severe, abrupt, and progressive respiratory insufficiency, which was associated with diffuse pulmonary infiltrates on imaging studies, similar to those rarely noted with bortezomib and other inhibitors of the 20S proteasome, culminating in death. Although the contribution of VLX1570s formulation to the pulmonary toxicity could not be ruled out, the severity and precipitous nature of the toxicity and the steep relationship between dose and toxicity, the study was discontinued. Despite the severe pulmonary toxicity noted with VLX1570, efforts directed at identifying DUB inhibitors with greater therapeutic indices appear warranted based on the unique mechanism of action, robustness of preclinical antitumor activity, and activity of the DUB inhibitors in MM resistant to PIs targeting the 20S proteasome subunit. Funding Agencies|Uppsala University; Vivolux, AB

Pancreatic ductal adenocarcinoma (PDAC) is resistant to PD1/PDL1 blocking antibodies. The purpose of this study was to evaluate if immunotherapy with LOAd703, an oncolytic adenovirus armed with trimerized CD40L and 4-1BBL, can sensitize PDAC to mAbs targeting PD1/PDL1. The high content of myeloid-derived suppressor cells (MDCSs) in the tumor microenvironment may in part explain the limited anti-tumor T cell response in PDAC. We have previously shown that gemcitabine can reduce MDSCs in patients with PDAC, with the T cell proliferative capacity remaining intact. Further, LOAd703 has been demonstrated to efficiently and robustly kill PDAC cells and stimulate the maturation of dendritic cells (DCs), which, in turn, induces T cell activation. Patients with advanced PDAC, colorectal, biliary, and ovarian cancer (NCT03225989) were treated with intratumoral injections of LOAd703 combined with appropriate chemotherapy such as gemcitabine ± nab-paclitaxel for PDAC patients, or combined with gemcitabine if there was no standard of care. Blood sampling was performed for immune cell profiling and anti-adenovirus antibodies (flow cytometry, ELISA). Tumor biopsies were also analyzed for mRNA expression (NanoString; PanCancer Immune Profiling Panel). The study was approved by the ethical review board and the Swedish Medical Products Agency. The dose of LOAd703 was escalated (5 × 10e10 VP, 1 × 10e11 VP, 5 × 10e11VP) in separate cohorts of new patients and administered every other week. To date, blood samples from 23 patients have been analyzed by flow cytometry to profile immune cells. The mean percentages of monocytic- and granulocytic MDSCs, Tregs and M2-like myeloid cells were significantly decreased after treatment initiation. The effector memory (EM) and central memory (CM) CD8+ T cells were significantly increased, while naïve and CM cells were increased among CD4 T cells. Both CD4 and CD8 T cells expressing ICOS were present post-treatment but only CD8 T cells showed significant elevation of PD1. The NanoString data were analyzed using published mRNA immune signatures (bioinformatics). LOAd703 in combination with chemotherapy inflamed tumor lesions as shown by increased markers of the T cell inflamed signature (16 genes), T effector cell signature (19 genes), expanded immune signature (25 genes) and IFNg-related gene signature (12 genes) (week 1 versus week 13). Anti-adenovirus antibody titers increased in all patients after treatment initiation. The antibody levels did not correlate with the dose of virus, radiological response to treatment, nor overall survival. The results presented herein show that LOAd703 combined with chemotherapy enhances immune reactivity in patients with immune cold tumors as demonstrated by increases in EM and CM T cells in the peripheral blood, while suppressive immune cells were decreased. Further, the tumor milieu was inflamed post treatment as shown by mRNA profiling. Hence, LOAd703 may sensitize immune cold tumors to mAbs targeting PD1/PDL1. Citation Format: Jessica Wenthe, Emma Eriksson, Linda Sandin, Tanja Lövgren, Justyna Leja Jarblad, Hanna Dahlstrand, Ulla Olsson-Strömberg, Aglaia Schiza, Anders Sundin, Sandra Irenaeus, Eric Rowinsky, Gustav Ullenhag, Angelica Loskog. Inflaming advanced solid tumors including pancreatic cancer using LOAd703, a TMZ-CD40L/4-1BBL-armed oncolytic virus [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-018.

Immunostimulatory gene therapy using oncolytic viruses - OV - for combined gene transfer and tumor-specific oncolysis is a promising immunotherapy for cancer. Due to neutralizing antibodies in the blood, OVs are commonly administered by intratumoral delivery. The immune activation is both local and systemic as activated dendritic cells in the injected lesion travel to lymph nodes to activate tumor-reactive T cells that can in turn act on both injected and non-injected lesions. In the present project, we demonstrate that OV-infected tumor cells released exosomes that cargo mRNA and protein coded by the human immunostimulatory genes expressed by the OV. Tumor cell-derived exosomes are commonly immunosuppressive because they cargo immunosuppressive molecules expressed by the tumor. We hypothesized that exosomes derived from OV-infected tumor cells may instead be immunostimulatory. To investigate the role of exosomes derived from OV-treated tumors, human melanoma Mel526 cells were infected by delolimogene mupadenorepvec -LOAd703 - which is an adenoviral serotype 5/35 OV armed with costimulatory molecules CD40L and 4-1BBL. LOAd703 could induce expression of CD40L and 4-1BBL on the melanoma cell surface and kill the cells by replication-induced oncolysis after 48-72 hours culture as evaluated by MTS. Exosomes were purified prior oncolysis and investigated for the presence of CD40L and 4-1BBL mRNA and protein, and for their capacity to stimulate immune responses. The results demonstrated that the exosomes can indeed cargo CD40L and 4-1BBL both as mRNA as shown by PCR, and protein as shown by ELISA, western blot, flow cytometry and electron microscopy post LOAd703 tumor cell infection. Furthermore, the exosomes from CD40L and 4-1BBL-expressing tumor cells could stimulate robust dendritic cell activation with enhanced levels of MHC class I and II as well as costimulatory molecules CD80, CD86 and CD83. In contrast, MHC class I and the costimulatory molecules were reduced on dendritic cells stimulated with exosomes derived from non-infected tumor cells, which is in line with previous knowledge of exosomes as immunosuppressants in the tumor. However, PD-L1 was increased on dendritic cells stimulated with the exosomes from CD40L and 4-1BBL-expressing tumor cells. Hence, LOAd703 therapy may benefit from combination with PD-L1 or PD-1 checkpoint blockade. In conclusion, tumor-derived exosomes released by OV-infected tumor cells can cargo the immunostimulatory transgenes expressed by the OVs and are then converted from immunosuppressive to immune activating exosomes as shown by their capacity to activate immune cells such as dendritic cells. Exosomes that cargo immunostimulatory transgenes from OV-infected tumor cells may support the systemic immunity gained by OVs after local intratumoral delivery. Citation Format: Alireza Labani-Motlagh, Sedigheh Naseri, Jessica Wenthe, Emma Eriksson, Angelica Loskog. Exosomes derived from tumor cells treated with immunostimulatory oncolytic virotherapy exert immune activation instead of immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1689.

The purpose was to evaluate the potential of diffusion-weighted-magnetic resonance imaging (DW-MRI) and 18F-fludeoxy-glucose-positron emission tomography integrated with CT (FDG-PET/CT) for prediction of overall survival (OS) following AdCD40L-immunotherapy in patients with metastatic malignant melanoma (MMM). Twenty-four patients with refractory MMM were treated with immunostimulatory AdCD40L gene therapy in a phase I/IIa study. Pre-therapeutic DW-MRI and FDG-PET/CT were performed and then repeated at 5 and 9 weeks post-treatment. Evaluation was conducted according to RECIST 1.1 and EORTC criteria. Apparent diffusion coefficient (ADC), true diffusion coefficient (D), maximum standardized uptake value (SUVmax) were measured in the injected lesions. Fold changes (F) in ADC (F ADC), D (F D), SUVmax (F SUVmax) were statistically assessed. F D ≥ 1 and F ADC ≥ 1 were associated with better OS in scans at week 5 and 9 respectively. F SUVmax was not correlated to OS. F ADC ≥ 1 in both post-treatment scans and F D ≥ 1 at week 5 were related to a significant decrease of size of the injected lesions. These results suggest that in patients with MMM treated with AdCD40l, functional parameters of DW-MRI are better early predictors of OS than the established metabolic and morphologic criteria for FDG-PET/CT and MRI, respectively.

Background and aims: The simultaneous measurement of many proteins is now possible using multiplex assays. In this pilot study we investigated a total of 124 proteins in plasma from chronic myeloid leukemia (CML) patients with the purpose of identifying proteins that are differently expressed at diagnosis and after tyrosine kinase inhibitor (TKI) treatment initiation. Methods: Samples were taken from 14 CML patients at diagnosis and after three months of TKI treatment (imatinib or dasatinib). Samples were analyzed by Mesoscale Discovery, Myriad RBM MAP technology and Olink Proseek. Results: Multiple plasma proteins were differentially expressed before and after initiation of TKI therapy. Protein patterns demonstrated a possible shift towards Th1-immunity and reduced angiogenic stimuli. Further, some plasma proteins were identified that can be of potential interest to study further for biologic, prognostic or therapeutic significance such as E-selectin, uPAR, growth hormone and carbonic anhydrase IX. Conclusions: Plasma proteomics seems feasible and useful in CML patients, both for studying patterns of protein expression and for identifying single proteins differentially expressed before and after treatment. Plasma proteomics may be useful to map disease activity and biological processes. Hence, plasma proteomics can be used to understand drug mechanisms and treatment responses in CML. (C) 2016 Elsevier Ltd. All rights reserved.

The purpose of this study was to evaluate the anti-tumor immune response using an oncolytic adenovirus encoding TMZ-CD40L and 4-1BBL (LOAd703) with or without PD-1 or PD-L1 checkpoint blockade. Oncolytic immunostimulatory viruses are currently being evaluated for their capacity to sensitize checkpoint blockade resistant patients to checkpoint blockade therapy since resistance is associated with a non-inflamed tumor phenotype. Herein we evaluated LOAd703, with or without PD-1 or PD-L1 blockade, in preclinical melanoma models. A murine version of LOAd703 (mLOAd703) expressing murine TMZ-CD40L and 4-1BBL was evaluated in the B16-hCD46/C57BL6 model. Immune activation was determined by flow cytometry and multiplex assays from Meso Scale Discovery. Note that adenoviruses do not replicate in murine cells, hence, the treatment effect of mLOAd703 in animal models is only due to expression of the immunostimulatory transgenes. Single and twin-tumor models were used to evaluate the effect of mLOAd703 (1 × 10e9 ffu/treatment) by repeated intravenous (i.v.) or intratumoral (i.t.) injections (one lesion). I.v. treatment did not control tumor growth likely due to the anti-adenovirus antibodies post LOAd703 exposure as evaluated by ELISA. In contrast, i.t. treatment could control growth of the injected tumor as well as hamper growth of the non-injected tumor demonstrating that it is more important to induce systemic immunity from a highly active tumor site (high virus load) than to reach all lesions with a small number of viral particles as shown by PCR. Further, mice with single or twin-tumors were treated with i.t. mLOAd703 (1 × 10e9 ffu), anti-PD-1 or anti- PD-L1 (intraperitoneal 5 mg/kg/injection), or a combination of either antibody and mLOAd703. Anti-PD-1 and PD-L1 monotherapy had only a limited effect on tumor growth in this model, whereas the combination with mLOAd703 could control the growth of the injected tumor and further delay the growth of the non-injected tumor compared to mLOAd703 alone. In agreement, flow cytometry analysis of the tumor biopsies showed an increase of CD8+ T cells in both tumors. In line with tumor inflammation, serum levels of T effector cytokines such as IFNg and TNFa were highest in animals treated with the combination. In conclusion, local mLOAd703 virotherapy induced a systemic anti-tumor immune response in the B16-hCD46 melanoma model. The abscopal effect was further increased by combining mLOAd703 with anti-PD-1 or anti-PD-L1. LOAd703 (i.t.) is currently in clinical development with and without PD-L1. Citation Format: Jessica Wenthe, Sedigheh Naseri, Ann-Charlotte Hellström, Rafael Moreno, Gustav Ullenhag, Ramon Alemany, Tanja Lövgren, Emma Eriksson, Angelica Loskog. Abscopal effect using intratumoral oncolytic virotherapy (LOAd703) is enhanced by anti-PD-1 or anti-PD-L1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 915.

716 Background: Pancreatic ductal adenocarcinoma (PDAC) has been highly resistant to immunotherapeutics to date. LOAd703, an oncolytic adenovirus with transgenes encoding TMZ-CD40L and 4-1BBL, has been shown to lyse tumor cells selectively, induce anti-tumor cytotoxic T-cell responses, reduce myeloid-derived suppressor cell (MDSC) infiltration, and induce tumor regression in preclinical studies. Methods: In this phase I/II trial, patients with unresectable or metastatic PDAC are treated with LOAd703 intratumoral injections and standard nab-paclitaxel/gemcitabine (nab-P/G) chemotherapy. Starting on cycle 1 day 15 of nab-P/G, LOAd703 is injected with image guidance into the primary pancreatic tumor or a metastasis every 2 weeks for 6 injections. In the event of sustained tumor control, subjects are eligible to receive 6 more injections. Three dose levels of LOAd703 are being investigated using a BOIN dose escalation design. Primary endpoints are safety and feasibility. Secondary endpoints include response rate and overall survival. Results: To date, 13 subjects are evaluable for safety and feasibility. Three patients were treated at dose 1 (5x10e10 VP), 4 subjects at dose 2 (1x10e11 VP), and 6 subjects at dose 3 (5x10e11 VP). The most common adverse events (AEs) attributed to LOAd703 have been fever, chills, nausea, and increased transaminases. AEs have been transient and grade 1-2, with the exception of a grade 3 transaminase elevation in 1 subject receiving dose 3 (the only dose-limiting toxicity observed thus far). During protocol treatment, circulating MDSCs decreased in 8/13 subjects while effector memory T-cells increased in 10/13. ELISPOT analyses showed a rise in tumor antigen-specific T-cells in 10/13 subjects. At the lowest dose level, best response was stable disease, and 6/10 patients who received higher LOAd703 doses have had partial responses. Only 1 patient has had progressive disease as best response. Conclusions: Adding LOAd703 to nab-P/G has been safe and feasible. Treatment-emergent immune responses have been demonstrated in most subjects, with a notable proportion having objective anti-tumor responses. Clinical trial information: NCT02705196.

IL-6 plays a role in cancer pathogenesis via its connection to proteins involved in the formation of desmoplastic stroma and to immunosuppression by driving differentiation of myeloid suppressor cells together with TGF-β. Inhibition of IL-6 signaling in the tumor microenvironment may, thus, limit desmoplasia and myeloid suppressor cell differentiation. CD40 signaling can further revert myeloid cell differentiation toward antitumor active phenotypes. Hence, the simultaneous use of IL-6 blockade with CD40 stimuli may tilt the tumor microenvironment to promote antitumor immune responses. In this paper, we evaluated the mechanisms of LOAd713, an oncolytic adenovirus designed to block IL-6R signaling and to provide myeloid cell activation via a trimerized membrane-bound isoleucine zipper (TMZ) CD40L. LOAd713-infected pancreatic cancer cells were killed by oncolysis, whereas infection of stellate cells reduced factors involved in stroma formation, including TGF-β-1 and collagen type I. Virus infection prevented IL-6/GM-CSF–mediated differentiation of myeloid suppressors, but not CD163 macrophages, whereas infection of dendritic cells led to upregulation of maturation markers, including CD83, CD86, IL-12p70, and IFN-γ. Further, IL-6R blockade prevented upregulation of programed death ligand 1 (PD-L1) and PD-1 on the stimulated dendritic cells. These results suggest that LOAd713 can kill infected tumor cells and has the capacity to affect the tumor microenvironment by stimulating stellate cells and myeloid suppressors with TMZ-CD40L and IL-6R blockade. Gene transfer of murine TMZ-CD40L prolonged survival in an animal model. LOAd713 may be an interesting therapeutic option for cancers connected to IL-6 signaling, such as pancreatic cancer.

Purpose: The chimeric antigen receptor (CAR) T-cell therapy has been effective for patients with CD19+ B-cell malignancies. Most studies have investigated the second-generation CARs with either CD28 or 4-1BB costimulatory domains in the CAR receptor. Here, we describe the first clinical phase I/IIa trial using third-generation CAR T cells targeting CD19 to evaluate safety and efficacy. Patients and Methods: Fifteen patients with B-cell lymphoma or leukemia were treated with CAR T cells. The patients with lymphoma received chemotherapy during CAR manufacture and 11 of 15 were given low-dose cyclophosphamide and fludarabine conditioning prior to CAR infusion. Peripheral blood was sampled before and at multiple time points after CAR infusion to evaluate the persistence of CAR T cells and for immune profiling, using quantitative PCR, flow cytometry, and a proteomic array. Results: Treatment with third-generation CAR T cells was generally safe with 4 patients requiring hospitalization due to adverse reactions. Six of the 15 patients had initial complete responses [4/11 lymphoma and 2/4 acute lymphoblastic leukemia (ALL)], and 3 of the patients with lymphoma were in remission at 3 months. Two patients are still alive. Best predictor of response was a good immune status prior to CAR infusion with high IL12, DC-Lamp, Fas ligand, and TRAIL. Responding patients had low monocytic myeloid-derived suppressor cells (MDSCs; CD14+CD33+HLA−DR−) and low levels of IL6, IL8, NAP3, sPDL1, and sPDL2. Conclusions: Third-generation CARs may be efficient in patients with advanced B-cell lymphoproliferative malignancy with only modest toxicity. Immune profiling pre- and posttreatment can be used to find response biomarkers.

Immune escape mechanisms promote tumor progression and are hurdles of cancer immunotherapy. Removing immunosuppressive cells before treatment can enhance efficacy. Tyrosine kinase inhibitors (TKI) may be of interest to combine with immunotherapy, as it has been shown that the inhibitor sunitinib reduces myeloid suppressor cells in patients with renal cell carcinoma and dasatinib promotes expansion of natural killer–like lymphocytes in chronic myeloid leukemia (CML). In this study, the capacity of dasatinib and imatinib to reduce myeloid suppressor cells and to induce immunomodulation in vivo was investigated ex vivo. Samples from CML patients treated with imatinib (n = 18) or dasatinib (n = 14) within a Nordic clinical trial (clinicalTrials.gov identifier: NCT00852566) were investigated for the presence of CD11b+CD14−CD33+ myeloid cells and inhibitory molecules (arginase I, myeloperoxidase, IL10) as well as the presence of natural killer cells, T cells (naïve/memory), and stimulatory cytokines (IL12, IFNγ, MIG, IP10). Both imatinib and dasatinib decreased the presence of CD11b+CD14−CD33+ myeloid cells as well as the inhibitory molecules and the remaining myeloid suppressor cells had an increased CD40 expression. Monocytes also increased CD40 after therapy. Moreover, increased levels of CD40, IL12, natural killer cells, and experienced T cells were noted after TKI initiation. The presence of experienced T cells was correlated to a higher IFNγ and MIG plasma concentration. Taken together, the results demonstrate that both imatinib and dasatinib tilted the immunosuppressive CML tumor milieu towards promoting immune stimulation. Hence, imatinib and dasatinib may be of interest to combine with cancer immunotherapy. Mol Cancer Ther; 14(5); 1181–91. ©2015 AACR.

The classical Hodgkin lymphoma (cHL) tumor microenvironment shows an ongoing inflammatory response consisting of varying degrees of infiltrating eosinophils, mast cells, macrophages, regulatory T lymphocytes (Tregs), and activated lymphocytes surrounding the malignant cells. Herein, different immune signatures are characterized and correlated with treatment outcome.Tumor-infiltrating leukocytes were phenotyped in biopsies from 459 patients with cHL. Time to progression (TTP) (primary progression, relapse, or death from cHL) and overall survival were analyzed using Cox proportional hazards regression.The leukocyte infiltration in the microenvironment was highly diverse between patients and was categorized in 4 immune signatures (active, anergic, innate, or mixed). A high proportion of Tregs (anergic) resulted in shorter TTP (median 12.9-year follow-up) in age-adjusted analyses (hazard ratio = 1.82; 95% confidence interval 1.05-3-15). Epstein-Barr virus (EBV)-positive cases had higher proportions of macrophages and activated lymphocytes than EBV negative, but neither of those leukocytes predicted prognosis.Abundant Tregs (anergic signature) indicate a shorter TTP, particularly in younger patients. This is probably due to a reduced ability of the immune system to attack the tumor cells. Our data warrant further investigation if these suggested immune signatures could predict outcome of immunotherapy such as immune checkpoint inhibitors.

Background Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Methods Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0–300 IU/mL) and length of expansion (10–20 days) on the phenotype of the T-cell products were analyzed. Results High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (TSCM, CD95+CD45RO−CD45RA+CD27+) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. Discussion The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing., published version, peerReviewed

Mebendazole (MBZ), a drug commonly used for helminitic infections, has recently gained substantial attention as a repositioning candidate for cancer treatment. However, the mechanism of action behind its anticancer activity remains unclear. To address this problem, we took advantage of the curated MBZ-induced gene expression signatures in the LINCS Connectivity Map (CMap) database. The analysis revealed strong negative correlation with MEK/ERK1/2 inhibitors. Moreover, several of the most upregulated genes in response to MBZ exposure were related to monocyte/macrophage activation. The MBZ-induced gene expression signature in the promyeloblastic HL-60 cell line was strongly enriched in genes involved in monocyte/macrophage pro-inflammatory (M1) activation. This was subsequently validated using MBZ-treated THP-1 monocytoid cells that demonstrated gene expression, surface markers and cytokine release characteristic of the M1 phenotype. At high concentrations MBZ substantially induced the release of IL-1β and this was further potentiated by lipopolysaccharide (LPS). At low MBZ concentrations, cotreatment with LPS was required for MBZ-stimulated IL-1β secretion to occur. Furthermore, we show that the activation of protein kinase C, ERK1/2 and NF-kappaB were required for MBZ-induced IL-1β release. MBZ-induced IL-1β release was found to be dependent on NLRP3 inflammasome activation and to involve TLR8 stimulation. Finally, MBZ induced tumor-suppressive effects in a coculture model with differentiated THP-1 macrophages and HT29 colon cancer cells. In summary, we report that MBZ induced a pro-inflammatory (M1) phenotype of monocytoid cells, which may, at least partly, explain MBZ's anticancer activity observed in animal tumor models and in the clinic.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) signaling pathway is in clinical use, but its effect on vascular function and the tumor microenvironment is poorly understood. Here, we investigate cross-talk between VEGF and proinflammatory TNF-α signaling in endothelial cells and its impact on leukocyte recruitment. We found that cotreatment with VEGF decreased TNF-α-induced Jurkat cell adhesion to human microvascular endothelial cells by 40%. This was associated with inhibition of TNF-α-mediated regulation of 86 genes, including 2 T-lymphocyte-attracting chemokines, CXCL10 and CXCL11 [TNF-α concentration 1 ng/ml; 50% inhibition/inhibitory concentration (IC50) VEGF, 3 ng/ml]. Notably, VEGF directly suppressed TNF-α-induced gene expression through negative cross-talk with the NF-κB-signaling pathway, leading to an early decrease in IFN regulatory factor 1 (IRF-1) expression and reduced phosphorylation of signal transducer and activator of transcription 1 (p-Stat1) at later times. Inhibition of VEGF signaling in B16 melanoma tumor-bearing mice by sunitinib treatment resulted in up-regulation of CXCL10 and CXCL11 in tumor vessels, accompanied by up to 18-fold increased infiltration of CD3(+) T-lymphocytes in B16 tumors. Our results demonstrate a novel role of VEGF in negative regulation of NF-κB signaling and endothelial activation in the tumor microenvironment and provide evidence that pharmacological inhibition of VEGF signaling enhances T-lymphocyte recruitment through up-regulation of chemokines CXCL10 and CXCL11.

Checkpoint blockade antibody therapy has revolutionized the treatment of metastatic melanoma. However, up to 50% of patients establish treatment resistance, which is frequently connected to a non-T cell inflamed phenotype. Poor T cell activation and infiltration is suggested to be caused by a lack of functional dendritic cells (DCs) that are crucial for T cell priming. Indeed, in progressing melanoma patients, immature DCs are often increased in the tumor lesion and draining lymph node. Herein, we investigated the effect of an immunostimulatory oncolytic adenovirus LOAd703 in a humanized melanoma-DC model. LOAd703 (serotype Ad5/35) carries two immunostimulatory transgenes; trimerized membrane-bound (TMZ)-CD40 ligand and 4-1BBL. Viral replication is restricted to tumor cells but transgene expression is controlled by a CMV promoter, which allows expression also in healthy cells of the surrounding tumor microenvironment (TME). LOAd703 efficiently infected and killed the human melanoma cell line 526-mel as evaluated by MTS viability assay. Infected 526-mel cells expressed both TMZ-CD40L and 4-1BBL as verified by flow cytometry. To investigate LOAd703’s effect on DC maturation, immature DCs were differentiated from CD14+ monocytes using GM-CSF and IL-4 and infected with LOAd703 or stimulated with MEGACD40L®, which is a highly active CD40L oligomer. Flow cytometry analysis revealed that LOAd703-infected DCs upregulated the expression of maturation markers and co-stimulatory molecules in a similar manner as high doses of MEGACD40L®. These included CD83, CD80, CD86, CCR7, ICAM-1 and MHC molecules. Interestingly, CD70 that is required for CD27 stimuli of T cells and lowers T cell receptor signaling threshold, was highly upregulated on the DCs using LOAd703, but not MEGACD40L®. Tumor cells are often immunosuppressive which may reduce the capacity of LOAd703 to activate DCs. To model this, immature DCs were co-cultured with LOAd703-infected 526-mel cells, which is also better mimicking the events in the TME. Co-cultured DCs upregulated the same markers as before, but this upregulation was twofold higher for CD83 and CD80. In agreement with the previous observation that CD40L signaling alone did not induce CD70 expression, CD70 expression on DCs was lower in the co-culture, indicating that a direct infection of the virus and potentially signaling through TLRs together with CD40L signaling is required to induce CD70 on DCs or that it may be inhibited by immunosuppressive factors released by the tumor. LOAd703-matured DCs also upregulated PD-L1 and thus it would be of interest to combine LOAd703 therapy with PD-L1/PD-1 blockade. In conclusion, LOAd703 killed human melanoma cells and induced the expression of TMZ-CD40L and 4-1BBL in infected cells. Moreover, LOAd703 infection activated DCs to express costimulatory molecules, as well as CCR7, which is essential for a systemic immune response. Citation Format: Jessica Wenthe, Tanja Lövgren, Mantas Šilanskas, Emma Eriksson, Angelica Loskog. Activation of dendritic cells by immunostimulatory CD40L/4-1BBL-encoding oncolytic virotherapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5018.

Despite the impressive effects of checkpoint blockade antibody therapy seen across indications, many patients remain resistant to treatment. PD-1/PD-L1 blockade aims to restore the function of anergic tumor-infiltrating T cells, thereby inducing an efficient anti-tumor response. Thus, a prerequisite to benefit from checkpoint blockade is the presence of T cells in the tumor. One promising strategy to prime non-inflamed tumors for checkpoint blockade is the use of oncolytic viruses, which can additionally be engineered to express immunostimulatory molecules to further induce immune activation and T cell infiltration. In this study, we are investigating the use of the oncolytic adenovirus mLOAd703 expressing a murine trimerized membrane-bound (TMZ)-CD40L and 4-1BBL in the syngeneic murine B16 melanoma model. Adenoviruses cannot replicate in murine cells, but the immunostimulatory effect of the murine transgenes TMZ-CD40L and 4-1BBL can still be evaluated. LOAd703 (serotype 5/35) targets human CD46 for viral entry. Thus, a B16 cell line expressing human CD46 was used to allow infection. Upon infection with mLOAd703, B16-hCD46 cells not only expressed the transgenes TMZ-CD40L and 4-1BBL, but also changed their phenotype to become more immunogenic by upregulating co-stimulatory molecules CD80 and CD86, as well as MHC molecules. Moreover, expression of the death receptor Fas was increased, altogether potentially making the tumor cells more susceptible to T cell-mediated killing. Strikingly, two markers (CD44 and beta-3 integrin) associated with metastasis were reduced upon virus infection. Previous in vivo studies in immunocompetent C57BL6 mice with a predecessor of mLOAd703, which only expresses murine TMZ-CD40L, led to an increase of CD8+ T cells and CD11b+MHCII+ antigen presenting cells in the tumors. However, these cells also upregulated PD-1 or PD-L1, respectively, which warrants a combination with P-D1/PD-L1 checkpoint blockade. To evaluate this combination, mice with established B16-hCD46 tumors were treated with mLOAd703 (intratumoral 6x, 1x10e9 IU/mouse/injection), checkpoint blockade antibody (anti-mouse PD-1 or PD-L1, i.p. 6x 5mg/kg/mouse/injection; BioXcell), or treated in combination with mLOAd703 plus anti-PD-1 or mLOAd703 plus anti-PD-L1. Monotherapy with checkpoint blockade antibodies did not reduce tumor growth in this model. However, mLOAd703 reduced tumor growth as a monotherapy, but this reduction was even further enhanced in combination with both anti-PD-1 and anti-PD-L1 therapy. In conclusion, mLOAd703 could infect B16 cells expressing CD46, leading to TMZ-CD40L and 4-1BBL transgene expression. In addition, infected tumor cells became more immunogenic and reduced molecules involved in metastasis. In vivo studies revealed that mLOAd703 therapy sensitized PD-1/PD-L1-resistant mice to checkpoint blockade therapy. Citation Format: Jessica Wenthe, Sedigheh Naseri, Ann-Charlotte Hellström, Emma Eriksson, Angelica Loskog. CD40L/4-1BBL virotherapy enhances efficacy of checkpoint blockade therapy in a resistant melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4092.

In recent years, cancer immunotherapies significantly advanced the clinical management of metastatic melanoma. In particular, treatment with immune checkpoint inhibitors increased survival rates compared with standard therapy. However, not all patients benefit from checkpoint blockade. Primary treatment resistance is connected to poor T-cell infiltration in the tumors. This can be due to limited activation of antigen-presentation cells such as dendritic cells (DCs) and the high threshold of activating low-affinity T-cells that may respond to the tumor antigens. CD27 signaling in T-cells during activation lowers the T-cell receptor signaling threshold, which may be important to activate tumor-targeting T-cells. Secondary resistance to checkpoint blockade therapy includes loss of MHC-I on the tumor cells. Hence, inducing natural killer (NK) cell activation in parallel to antitumor T-cells may be crucial. LOAd703 was designed to optimize antitumor immune activation. LOAd703 is an oncolytic adenovirus (serotype Ad5/35) carrying two immunostimulatory transgenes; a trimerized membrane-bound CD40 ligand (TMZ-CD40L) and the full-length 4-1BB ligand (4-1BBL) (patent filed: PCT/EP2015/057489). The viral replication is restricted to tumor cells with a hyperphosphorylated retinoblastoma pathway due to a deletion in E1A, but transgenes are expressed under the control of a cytomegalovirus (CMV) promoter, which enables transgene expression even in the surrounding tumor microenvironment. Herein, we investigated LOAd703 in a melanoma model and its immunostimulatory effect on DC maturation to induce antigen-specific T-cell responses. LOAd703 infected the human melanoma cell line 526-mel and efficiently induced tumor cell death as evaluated by MTS viability assay. The viability of infected cells was reduced to 15% at 72 hours post infection compared to uninfected cells. Transgene expression of both TMZ-CD40L and 4-1BBL was confirmed by flow cytometry post infection. To evaluate the immunostimulatory capacity of LOAd703, immature DCs were differentiated from CD14+ monocytes using granulocyte-macrophage colony-stimulating factor and interleukin-4 and infected with virus. LOAd703-infected DCs upregulated the expression of maturation markers, such as CD83, CD86 and MHC molecules as analyzed by flow cytometry. Interestingly, CD70 that is required for CD27 stimuli of T-cells was highly upregulated on the DCs using LOAd703. Furthermore, the chemokine receptor CCR7 and the adhesion molecule ICAM-1 were increased upon LOAd703 infection, which are crucial for lymph node homing and the initiation of a systemic response. Next, the functional capacity of LOAd703-matured DCs to induce antigen-specific T-cell responses was assessed in a CMV model, in which CMV-peptide pulsed DCs are utilized to induce expansion of CMV-specific T-cells. LOAd703-matured DCs from CMV+ donors were pulsed with CMV-peptide and co-cultured with autologous peripheral blood mononuclear cells for 11 days. LOAd703-matured DCs were able to expand CMV-specific T-cells, but to a lower degree than the positive control Poly I:C matured DCs. However, the CMV-specific T-cells expanded from the positive control had higher expression of PD-1 compared to the LOAd703 group, indicating that LOAd703 leads to the expansion of less exhausted and potentially more functional T-cells. Moreover, LOAd703 also induced a massive expansion of NK cells, which is probably driven by 4-1BBL. In conclusion, LOAd703 killed human melanoma cells by oncolysis and induced the expression of TMZ-CD40L and 4-1BBL in infected cells. Furthermore, LOAd703 infection activated DCs to express costimulatory molecules including high levels of CD70 and CCR7, which in turn could promote the expansion of antigen-specific T-cells with low PD-1 expression, as well as the potent expansion of NK cells. Citation Format: Jessica Wenthe, Mantas Šilanskas, Emma Eriksson, Angelica Loskog. Combating primary and secondary checkpoint blockade resistance using immunostimulatory CD40L/4-1BBL-encoding oncolytic virotherapy for melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A119.

Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for multiple sclerosis (MS) with now more than 600 documented cases in the medical literature. Long-term remission can be achieved with this therapy, but when is it justified to claim that a patient is cured from MS? In attempt to answer this question, the outcome of the Swedish patients is described, mechanisms behind the therapeutic effect are discussed and new tools for demonstration of absence of disease have been developed.In Swedish patients treated with HSCT for aggressive MS, disease free survival was 68 % at five years, and no patient progressed after three years of stable disease. Presence of gadolinium enhancing lesions prior to HSCT was associated with a favorable outcome (disease free survival 79 % vs 46 %, p=0.028). There was no mortality and no patient required intensive care.The immune system of twelve of these patients was investigated further. In most respects HSCT-treated patients were similar to healthy controls, demonstrating normalization. In the presence of a potential antigen, leukocytes from HSCT-treated patients ceased producing pro-inflammatory IL-17 and increased production of the inhibitory cytokine TGF-β1 suggesting restoration of tolerance.Cytokine levels and biomarkers of tissue damage were investigated in cerebrospinal fluid from a cohort of MS patients. The levels were related to clinical and imaging findings. A cytokine signature of patients with relapsing-remitting MS could be identified, characterized by increased levels of CCL22, CXCL10, sCD40L, CXCL1 and CCL5 as well as down-regulation of CCL2. Further, we could demonstrate that active inflammation in relapsing-remitting MS is a tissue damaging process, with increased levels of myelin basic protein and neurofilament light. Importantly, relapsing-remitting MS patients in remission displayed no tissue damage. In secondary progressive MS, moderate tissue damage was present without signs of active inflammation.From a clinical vantage point, it seems that we confidently can claim cure of relapsing-remitting MS patients after five years absence of disease activity. The new tools for evaluation of disease can strengthen this assertion and may enable earlier prediction of outcome.

T-cell immunotherapy is a promising approach to treat disseminated cancer. However, it has been limited by the ability to isolate and expand T cells restricted to tumour-associated antigens. Using ex vivo gene transfer, T cells from patients can be genetically engineered to express a novel T cell receptor or chimeric antigen receptor to specifically recognize a tumour-associated antigen and thereby selectively kill tumour cells. Indeed, genetically engineered T cells have recently been successfully used for cancer treatment in a small number of patients. Here we review the recent progress in the field, and summarize the challenges that lie ahead and the strategies being used to overcome them.