Lotta Vassilev

Serotype chimeric oncolytic adenovirus coding for GM-CSF for treatment of sarcoma in rodents and humans.

Sarcomas are a relatively rare cancer, but often incurable at the late metastatic stage. Oncolytic immunotherapy has gained attention over the past years, and a wide range of oncolytic viruses have been delivered via intratumoral injection with positive safety and promising efficacy data. Here, we report preclinical and clinical results from treatment of sarcoma with oncolytic adenovirus Ad5/3‐D24‐GMCSF (CGTG‐102). Ad5/3‐D24‐GMCSF is a serotype chimeric oncolytic adenovirus coding for human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). The efficacy of Ad5/3‐D24‐GMCSF was evaluated on a panel of soft‐tissue sarcoma (STS) cell lines and in two animal models. Sarcoma specific human data were also collected from the Advanced Therapy Access Program (ATAP), in preparation for further clinical development. Efficacy was seen in both in vitro and in vivo STS models. Fifteen patients with treatment‐refractory STS (13/15) or primary bone sarcoma (2/15) were treated in ATAP, and treatments appeared safe and well‐tolerated. A total of 12 radiological RECIST response evaluations were performed, and two cases of minor response, six cases of stable disease and four cases of progressive disease were detected in patients progressing prior to virus treatment. Overall, the median survival time post treatment was 170 days. One patient is still alive at 1,459 days post virus treatment. In summary, Ad5/3‐D24‐GMCSF appears promising for the treatment of advanced STS; a clinical trial for treatment of refractory injectable solid tumors including STS is ongoing.© 2013 UICC

Repeated intratumoral administration of ONCOS-102 leads to systemic antitumor CD8+ T-cell response and robust cellular and transcriptional immune activation at tumor site in a patient with ovarian cancer

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.

Oncolytic adenovirus and doxorubicin-based chemotherapy results in synergistic antitumor activity against soft-tissue sarcoma

Despite originating from several different tissues, soft‐tissue sarcomas (STS) are often grouped together as they share mesenchymal origin and treatment guidelines. Also, with some exceptions, a common denominator is that when the tumor cannot be cured with surgery, the efficacy of current therapies is poor and new treatment modalities are thus needed. We have studied the combination of a capsid‐modified oncolytic adenovirus CGTG‐102 (Ad5/3‐D24‐GMCSF) with doxorubicin, with or without ifosfamide, the preferred first‐line chemotherapeutic options for most types of STS. We show that CGTG‐102 and doxorubicin plus ifosfamide together are able to increase cell killing of Syrian hamster STS cells over single agents, as well as upregulate immunogenic cell death markers. When tested in vivo against established STS tumors in fully immunocompetent Syrian hamsters, the combination was highly effective. CGTG‐102 and doxorubicin (without ifosfamide) resulted in synergistic antitumor efficacy against human STS xenografts in comparison with single agent treatments. Doxorubicin increased adenoviral replication in human and hamster STS cells, potentially contributing to the observed therapeutic synergy. In conclusion, the preclinical data generated here support clinical translation of the combination of CGTG‐102 and doxorubicin, or doxorubicin plus ifosfamide, for the treatment of STS, and provide clues on the mechanisms of synergy.

Treatment of melanoma with a serotype 5/3 chimeric oncolytic adenovirus coding for GM-CSF: Results in vitro, in rodents and in humans.

Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune‐checkpoint‐inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno‐virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3‐D24‐GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma‐specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK‐MEL‐28 melanoma xenografts in nude mice when combined with low‐dose cyclophosphamide. Furthermore, virally‐expressed granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well‐tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3‐D24‐GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma.

Local treatment of a pleural mesothelioma tumor with ONCOS-102 induces a systemic antitumor CD8 + T-cell response, prominent infiltration of CD8 + lymphocytes and Th1 type polarization

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 patients 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.

Intravenously usable fully serotype 3 oncolytic adenovirus coding for CD40L as an enabler of dendritic cell therapy

ABSTRACT Vaccination with dendritic cells (DCs), the most potent professional antigen-presenting cells in the body, is a promising approach in cancer immunotherapy. However, tumors induce immunosuppression in their microenvironment that suppresses and impairs the function of DCs. Therefore, human clinical trials with DC therapy have often been disappointing. To improve the therapeutic efficacy and to overcome the major obstacles of DC therapy, we generated a novel adenovirus, Ad3-hTERT-CMV-hCD40L, which is fully serotype 3 and expresses hCD40L for induction of antitumor immune response. The specific aim is to enhance DCs function. Data from a human cancer patient indicated that this capsid allows effective transduction of distant tumors through the intravenous route. Moreover, patient data suggested that virally produced hCD40L can activate DCs in situ. The virus was efficient in vitro and had potent antitumor activity in vivo. In a syngeneic model, tumors treated with Ad5/3-CMV-mCD40L virus plus DCs elicited greater antitumor effect as compared with either treatment alone. Moreover, virally coded CD40L induced activation of DCs, which in turn, lead to the induction of a Th1 immune response and increased tumor-specific T cells. In conclusion, Ad3-hTERT-CMV-hCD40L is promising for translation into human trials. In particular, this virus could enable successful dendritic cell therapy in cancer patients.

Oncolytic virotherapy for treatment of breast cancer, including triple-negative breast cancer

ABSTRACT Breast cancer is a heterogeneous disease, characterized by several distinct biological subtypes, among which triple-negative breast cancer (TNBC) is one associated with a poor prognosis. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules to boost virus triggered antitumoral immune responses. Cyclophosphamide (CP) is a chemotherapy drug that is associated with cytotoxicity and immunosuppression at higher doses, whereas immunostimulatory and anti-angiogenic properties are observed at low continuous dosage. Therefore, the combination of oncolytic immuno-virotherapy with low-dose CP is an appealing approach. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a TNBC cell line and in vivo in an orthotopic xenograft mouse model, in combination with low-dose CP or its main active metabolite 4-hydroperoxycyclophosphamide (4-HP-CP). Furthermore, we summarized the breast cancer-specific human data on this virus from the Advanced Therapy Access Program (ATAP). Low-dose CP increased the efficacy of Ad5/3-D24-GMCSF in vitro and in a TNBC mouse model. In ATAP, treatments appeared safe and well-tolerated. Thirteen out of 16 breast cancer patients treated were evaluable for possible benefits with modified RECIST 1.1 criteria: 1 patient had a minor response, 2 had stable disease (SD), and 10 had progressive disease (PD). One patient is alive at 1,771 d after treatment. Ad5/3-D24-GMCSF in combination with low-dose CP showed promising efficacy in preclinical studies and possible antitumor activity in breast cancer patients refractory to other forms of therapy. This preliminary data supports continuing the clinical development of oncolytic adenoviruses for treatment of breast cancer, including TNBC.

Chronic Activation of Innate Immunity Correlates With Poor Prognosis in Cancer Patients Treated With Oncolytic Adenovirus

Despite many clinical trials conducted with oncolytic viruses, the exact tumor-level mechanisms affecting therapeutic efficacy have not been established. Currently there are no biomarkers available that would predict the clinical outcome to any oncolytic virus. To assess the baseline immunological phenotype and find potential prognostic biomarkers, we monitored mRNA expression levels in 31 tumor biopsy or fluid samples from 27 patients treated with oncolytic adenovirus. Additionally, protein expression was studied from 19 biopsies using immunohistochemical staining. We found highly significant changes in several signaling pathways and genes associated with immune responses, such as B-cell receptor signaling (P < 0.001), granulocyte macrophage colony-stimulating factor (GM-CSF) signaling (P < 0.001), and leukocyte extravasation signaling (P < 0.001), in patients surviving a shorter time than their controls. In immunohistochemical analysis, markers CD4 and CD163 were significantly elevated (P = 0.020 and P = 0.016 respectively), in patients with shorter than expected survival. Interestingly, T-cell exhaustion marker TIM-3 was also found to be significantly upregulated (P = 0.006) in patients with poor prognosis. Collectively, these data suggest that activation of several functions of the innate immunity before treatment is associated with inferior survival in patients treated with oncolytic adenovirus. Conversely, lack of chronic innate inflammation at baseline may predict improved treatment outcome, as suggested by good overall prognosis.

Biodistribution Analysis of Oncolytic Adenoviruses in Patient Autopsy Samples Reveals Vascular Transduction of Noninjected Tumors and Tissues.

In clinical trials with oncolytic adenoviruses, there has been no mortality associated with treatment vectors. Likewise, in the Advanced Therapy Access Program (ATAP), where 290 patients were treated with 10 different viruses, no vector-related mortality was observed. However, as the patient population who received adenovirus treatments in ATAP represented heavily pretreated patients, often with very advanced disease, some patients died relatively soon after receiving their virus treatment mandating autopsy to investigate cause of death. Eleven such autopsies were performed and confirmed disease progression as the cause of death in each case. The regulatory requirement for investigating the safety of advanced therapy medical products presented a unique opportunity to study tissue samples collected as a routine part of the autopsies. Oncolytic adenoviral DNA was recovered in a wide range of tissues, including injected and noninjected tumors and various normal tissues, demonstrating the ability of the vector to disseminate through the vascular route. Furthermore, we recovered and cultured viable virus from samples of noninjected brain metastases of an intravenously treated patient, confirming that oncolytic adenovirus can reach tumors through the intravascular route. Data presented here give mechanistic insight into mode of action and biodistribution of oncolytic adenoviruses in cancer patients.

Synergistic anti‐tumor efficacy of immunogenic adenovirus ONCOS‐102 (Ad5/3‐D24‐GM‐CSF) and standard of care chemotherapy in preclinical mesothelioma model

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.