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PDF file - 48K, Description of disease at the time of first treatment.

PDF file - 1MB, PBMCs of a healthy donor was stimulated with supernatant collected from virus (CGTG-401 or control virus not coding CD40L) infected A549 lung cancer cells and IL-8 production was assessed 12, 24, 48, and 72 h later with FACSArray. The increase demonstrated at later time points indicates biological activity of the virus produced CD40L.

PDF file - 1.1MB, (A) Patient serum samples were analyzed for Th1 induced cytokines interferon-� (IFN-�), tumor necrosis factor-� (TNF-�) and interleukin-2 (IL-2) and Th2 cytokines interleukin-4 (IL-4), interleukin-5 (IL-5) and Interleukin 10 (IL-10). The ratio between Th1 and Th2 type cytokines was assessed. Cytokines levels are reported relative to their baseline level, which was set as 1 and a ratio between Th1/Th2 was calculated for each time point. (B) Malignant ascites of patient R8 and pleural effusion of patient T181 were analyzed for Th1 induced cytokines IFN-� and IL-2, and for Th2 induced IL-10 before and after treatment.

PDF file - 1.2MB, Blood samples for all 9 patients were collected before (baseline) and at several time points after the treatment. Soluble CD40L (sCD40L) and RANTES concentrations in the serum were assessed. Data are presented as median � SD.

PDF file - 64K, Adverse reactions. 9 patients total, 6 received concomitant low-lose cyclophosphamide 50 mg/d, 3 did not. Numbers of patients experiencing the reaction at any time during their treatment (including 28d follow-up) is shown. The data is shown for the two subgroups separately in parentheses (cyclophosphamide/none).

PDF file - 41K, Response evaluation of injected and non-injected lesions.

PDF file - 58K, Prior therapies.

PDF file - 2MB, Serum levels for (A) IL-6, (B) IL-8, (C) IL-10, (D) IL-12, (E) TNF-alpha, and (F) IFN-gamma was assessed before and after the treatment. Data are presented separately for patients receiving virus treatment with or without concomitant low dose cyclophosphamide (50 mg/d) and shown as median � SD. NA, sample not available.

PDF file - 69K, Description of historical controls (Pesonen et al. Gene Ther 2010;17:892-904) treated with unarmed oncolytic adenovirus Ad5/3-cox2L-D24.

Malignant mesothelioma (MM) is a rare cancer type caused mainly by asbestos exposure. The median overall survival time of a mesothelioma cancer patient is less than 1-year from diagnosis. Currently there are no curative treatment modalities for malignant mesothelioma, however treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase life expectancy. Pemetrexed-Cisplatin is the only standard of care (SoC) chemotherapy for malignant mesothelioma, but the median PFS/OS (progression-free survival/overall survival) from the initiation of treatment is only up to 12 months. Therefore, new treatment strategies against malignant mesothelioma are in high demand. ONCOS-102 is a dual targeting, chimeric oncolytic adenovirus, coding for human GM-CSF. The safety and immune activating properties of ONCOS-102 have already been assessed in phase 1 study (NCT01598129). In this preclinical study, we evaluated the antineoplastic activity of combination treatment with SoC chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and ONCOS-102 in xenograft BALB/c model of human malignant mesothelioma. We demonstrated that ONCOS-102 is able to induce immunogenic cell death of human mesothelioma cell lines in vitro and showed anti-tumor activity in the treatment of refractory H226 malignant pleural mesothelioma (MPM) xenograft model. While chemotherapy alone showed no anti-tumor activity in the mesothelioma mouse model, ONCOS-102 was able to slow down tumor growth. Interestingly, a synergistic anti-tumor effect was seen when ONCOS-102 was combined with chemotherapy regimens. These findings give a rationale for the clinical testing of ONCOS-102 in combination with first-line chemotherapy in patients suffering from malignant mesothelioma.

Multiple oncolytic viruses (OV) are currently in clinical development as cancer immunotherapy candidates, due to their high immunogenicity and immune activation capacity. However, only anecdotal clinical evidence for OV-mediated tumor-specific immune responses exists. Peptide vaccines are highly tumor-specific, but the low immunogenicity and lack of CD8+ T-cell responses limit the clinical utility of this class of cancer immunotherapy. We have developed a unique cancer immunotherapy platform, PeptiCRAd™, which utilizes a highly immunogenic genetically engineered oncolytic adenovirus to deliver tumor-specific peptides to the immune system. This creates robust Th1 biased cellular immune responses against multiple tumor-specific targets simultaneously. This study compares the immune activation characteristics of intratumorally administered PeptiCRAd-1 to intratumoral OV treatment and standard intradermal peptide vaccination in a humanized mouse melanoma model. PeptiCRAd™ is an immunotherapy platform where multiple tumor-specific peptides are adsorbed onto the negatively charged capsid of genetically engineered oncolytic adenovirus via electrostatic interactions. Our lead clinical candidate PeptiCRAd-1 is based on a state-of-the-art oncolytic adenovirus coding for human CD40L and OX40L transgenes, coated with poly-lysine extended NY-ESO-1 and MAGE-A3 peptides. NOD/Shi-scid/IL-2Rγnull immunodeficient mice were humanized using hematopoietic stem cells (CD34+, HLA-B35+) isolated from human cord blood. A375 melanoma tumors were implanted subcutaneously and treated either with PeptiCRAd-1 or the naked virus. Peptide vaccines were given intradermally with Poly-IC as an adjuvant. Secondary tumors were implanted into the contralateral flank two days after the treatments were stopped. No treatments for secondary tumors were given. Peripheral blood mononuclear cells (PBMCs) and tumor infiltrating CD8+ lymphocytes (TILs) were analyzed for NY-ESO-1 and MAGE-A3 specific CD8+ T-cells by flow cytometry with dextramer analysis. Different immune cell subsets among PBMCs and TILs were assessed. All active treatments increased the number of immune cells in primary tumors in comparison to mock treated animals. Both OV and PeptiCRAd-1 treated animals showed more T-cells (CD3, CD4, CD8) in primary tumors in comparison to peptide vaccine or mock treated animals post treatment. Furthermore, the number of T regulatory cells (CD3+/CD4+/FoxP3+) was smaller in OV and PeptiCRAd-1 treated primary tumors in comparison to primary tumors from peptide vaccine or mock treated animals. This suggests that intratumorally administered immunogenic adenovirus (either naked virus or PeptiCRAd-1) modulates the tumor microenvironment by reducing local immune-suppression. Unlike OV treated animals, PeptiCRAd-1 treated animals showed more CD4+ and CD8+ T-cells in untreated secondary tumors than in treated primary tumors, suggesting that tumor-targeting via peptide-coating of the virus was critically important for the induction of an effect in distant untreated tumors. Furthermore, PeptiCRAd-1 treated animals had more NYESO-specific CD8+ T-cells in blood post priming (mean= 4.3% of total CD8+ cells) in comparison to OV treated (mean= 0.6%) or peptide vaccine treated (mean= 0.6%) animals. PeptiCRAd™ is superior to naked oncolytic adenovirus or standard peptide vaccination in triggering systemic tumor-targeted CD8+ T-cell responses and infiltration of CD8+ TILs into untreated distant tumors. The data suggest that PeptiCRAd improves the tumor targeting specificity of a standard OV. This easily adaptable technology could potentially be used to improve the tumor-focused immune responses generated by any oncolytic adenovirus currently in development. Importantly, PeptiCRAd™ could potentially be used for personalized tumor vaccination by delivering patient-specific neoantigens. A phase I clinical trial with PeptiCRAd-1 in several solid tumor indications is under preparation. Citation Format: Erkko Ylosmaki, Tuuli Ranki, Petri Priha, Charlotta Backman, Matthew Vaughn, Vincenzo Cerullo, Sari Pesonen. Local treatment with PeptiCRAd-1, a novel cancer immunotherapy approach, mediates a systemic antitumour CD8+ T-cell response and infiltration of CD8+ and CD4+ T-cells into distant untreated tumors in a clinically relevant humanized mouse melanoma model [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 B123.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

We conducted a phase I study with a granulocyte macrophage colony stimulating factor (GMCSF)-expressing oncolytic adenovirus, ONCOS-102, in patients with solid tumors refractory to available treatments. The objectives of the study were to determine the optimal dose for further use and to assess the safety, tolerability and adverse event (AE) profile of ONCOS-102. Further, the response rate and overall survival were evaluated as well as preliminary evidence of disease control. As an exploratory endpoint, the effect of ONCOS 102 on biological correlates was examined. The study was conducted using a classic 3 + 3 dose escalation study design involving 12 patients. Patients were repeatedly treated intratumorally with ONCOS-102 plus daily low-dose oral cyclophosphamide (CPO). Tumor response was evaluated with diagnostic positron emission tomography (PET) and computed tomography (CT). Tumor biopsies were collected at baseline and after treatment initiation for analysis of immunological correlates. Peripheral blood mononuclear cells (PBMCs) were collected at baseline and during the study to assess antigen specificity of CD8+ T cells by interferon gamma (IFNγ) enzyme linked immunospot assay (ELISPOT). No dose limiting toxicity (DLT) or maximum tolerated dose (MTD) was identified for ONCOS-102. Four out of ten (40 %) evaluable patients had disease control based on PET/CT scan at 3 months and median overall survival was 9.3 months. A short-term increase in systemic pro-inflammatory cytokines and a prominent infiltration of TILs to tumors was seen post-treatment in 11 out of 12 patients. Two patients showed marked infiltration of CD8+ T cells to tumors and concomitant systemic induction of tumor-specific CD8+ T cells. Interestingly, high expression levels of genes associated with activated TH1 cells and TH1 type immune profile were observed in the post-treatment biopsies of these two patients. ONCOS-102 is safe and well tolerated at the tested doses. All three examined doses may be used in further development. There was evidence of antitumor immunity and signals of clinical efficacy. Importantly, treatment resulted in infiltration of CD8+ T cells to tumors and up-regulation of PD-L1, highlighting the potential of ONCOS-102 as an immunosensitizing agent for combinatory therapies with checkpoint inhibitors. NCT01598129 . Registered 19/04/2012

Malignant mesothelioma (MM) is a rare cancer type caused mainly by asbestos exposure. The median overall survival time of a mesothelioma cancer patient is less than 1-year from diagnosis. Currently there are no curative treatment modalities for malignant mesothelioma, however treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase life expectancy. Pemetrexed-Cisplatin is the only standard of care (SoC) chemotherapy for malignant mesothelioma, but the median PFS/OS (progression-free survival/overall survival) from the initiation of treatment is only up to 12 months. Therefore, new treatment strategies against malignant mesothelioma are in high demand. ONCOS-102 is a dual targeting, chimeric oncolytic adenovirus, coding for human GM-CSF. The safety and immune activating properties of ONCOS-102 have already been assessed in phase 1 study (NCT01598129). In this preclinical study, we evaluated the antineoplastic activity of combination treatment with SoC chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and ONCOS-102 in xenograft BALB/c model of human malignant mesothelioma. We demonstrated that ONCOS-102 is able to induce immunogenic cell death of human mesothelioma cell lines in vitro and showed anti-tumor activity in the treatment of refractory H226 malignant pleural mesothelioma (MPM) xenograft model. While chemotherapy alone showed no anti-tumor activity in the mesothelioma mouse model, ONCOS-102 was able to slow down tumor growth. Interestingly, a synergistic anti-tumor effect was seen when ONCOS-102 was combined with chemotherapy regimens. These findings give a rationale for the clinical testing of ONCOS-102 in combination with first-line chemotherapy in patients suffering from malignant mesothelioma.

Malignant mesothelioma is a rare cancer type with no effective treatment therapies so far. Most of the mesothelioma cases are caused due to asbestos exposure. The median survival time of mesothelioma cancer patient is short, around one year from diagnosis and conventional treatments such as surgery, chemotherapy and radiotherapy can help only to improve mesothelioma prognosis and finally increase patient's life expectancy. Unfortunately, currently there is no cure for mesothelioma, and new treatment strategies against mesothelioma are in high demand. ONCOS-102 is a double targeted, chimeric oncolytic adenovirus, coding for human GM-CSF. Its potency as an antitumor agent has been reported in pre-clinical and clinical studies. In this study we evaluated the anticancer activity of combined therapy: first-line chemotherapy (Pemetrexed, Cisplatin, Carboplatin) with ONCOS-102 in a human malignant mesothelioma xenograft model in BALB/c mice. ONCOS-102 was able to kill all human mesothelioma cell lines in vitro and exhibited anti-tumor activity in treatment refractory H226 mesothelioma xenograft model. Current standard of care chemotherapy regimens for malignant mesothelioma presented no anti-tumor efficacy in tested mesothelioma model. Importantly, a synergistic anti-tumor effect was shown when ONCOS-102 was combined with chemotherapy regimens. Our findings support the clinical use of ONCOS-102 with first-line chemotherapy against malignant mesothelioma.

The immunosuppressive environment of advanced tumors is a primary obstacle to the efficacy of immunostimulatory and vaccine approaches. Here, we report an approach to arm an oncolytic virus with CD40 ligand (CD40L) to stimulate beneficial immunologic responses in patients. A double-targeted chimeric adenovirus controlled by the hTERT promoter and expressing CD40L (CGTG-401) was constructed and nine patients with progressing advanced solid tumors refractory to standard therapies were treated intratumorally. No serious adverse events resulting in patient hospitalization occurred. Moderate or no increases in neutralizing antibodies were seen, suggesting effective Th1 immunologic effects. An assessment of the blood levels of virus indicated 17.5% of the samples (n = 40) were positive at a low level early after treatment, but not thereafter. In contrast, high levels of virus, CD40L, and RANTES were documented locally at the tumor. Peripheral blood mononuclear cells were analyzed by IFN-γ ELISPOT analysis and induction of both survivin-specific and adenovirus-specific T cells was seen. Antitumor T-cell responses were even more pronounced when assessed by intracellular cytokine staining after stimulation with tumor type–specific peptide pools. Of the evaluable patients, 83% displayed disease control at 3 months and in both cases in which treatment was continued the effect was sustained for at least 8 months. Injected and noninjected lesions responded identically. Together, these findings support further clinical evaluation of CGTG-401. Cancer Res; 72(7); 1621–31. ©2012 AACR.

Augmenting antitumor immunity is a promising way to enhance the potency of oncolytic adenoviral therapy. Granulocyte-macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific CD8(+) cytotoxic T-lymphocytes. Serotype 5 adenoviruses (Ad5) are commonly used in cancer gene therapy. However, expression of the coxsackie-adenovirus receptor is variable in many advanced tumors and preclinical data have demonstrated an advantage for replacing the Ad5 knob with the Ad3 knob. Here, a 5/3 capsid chimeric and p16-Rb pathway selective oncolytic adenovirus coding for GMCSF was engineered and tested preclinically. A total of 21 patients with advanced solid tumors refractory to standard therapies were then treated intratumorally and intravenously with Ad5/3-D24-GMCSF, which was combined with low-dose metronomic cyclophosphamide to reduce regulatory T cells. No severe adverse events occurred. Analysis of pretreatment samples of malignant pleural effusion and ascites confirmed the efficacy of Ad5/3-D24-GMCSF in transduction and cell killing. Evidence of biological activity of the virus was seen in 13/21 patients and 8/12 showed objective clinical benefit as evaluated by radiology with Response Evaluation Criteria In Solid Tumors (RECIST) criteria. Antiadenoviral and antitumoral immune responses were elicited after treatment. Thus, Ad5/3-D24-GMCSF seems safe in treating cancer patients and promising signs of efficacy were seen.

Cancer gene therapy consists of numerous approaches where the common denominator is utilization of vectors for achieving therapeutic effect. A particularly potent embodiment of the approach is virotherapy, in which the replication potential of an oncolytic virus is directed towards tumor cells to cause lysis, while normal cells are spared. Importantly, the therapeutic effect of the initial viral load is amplified through viral replication cycles and production of progeny virions. All cancer gene therapy approaches rely on a sufficient level of delivery of the anticancer agent into target cells. Thus, enhancement of delivery to target cells, and reduction of delivery to non-target cells, in an approach called transductional targeting, is attractive. Both genetic and non-genetic retargeting strategies have been utilized. However, in the context of oncolytic viruses, it is beneficial to have the specific modification included in progeny virions and hence genetic modification may be preferable. Serotype chimerism utilizes serotype specific differences in receptor usage, liver tropism and seroprevalence in order to gain enhanced infection of target tissue. This review will focus on serotype chimeric adenoviruses for cancer gene therapy applications.

It has been proposed that human tumors contain stem cells that have a central role in tumor initiation and posttreatment relapse. Putative breast cancer stem cells may reside in the CD44+CD24−/low population. Oncolytic adenoviruses are attractive for killing of these cells because they enter through infection and are therefore not susceptible to active and passive mechanisms that render stem cells resistant to many drugs. Although adenoviruses have been quite safe in cancer trials, preclinical work suggests that toxicity may eventually be possible with more active agents. Therefore, restriction of virus replication to target tissues with tissues-specific promoters is appealing for improving safety and can be achieved without loss of efficacy. We extracted CD44+CD24−/low cells from pleural effusions of breast cancer patients and found that modification of adenovirus type 5 tropism with the serotype 3 knob increased gene delivery to CD44+CD24−/low cells. α-Lactalbumin, cyclo-oxygenase 2, telomerase, and multidrug resistance protein promoters were studied for activity in CD44+CD24−/low cells, and a panel of oncolytic viruses was subsequently constructed. Each virus featured 5/3 chimerism of the fiber and a promoter controlling expression of E1A, which was also deleted in the Rb binding domain for additional tumor selectivity. Cell killing assays identified Ad5/3-cox2L-d24 and Ad5/3-mdr-d24 as the most active agents, and these viruses were able to completely eradicate CD44+CD24−/low cells in vitro. In vivo, these viruses had significant antitumor activity in CD44+CD24−/low–derived tumors. These findings may have relevance for elimination of cancer stem cells in humans. [Cancer Res 2008;68(14):5533–9]

Renal cancer is a common and deadly disease that lacks curative treatments when metastatic. Here, we have used oncolytic adenoviruses, a promising developmental approach whose safety has recently been validated in clinical trials. Although preliminary clinical efficacy data exist for selected tumor types, potency has generally been less than impressive. One important reason may be that expression of the primary receptor, coxsackie-adenovirus receptor, is often low on many or most advanced tumors, although not evaluated in detail with renal cancer. Here, we tested if fluorescence-assisted cell sorting could be used to predict efficacy of a panel of infectivity-enhanced capsid-modified marker gene expressing adenoviruses in renal cancer cell lines, clinical specimens, and subcutaneous and orthotopic murine models of peritoneally metastatic renal cell cancer. The respective selectively oncolytic adenoviruses were tested for killing of tumor cells in these models, and biodistribution after locoregional delivery was evaluated. In vivo replication was analyzed with noninvasive imaging. Ad5/3-Δ24, Ad5-Δ24RGD, and Ad5.pK7-Δ24 significantly increased survival of mice compared with mock or wild-type virus and 50% of Ad5/3-Δ24 treated mice were alive at 320 days. Because renal tumors are often highly vascularized, we investigated if results could be further improved by adding bevacizumab, a humanized antivascular endothelial growth factor antibody. The combination was well tolerated but did not improve survival, suggesting that the agents may be best used in sequence instead of together. These results set the stage for clinical testing of oncolytic adenoviruses for treatment of metastatic renal cancer currently lacking other treatment options. [Mol Cancer Ther 2007;6(10):2728–36]

Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.

Background Adenoviruses can cause severe toxicity in immunocompromised individuals. Although clinical trials have confirmed the potency and safety of selectively oncolytic adenoviruses for treatment of advanced cancers, increasingly effective agents could result in more toxicity and therefore it would be useful if replication could be abrogated if necessary. Methods We analyzed the effect of chlorpromazine, an inhibitor of clathrin-dependent endocytosis and apigenin, a cell cycle regulator, on adenovirus replication and toxicity. First, we evaluated the in vitro replication of a tumor targeted Rb-p16 pathway selective oncolytic adenovirus (Ad5/3-Δ24) and a wild-type adenovirus in normal cells, fresh liver samples and in ovarian cancer cell lines. Further, we analyzed the in vitro cell killing efficacy of adenoviruses in the presence and absence of the substances. Moreover, the effect on in vivo efficacy, replication and liver toxicity of the adenoviruses was evaluated. Results We demonstrate in vitro and in vivo reduction of adenovirus replication and associated toxicity with chlorpromazine and apigenin. Effective doses were well within what would be predicted safe in humans. Conclusions Chlorpromazine and apigenin might reduce the replication of adenovirus, which could provide a safety switch in case replication-associated side effects are encountered in patients. In addition, these substances could be useful for the treatment of systemic adenoviral infections in immunosuppressed patients. Copyright © 2006 John Wiley & Sons, Ltd.

Conditionally replicating oncolytic adenoviruses represent a promising developmental strategy for the treatment of cancer refractory to current treatments, such as hormone refractory metastatic breast cancer. In clinical cancer trials, adenoviral agents have been well tolerated, but gene transfer has been insufficient for clinical benefit. One of the main reasons may be the deficiency of the primary adenovirus receptor, and therefore viral capsid modifications have been employed. Another obstacle to systemic delivery is rapid clearance of virus by hepatic Kupffer cells and subsequent inadequate bioavailability. In this study, we compared several capsid-modified oncolytic adenoviruses for the treatment of breast cancer with and without Kupffer cell inactivation. Replication deficient capsid-modified viruses were analyzed for their gene transfer efficacy in vitro in breast cancer cell lines and clinical samples and in vivo in orthotopic models of breast cancer. The effect of Kupffer cell depleting agents on gene transfer efficacy in vivo was evaluated. An aggressive lung metastatic model was developed to study the effect of capsid-modified oncolytic adenoviruses on survival. Capsid-modified viruses displayed increased gene transfer and cancer cell killing in vitro and resulted in increased survival in an orthotopic model of lung metastatic breast cancer in mice. Biodistribution of viruses was favorable, tumor burden and treatment response could be monitored repeatedly. Kuppfer cell inactivation led to enhanced systemic gene delivery, but did not increase the survival of mice. These results facilitate clinical translation of oncolytic adenoviruses for the treatment of hormone refractory metastatic breast cancer.

Purpose: Metastatic gastric cancer remains a common and devastating disease without curative treatment. Recent proof-of-concept clinical trials have validated gene therapy with adenoviruses as an effective and safe modality for the treatment of cancer. However, expression of the primary coxsackie-adenovirus receptor is variable in advanced cancers, and therefore, the use of heterologous receptors could be advantageous.Experimental Design: Here, we used capsid-modified adenoviruses for increasing the transduction and subsequent antitumor efficacy. 5/3 chimeric viruses have a serotype 3 knob which allows binding to a receptor distinct from coxsackie-adenovirus receptor. The fiber of Ad5lucRGD is modified with an integrin-targeted motif. Polylysine motifs, pK7 and pK21, bind to heparan sulfates. Oncolytic adenoviruses replicate in and kill tumor cells selectively. Gastric cancer cell lines and fresh clinical samples from patients were infected with transductionally targeted viruses. Capsid-modified oncolytic adenoviruses were used in cell killing experiments. To test viral transduction and therapeutic efficacy in vivo, we developed orthotopic mouse models featuring i.p. disseminated human gastric cancer, which allowed the evaluation of biodistribution and antitumor efficacy in a system similar to humans.Results: Capsid modifications benefited gene transfer efficiency and cell killing in gastric cancer cell lines and clinical samples in vitro and in vivo. Modified oncolytic adenoviruses significantly increased the survival of mice with orthotopic gastric cancer.Conclusions: These preclinical data set the stage for the clinical evaluation of safety and efficacy in patients with disease refractory to current modalities.

Conditionally replicating adenoviruses (CRAds) represent a novel approach for the treatment of cancers resistant to conventional therapies. The efficacy of CRAds might be further improved by using chemotherapeutic agents in a multimodal antitumor approach. We have evaluated the use of Ad5/3-Delta24, a serotype 3 receptor targeted Rb/p16 pathway selective CRAd, in combination with gemcitabine against human ovarian adenocarcinoma. The combination of these agents showed synergistic cell killing in vitro compared to single treatments. However, the effect was dependent on dose and sequencing of the agents. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Delta24 replication without affecting the total amount of virus produced. Possible reasons for synergy between Ad5/3-Delta24 and gemcitabine include the chemosensitizing activity of E1A and/or altered replication kinetics. In an orthotopic murine model of peritoneally disseminated ovarian cancer, the combination increased the survival of mice over either agent alone, and almost 60% of treated mice were cured. Sequencing of the agents was critical for toxicity versus efficacy. Mice remained free from intraperitoneal disease, but some succumbed to treatment-related hepatic or bone marrow toxicity. This suggests that improved efficacy may uncover treatment-related toxicity, which needs to be monitored closely in clinical trials.

In clinical trials with cancer patients, the safety of conditionally replicating adenoviruses (CRAds) has been good. However, marginal data are available on the persistence or antitumor efficacy of these agents. The oncolytic potency of CRAds is determined by their capacity for entering target cells. Consequently, we constructed a retargeted CRAd featuring a secreted marker protein, soluble human carcinoembryogenic antigen (hCEA), which can be measured in growth medium or plasma. We found that virus replication closely correlated with hCEA secretion both in vitro and in vivo. Further, antitumor efficacy and the persistence of the virus could be deduced from plasma hCEA levels. Finally, using in vivo bioluminescence imaging, we were able to detect effective tumor cell killing by the virus, which led to enhanced therapeutic efficacy.

Late stage cancer is often associated with reduced immune recognition and a highly immunosuppressive tumor microenvironment. The presence of tumor infiltrating lymphocytes (TILs) and specific gene-signatures prior to treatment are linked to good prognosis, while the opposite is true for extensive immunosuppression. The use of adenoviruses as cancer vaccines is a form of active immunotherapy to initialise a tumor-specific immune response that targets the patient's unique tumor antigen repertoire. We report a case of a 68-year-old male with asbestos-related malignant pleural mesothelioma who was treated in a Phase I study with a granulocyte-macrophage colony‑stimulating factor (GM-CSF)-expressing oncolytic adenovirus, Ad5/3-D24-GMCSF (ONCOS-102). The treatment resulted in prominent infiltration of CD8+ lymphocytes to tumor, marked induction of systemic antitumor CD8+ T-cells and induction of Th1-type polarization in the tumor. These results indicate that ONCOS-102 treatment sensitizes tumors to other immunotherapies by inducing a T-cell positive phenotype to an initially T-cell negative tumor.

INTRODUCTION: ONCOS-102 is a serotype 5 adenovirus, comprising a chimeric capsid for enhanced gene delivery to cancer cells and a 24 bp deletion in Rb binding site of E1A region for cancer cell restricted replication. ONCOS-102 is armed with granulocyte-macrophage colony-stimulating factor (GM-CSF) for an enhanced immunostimulatory effect. ONCOS-102 treatment is a promising immunotherapy strategy for advanced cancer by directly recruiting antigen presenting cells (APC) at tumor site leading to an induction of adaptive tumor-specific CD8+ T cell response. It's immunological activity has already been demonstrated in Phase I clinical study. In this phase 1 study, local treatment of pleural mesothelioma with ONCOS-102 induced a systemic antitumor CD8+ T-cell response, prominent infiltration of CD8+ lymphocytes and Th1 type polarization.PURPOSE OF THE STUDY: The aim of this study was to evaluate the toxicity profile and biodistribution of ONCOS-102 after intracardial and repeated intraperitoneal or subcutaneous administration in Syrian hamsters.METHOD: This study was performed in compliance with the OECD principles of GLP. 300 Syrian hamsters were divided into nine groups, each group containing males and females in the same ratio. ONCOS-102 was administered in NaCl solution by intracardial, intraperitoneal or subcutaneous injections. The control animals were administered with NaCl solution without ONCOS-102 in the same volume and the same way. Hamsters were checked daily for mortality and clinical signs. Body weight and food consumption were recorded weekly. For bio-distribution analysis, hamsters were euthanized on days 0, 3, 29, and 183 and tissues and organs were collected for assessment of viral DNA. Gross necropsy and histological examination of tissues and organs were performed on days 29 and 190.RESULTS: Treatment with ONCOS-102 had no effects on body weight and food consumption. No treatment related premature deaths occurred. Apart from some findings that were not related to treatment, none of the hamsters showed signs of intoxication in the course of the first four weeks of the study. No considerable differences in hematology and clinical chemistry parameters were observed in any of the treatment groups when compared to control animals after 26 weeks of the study and recovery period. ONCOS-102 did not cause gross or histopathological changes in liver and kidneys of animals indicative of a toxic effect. The highest accumulation of viral DNA was seen in heart, liver, lungs, spleen, kidneys and bone marrow. An overall decrease of concentration of viral DNA was observed from Day 3 to Day 29 and Day 183. No adverse effects of repeated administration of ONCOS-102 on clinical signs, hematology and clinical chemistry parameters or histopathology were observed in the course of 6-month administration and following 3- month recovery period.CONCLUSIONS: Under the conditions of the study, administration of hamsters with ONCOS 102 was well tolerated, without significant signs indicating toxic effects

Immunosuppression of established tumors is a major obstacle hindering the efficacy of anti-tumor T cells. Antagonistic mAbs targeting the PD-1/PD-L1 axis represent an effective way to decrease T cell exhaustion and/or anergy, but the efficacy of such therapies seems to be limited to specific cohorts of patients with ongoing immune responses against the tumor. Thus, we hypothesized that we could boost the efficacy of PD-L1 blockade by using an oncolytic cancer vaccine PeptiCrad, which has previously been shown to elicit potent tumor-specific T cell responses by stimulating antigen-presenting cells. First, we demonstrated that the outcome of therapy may depend on the treatment schedule as mice with high B16.OVA tumor burden were refractory to combination of PD-L1 blockade and oncolytic vaccine PeptiCrad targeting the tumor antigen OVA. In fact, mice receiving combination therapy showed only a modest increase in median survival compared to control groups. However, immunological analyses revealed that combination therapy elicited strong anti-tumor immune responses, observed both by tetramer analysis and IFN γ ELISPOT. Hence, we hypothesized that a different treatment schedule could be beneficial in the case of aggressive melanoma such as B16.OVA. In a subsequent experiment, we started the treatments earlier with an overlapping schedule, allowing the PD-L1 blockade to affect the newly primed T cells induced by PeptiCrad. With this regimen, combination treatment notably inhibited tumor growth and a significant increase in median survival of mice was observed when compared to groups receiving monotherapies. Moreover, 37.5% of combination treated mice were cured, suggesting that PeptiCrad can synergize with PD-L1 blockade. In order to eradicate the residual immunoedited tumors, we utilized the major advantage of PeptiCrad technology by treating the survivor mice with a single PeptiCRAd targeting two endogenous melanoma antigens (TRP-2 and gp100, respectively). As a result, the combination therapy was able to slow down the growth of the residual tumors, suggesting that targeting multiple cancer antigens simultaneously could be a feasible approach also in terms of immune checkpoint blockade. Further analyses on the effect of the combination therapy on antigen-specific T cell subsets and on PD-L1 expressing immune cells (such as MDSCs) are ongoing. Citation Format: Cristian Capasso, Siri Tähtinen, Federica Frascaro, Sara Carpi, Manlio Fusciello, Davide Cardella, Daniela Cropp, Karita Peltonen, Beatriz Martins, Madeleine Sjöberg, Sari Pesonen, Tuuli Ranki, Lukasz Kuryk, Erkko Ylösmäki, Vincenzo Cerullo. Boosting the efficacy of PD-L1 blockade with oncolytic vaccine for improved antitumor responses in melanoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A034.

3085 Background: Clinical studies consistently show how adenovirus, unlike other viral vectors, can both prime and boost immune responses. Consequently, adenovirus is used to develop vaccines again...

Adenoviruses are excellent immunotherapeutic agents with a unique ability to prime and boost immune responses. Recombinant adenoviruses cause immunogenic cancer cell death and subsequent release of tumor antigens for antigen presenting cells, resulting in the priming of potent tumor-specific immunity. This effect may be further enhanced by immune-stimulating transgenes expressed by the virus. We report a case of a 38-year-old female with Stage 3 metastatic micropapillary serous carcinoma of the ovary. She was treated in a Phase I study with a granulocyte-macrophage colony stimulating factor (GMCSF)-expressing oncolytic adenovirus, Ad5/3-D24-GMCSF (ONCOS-102). The treatment resulted in progressive infiltration of CD8+ lymphocytes into the tumor and concomitant systemic induction of several tumor-specific CD8+ T-cell populations. The patient was alive at the latest follow up more than 20 months after initiation of the study.

Meeting abstracts With the increasing excitement around new immunotherapeutic approaches, there has been a shift in the way viral cancer therapy is regarded from providing mainly oncolysis towards being a cancer immunotherapy. Adenoviruses have a unique ability to prime and boost immune responses.

BACKGROUND. Variable expression of the coxsackie and adenovirus receptor (CAR) has limited gene transfer efficacy to many types of tumors. Consequently, tropism-modified adenoviruses have been developed for enhanced infectivity. To the authors' knowledge, targeting approaches for nonsmall cell lung cancer (NSCLC) have not been comprehensively evaluated. The current hypothesis was that modified adenoviruses could be used for increasing gene transfer to and killing of NSCLC cells in vitro and in vivo. METHODS. Ten NSCLC cell lines were analyzed to represent the different NSCLC histologies. Because clinical tumors may differ from established cell lines, 6 clinical specimens fresh from patients were analyzed. For in vivo studies, a novel orthotopic murine model of advanced lung cancer was developed. Because tumor response is difficult to quantitate in orthotopic models, noninvasive imaging of green fluorescent protein (GFP) was utilized as a surrogate for tumor size measurements. RESULTS. Adenoviruses whose capsids were modified with RGD-4C, the serotype 3 knob, or polylysine displayed increased gene transfer to NSCLC cell lines and clinical samples in comparison to serotype 5 viruses. Conditionally replicating oncolytic adenoviruses (CRAds) with the same modifications showed enhanced therapeutic efficiency in vitro and in vivo. The median survival of mice treated with Ad5.pK7-Δ24 or Ad5-Δ24RGD increased 37% (P

Conditionally replicating adenoviruses (CRAds) that replicate in tumor but less in normal cells are promising anticancer agents. A major determinant of their potency is their capacity for infecting target cells. The primary receptor for serotype 5 adenovirus (Ad5), the most widely used serotype in gene therapy, is the coxsackie-adenovirus receptor (CAR). CAR is expressed variably and often at low levels in various tumor types including advanced breast cancer. We generated a novel p16/retinoblastoma pathway-dependent CRAd, Ad5.pK7-Delta24, with a polylysine motif in the fiber C-terminus, enabling CAR-independent binding to heparan sulfate proteoglycans (HSPG). Ad5.pK7-Delta24 mediated effective oncolysis of all breast cancer cell lines tested. Further, we utilized noninvasive, fluorescent imaging for analysis of antitumor efficacy in an orthotopic model of advanced hormone refractory breast cancer. A therapeutic benefit was seen following both intratumoral and intravenous delivery. Murine biodistribution similar to Ad5, proven safe in trials, suggests feasibility of clinical safety testing. Interestingly, upregulation of CAR was seen in low-CAR M4A4-LM3 breast cancer cells in vivo, which resulted in better than expected efficacy also with an isogenic CRAd with an unmodified capsid. These results suggest utility of Ad5.pK7-Delta24 and the orthotopic model for further translational studies.

e13035 Background: Following preclinical testing CGTG-102, a 5/3 chimeric oncolytic adenovirus armed with human GMCSF, has been used to treat 115 refractory cancer patients. Methods: Eligibility criteria included refractory advanced solid tumors, no major organ deficiencies and written informed consent. Patients were treated with either a single treatment or serial treatments with one or more viruses. Intra tumoral administration was performed under ultrasound guidance. The initial dose, 8 x 1010 Viral Particles (VP), was based on published safety results and preclinical testing and escalated in later patients. A routinely tolerated dose of 3 x 1011 VP was deemed optimal and is the target dose for clinical development. To reduce regulatory T-cells, low-dose cyclophosphamide 50 mg/day was given. Adverse Reactions (AR) were scored according to CTCAE 3.0. Imaging was done by CT before and ~2 months after treatment. Response was scored according to RECIST 1.1, including injected and non-injected lesions. Decrease not fulfilling PR was scored as minor response (MR). Results: The most common ARs were pain (82%), fever (81%), fatigue (79%), nausea (54%) and hemoglobin decrease (48%). Pain is mostly tumor pain or pain in the injected tumor, which may be causally related to the MOA of the therapy. Most ARs were G1 or G2; 6 were G4: 2 Hb decrease, 2 pulmonary embolism and single reports of thrombocytopenia and pericardial effusion, most probably due to the underlying disease.Imaging was performed when clinically useful. 65/115 are evaluable by imaging: 3% PR, 11% MR, 40% SD and 46% PD. Best results were obtained in Breast Cancer, Melanoma, Soft Tissue Sarcoma, Mesothelioma and Ovarian Cancer. Median survival in this heavily pre-treated refractory population is 164d, 95% CI 122d – 206d. Mean survival is 281d reflecting that approx. 30% survive more than 300d and 15% up to 600d. A wide range of samples are being analyzed to further characterize the viral and immunological aspects of the therapy. Conclusions: CGTG-102 is a novel oncolytic adenovirus with good safety profile and encouraging signs of efficacy. Formal clinical studies are underway in several tumor types in both US and EU.

Top of pageAbstract Oncolytic adenoviruses a.k.a. conditionally replicating adenoviruses (CRAds) represent a promising approach for treatment of cancers resistant to conventional therapies. Our hypothesis was that efficacy of CRAds might be further improved by using chemotherapeutic agents in a multimodal antitumor approach. Oncolytic tumor killing differs mechanistically from conventional therapies and therefore an additive or synergistic antitumor effect is possible. The toxicity profiles of the agents may be different and could result in enhanced efficacy without increased side effects. Finally, it may be possible to use lower treatment doses and still achieve a greater efficacy than with single agents. We evaluated Ad5/3-|[Delta]|24 in combination with deoxycytidine analogue gemcitabine against human ovarian adenocarcinoma. Ad5/3-|[Delta]|24 is a serotype 3 receptor targeted CRAd containing a 24 bp deletion in the E1A gene. As a result the E1A protein is unable to bind the retinoblastoma (Rb) protein for induction of viral replication. Therefore the virus replicates selectively in cells dysfunctional in the Rb/p16 pathway, including most if not all cancer cells. Most CRAds are based on adenovirus serotype 5 (Ad5). However, the expression of the Ad5 primary receptor, coxsackie-adenovirus receptor (CAR), is highly variable on ovarian cancer cells. The lack of CAR can be circumvented by substituting the knob domain of Ad5 with the corresponding domain of serotype 3 (Ad3). Ad3 has a distinct receptor which is expressed to high degree on ovarian cancer cells. For evaluation of the potential interaction between Ad5/3-|[Delta]|24 and gemcitabine in vitro, the cell killing effect of the combination treatment was compared to single treatments on established ovarian adenocarcinoma cell lines. The combination resulted in synergistic cell killing as determined by Chou and Talalay's synergy analysis. However, the effect was dependent on dose and sequencing of the agents. In an orthotopic murine model of aggressive peritoneally disseminated ovarian cancer, the combination of Ad5/3-|[Delta]|24 and gemcitabine increased the survival of mice over either agent alone, and almost 60 % of treated mice were cured. However, sequencing of the agents was critical for toxicity versus efficacy. Mice remained free from intraperitoneal disease, but some succumbed to treatment related toxicity. Possible reasons for the interaction between Ad5/3-|[Delta]|24 and gemcitabine include the chemosensitizing activity of E1A and/or altered replication kinetics. Our results indicate that gemcitabine reduces the initial rate of Ad5/3-|[Delta]|24 replication without affecting the total amount of virus produced.

Background: Low or variable expression of the coxsackie and adenovirus receptor (CAR) has limited gene transfer efficacy to many types of cancer cells. To circumvent the problem, adenoviruses (Ads) have been modified by improving their ability to attach to heterologous receptors more regularly present on tumor cells. In the context of lung cancer, the most common cause of cancer associated mortality worldwide, targeting approaches have not been comprehensively evaluated. Our hypothesis was that we could use previously described targeting moieties for increasing gene transfer to NSCLC cells and clinical samples in vitro and in vivo. Ad5lucRGD is modified with an RGD-4C motif allowing binding to integrins. Ad5/3luc1 has a chimeric fiber protein featuring the knob domain derived from Ad3 and it uses the serotype 3 receptor for entry. One can also add a polylysine tail to the C-terminal end of the fiber. Polylysins bind to heparan sulfate proteoglygans (HSPGs). For improving tumor penetration and subsequent oncolysis, conditionally replicating adenoviruses (CRAds) have been constructed. In this study, we used CRAds with same targeting moieties as in the non-replicating Ads.

Top of pageAbstract Background: Serotype 5 adenovirus (Ad5) displays high gene transfer efficacy to cells with abundant coxsackie-adenovirus receptor (CAR) expression. However, CAR is variably and often poorly expressed in many tumor types. This has not been comprehensively studied in the context of breast cancer, the most common cancer in women. Fortunately, adenoviruses can be modified to attach to other cellular receptors in order to circumvent the dependence on CAR and to achieve enhanced gene transfer to tumors. Ad5.pK7 (GL) (contains GFP- and luciferase genes) is modified with a polylysine motif allowing CAR independent Ad binding to cell-surface receptors with polyanionic motifs such as heparan sulphate. Ad5lucRGD contains an RGD-4C motif allowing binding to cellular integrins, Ad5.RGD.pK7 (GL) contains both of these modifications and Ad5/3luc1 has a chimeric fiber with the serotype 3 knob and binds to a yet unknown receptor distinct from CAR. Our hypothesis was that we could enhance the infectivity of breast cancer cell lines, clinical samples and orthotopic xenografts by using these targeting moieties. Methods: Gene transfer was studied with firefly luciferase expressing viruses. We also studied cell killing by the respective oncolytic, conditionally replicating adenoviruses (CRAds), featuring a 24 bp deletion in the E1A region, which leads to selective replication in Rb-p16 pathway defective cells. An orthotopic murine model was created by injections of GFP positive MDA-MB-435 cells into mammary fat pads of mice. Results: Some of the modified viruses displayed improved gene transfer to cancer cells in vitro and showed enhanced oncolytic activity over a virus with a wild type capsid. Capsid modified viruses also showed enhanced gene transfer to tumors in a murine model of advanced breast cancer. Conclusions: Mature in vitro and in vivo data will be presented and discussed. In vivo data will include imaging in an orthotopic murine model of advanced breast cancer.

Background: Gene therapy with adenoviral (Ad) vectors is a promising modality for treatment of cancer. The primary limiting factor for Ad vectors is their dependence on the coxsackie and adenovirus receptor (CAR), which is down-regulated in many cancers. However, this has not been studied in the context of gastric cancer, the fifth most common cause of cancer mortality worldwide. Native Ad5 tropism can be modified to circumvent CAR deficiency. For instance, Ad5/3luc1 with serotype 3 fiber knob binds to a receptor distinct from CAR. The fiber of Ad5lucRGD is modified with an integrin-targeted arginine-glycine-aspartic acid motif. Polylysine motifs pK7 and pK21 bind to heparan sulphates. The oncolytic potency of replicating agents is partly determined by their capability for infecting cells. Conditionally replicating adenoviruses (CRAds) are replication competent in tumor but not in non-cycling normal cells. |[Delta]|24 based CRAds with a 24 bp deletion in E1A region take advantage of tumor specific changes in the Rb pathway. Our hypothesis was, that capsid modified adenoviruses possess enhanced ability for transduction of gastric cancer cells and primary tumor tissues, and that this targeting would also benefit the oncolytic potency of CRAds.

Because of the high coverage of international vaccination programs, most people worldwide have been vaccinated against common pathogens, leading to acquired pathogen-specific immunity with a robust memory T-cell repertoire. Although CD8+ antitumor cytotoxic T lymphocytes (CTL) are the preferred effectors of cancer immunotherapy, CD4+ T-cell help is also required for an optimal antitumor immune response to occur. Hence, we investigated whether the pathogen-related CD4+ T-cell memory populations could be reengaged to support the CTLs, converting a weak primary antitumor immune response into a stronger secondary one. To this end, we used our PeptiCRAd technology that consists of an oncolytic adenovirus coated with MHC-I–restricted tumor-specific peptides and developed it further by introducing pathogen-specific MHC-II–restricted peptides. Mice preimmunized with tetanus vaccine were challenged with B16.OVA tumors and treated with the newly developed hybrid TT-OVA-PeptiCRAd containing both tetanus toxoid- and tumor-specific peptides. Treatment with the hybrid PeptiCRAd significantly enhanced antitumor efficacy and induced TT-specific, CD40 ligand-expressing CD4+ T helper cells and maturation of antigen-presenting cells. Importantly, this approach could be extended to naturally occurring tumor peptides (both tumor-associated antigens and neoantigens), as well as to other pathogens beyond tetanus, highlighting the usefulness of this technique to take full advantage of CD4+ memory T-cell repertoires when designing immunotherapeutic treatment regimens. Finally, the antitumor effect was even more prominent when combined with the immune checkpoint inhibitor anti–PD-1, strengthening the rationale behind combination therapy with oncolytic viruses. Significance: These findings establish a novel technology that enhances oncolytic cancer immunotherapy by capitalizing on pre-acquired immunity to pathogens to convert a weak antitumor immune response into a much stronger one.

Advanced tumors are often immunosuppressive. Intratumoral administration of adenovirus activates Toll-like receptor signalling leading to production of pro-inflammatory cytokines and activation of the innate immune system. Oncolytic adenovirus causes immunogenic cancer cell death and creates a danger signal at tumors, thus undermining immunological tolerance towards tumors. The release of tumor antigens from dying cancer cells results in priming of a potent anti-tumor immune response. This effect may be enhanced by the local production of immunostimulatory molecules coded by the virus. We present results of gene expression analysis of tumors from a Phase I study with ONCOS-102, an oncolytic adenovirus coding for GMCSF, in 12 patients with refractory solid tumors. A total of 9 intratumoral injections of ONCOS-102 were given to each patient. Biopsies were collected at baseline and 1 and 2 months after treatment initiation. RNA was extracted from fresh-frozen biopsies using standard methods. Gene expression profiling was carried out using the Illumina BeadChip platform. Data was normalized by quantile method, probes presenting the same genes were averaged, and the batch effects were adjusted using ComBat pipeline, as implemented in Chipster (http://chipster.csc.fi/). Finally, log2 fold-changes were computed by subtracting baseline data from after treatment data. Network and pathway analyses were conducted through the use of IPA (Ingenuity ®Systems, http://www.ingenuity.com). A cutoff value of 2-fold expression change was used to filter differentially expressed genes and run core analysis to identify underlying signaling networks and deregulated molecules. A significant enrichment of genes involved in immune cell trafficking, inflammatory response and antigen presentation were detected post treatment. A mesothelioma patient showed a prominent post-treatment induction of MAGE3-specific CD8+ T-cells in peripheral blood in IFNγ ELISPOT. Furthermore, a late decrease in metabolic activity was observed in PET imaging 7.5 months after treatment initiation during the follow-up period, measured as a 47% decrease in total lesion glycolysis in comparison to the imaging at 6 months. In the same patient, upstream regulators driving differentially expressed genes detected in the post treatment biopsy included cytokines (INFG, TNF, IL1B, CCL2, CXCL10, IL-17A, CD40LG), enzymes (FN1, NOS2, PTGS2, PARP9), growth factors (BMP2, LEP), kinases (CHUK, CRKL, IKBKB, IKBKG, ITK, STK11, SYK), transcriptional regulators (IRF1, NFATC2, NFKBIA, STAT1, STAT3, ZEB1, RELA), and transmembrane receptors (B2M, CD40, IL6R, TLR2-4, TNFSF1B). Likely, these events collectively induced a higher CD8+ mediated cytotoxic T cell response (GZMB, PRF1) post treatment. Detailed analysis per patient will be presented at the meeting.