Suvi Parviainen

Immune Response Is an Important Aspect of the Antitumor Effect Produced by a CD40L-Encoding Oncolytic Adenovirus

Oncolytic adenovirus is an attractive platform for immunotherapy because virus replication is highly immunogenic and not subject to tolerance. Although oncolysis releases tumor epitopes and provides costimulatory danger signals, arming the virus with immunostimulatory molecules can further improve efficacy. CD40 ligand (CD40L, CD154) induces apoptosis of tumor cells and triggers several immune mechanisms, including a T-helper type 1 (T(H)1) response, which leads to activation of cytotoxic T cells and reduction of immunosuppression. In this study, we constructed a novel oncolytic adenovirus, Ad5/3-hTERT-E1A-hCD40L, which features a chimeric Ad5/3 capsid for enhanced tumor transduction, a human telomerase reverse transcriptase (hTERT) promoter for tumor selectivity, and human CD40L for increased efficacy. Ad5/3-hTERT-E1A-hCD40L significantly inhibited tumor growth in vivo via oncolytic and apoptotic effects, and (Ad5/3-hTERT-E1A-hCD40L)-mediated oncolysis resulted in enhanced calreticulin exposure and HMGB1 and ATP release, which were suggestive of immunogenicity. In two syngeneic mouse models, murine CD40L induced recruitment and activation of antigen-presenting cells, leading to increased interleukin-12 production in splenocytes. This effect was associated with induction of the T(H)1 cytokines IFN-γ, RANTES, and TNF-α. Tumors treated with Ad5/3-CMV-mCD40L also displayed an enhanced presence of macrophages and cytotoxic CD8(+) T cells but not B cells. Together, our findings show that adenoviruses coding for CD40L mediate multiple antitumor effects including oncolysis, apoptosis, induction of T-cell responses, and upregulation of T(H)1 cytokines.

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

CD40 ligand and tdTomato-armed vaccinia virus for induction of antitumor immune response and tumor imaging

Oncolytic vaccinia virus is an attractive platform for immunotherapy. Oncolysis releases tumor antigens and provides co-stimulatory danger signals. However, arming the virus can improve efficacy further. CD40 ligand (CD40L, CD154) can induce apoptosis of tumor cells and it also triggers several immune mechanisms. One of these is a T-helper type 1 (Th1) response that leads to activation of cytotoxic T-cells and reduction of immune suppression. Therefore, we constructed an oncolytic vaccinia virus expressing hCD40L (vvdd-hCD40L-tdTomato), which in addition features a cDNA expressing the tdTomato fluorochrome for detection of virus, potentially important for biosafety evaluation. We show effective expression of functional CD40L both in vitro and in vivo. In a xenograft model of bladder carcinoma sensitive to CD40L treatment, we show that growth of tumors was significantly inhibited by the oncolysis and apoptosis following both intravenous and intratumoral administration. In a CD40-negative model, CD40L expression did not add potency to vaccinia oncolysis. Tumors treated with vvdd-mCD40L-tdtomato showed enhanced efficacy in a syngenic mouse model and induced recruitment of antigen-presenting cells and lymphocytes at the tumor site. In summary, oncolytic vaccinia virus coding for CD40L mediates multiple antitumor effects including oncolysis, apoptosis and induction of Th1 type T-cell responses.

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.

GMCSF-armed vaccinia virus induces an antitumor immune response

Oncolytic Western Reserve strain vaccinia virus selective for epidermal growth factor receptor pathway mutations and tumor‐associated hypermetabolism was armed with human granulocyte‐macrophage colony‐stimulating factor (GMCSF) and a tdTomato fluorophore. As the assessment of immunological responses to human transgenes is challenging in the most commonly used animal models, we used immunocompetent Syrian golden hamsters, known to be sensitive to human GMCSF and semipermissive to vaccinia virus. Efficacy was initially tested in vitro on various human and hamster cell lines and oncolytic potency of transgene‐carrying viruses was similar to unarmed virus. The hGMCSF‐encoding virus was able to completely eradicate subcutaneous pancreatic tumors in hamsters, and to fully protect the animals from subsequent rechallenge with the same tumor. Induction of specific antitumor immunity was also shown by ex vivo co‐culture experiments with hamster splenocytes. In addition, histological examination revealed increased infiltration of neutrophils and macrophages in GMCSF‐virus‐treated tumors. These findings help clarify the mechanism of action of GMCSF‐armed vaccinia viruses undergoing clinical trials.

Favorable Alteration of Tumor Microenvironment by Immunomodulatory Cytokines for Efficient T-Cell Therapy in Solid Tumors

Unfavorable ratios between the number and activation status of effector and suppressor immune cells infiltrating the tumor contribute to resistance of solid tumors to T-cell based therapies. Here, we studied the capacity of FDA and EMA approved recombinant cytokines to manipulate this balance in favor of efficient anti-tumor responses in B16.OVA melanoma bearing C57BL/6 mice. Intratumoral administration of IFN-α2, IFN-γ, TNF-α, and IL-2 significantly enhanced the anti-tumor effect of ovalbumin-specific CD8+ T-cell (OT-I) therapy, whereas GM-CSF increased tumor growth in association with an increase in immunosuppressive cell populations. None of the cytokines augmented tumor trafficking of OT-I cells significantly, but injections of IFN-α2, IFN-γ and IL-2 increased intratumoral cytokine secretion and recruitment of endogenous immune cells capable of stimulating T-cells, such as natural killer and maturated CD11c+ antigen-presenting cells. Moreover, IFN-α2 and IL-2 increased the levels of activated tumor-infiltrating CD8+ T-cells concomitant with reduction in the CD8+ T-cell expression of anergy markers CTLA-4 and PD-1. In conclusion, intratumoral administration of IFN-α2, IFN-γ and IL-2 can lead to immune sensitization of the established tumor, whereas GM-CSF may contribute to tumor-associated immunosuppression. The results described here provide rationale for including local administration of immunostimulatory cytokines into T-cell therapy regimens. One appealing embodiment of this would be vectored delivery which could be advantageous over direct injection of recombinant molecules with regard to efficacy, cost, persistence and convenience.

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.

Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma

Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of (111)In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies.

Effects of capsid-modified oncolytic adenoviruses and their combinations with gemcitabine or silica gel on pancreatic cancer.

Conventional cancer treatments often have little impact on the course of advanced pancreatic cancer. Although cancer gene therapy with adenoviruses is a promising developmental approach, the primary receptor is poorly expressed in pancreatic cancers which might compromise efficacy and thus targeting to other receptors could be beneficial. Extended stealth delivery, combination with standard chemotherapy or circumvention of host antiadenoviral immune response might improve efficacy further. In this work, capsid‐modified adenoviruses were studied for transduction of cell lines and clinical normal and tumor tissue samples. The respective oncolytic viruses were tested for oncolytic activity in vitro and in vivo. Survival was studied in a peritoneally disseminated pancreas cancer model, with or without concurrent gemcitabine while silica implants were utilized for extended intraperitoneal virus delivery. Immunocompetent mice and Syrian hamsters were used to study the effect of silica mediated delivery on antiviral immune responses and subsequent in vivo gene delivery. Capsid modifications selectively enhanced gene transfer to malignant pancreatic cancer cell lines and clinical samples. The respective oncolytic viruses resulted in increased cell killing in vitro, which translated into a survival benefit in mice. Early proinfammatory cytokine responses and formation of antiviral neutralizing antibodies was partially avoided with silica implants. The implant also shielded the virus from pre‐existing neutralizing antibodies, while increasing the pancreas/liver gene delivery ratio six‐fold. In conclusion, capsid modified adenoviruses would be useful for testing in pancreatic cancer trials. Silica implants might increase the safety and efficacy of the approach.

Syngeneic Syrian hamster tumors feature tumor-infiltrating lymphocytes allowing adoptive cell therapy enhanced by oncolytic adenovirus in a replication permissive setting

ABSTRACT Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown promising yet sometimes suboptimal results in clinical trials for advanced cancer, underscoring the need for approaches improving efficacy and safety. Six implantable syngeneic tumor cell lines of the Syrian hamster were used to initiate TIL cultures. TIL generated from tumor fragments cultured in human interleukin-2 (IL-2) for 10 d were adoptively transferred into tumor-bearing hamsters with concomitant intratumoral injections of oncolytic adenovirus (Ad5-D24) for the assessment of antitumor efficacy. Pancreatic cancer (HapT1) and melanoma (RPMI 1846) TIL exhibited potent and tumor-specific cytotoxicity in effector-to-target (E/T) assays. MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, indicating cytotoxic CD8+ T-cell activity. When TIL were combined with Ad5-D24 in vitro, HapT1 tumor cell killing was significantly enhanced over single agents. In vivo, the intratumoral administration of HapT1 TIL and Ad5-D24 resulted in improved tumor growth control compared with either treatment alone. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes, suggesting that systemic antitumor immunity was induced. For the first time, TIL of the Syrian hamster have been cultured, characterized and used therapeutically together with oncolytic adenovirus for enhancing the efficacy of TIL therapy. Our results support human translation of oncolytic adenovirus as an enabling technology for adoptive T-cell therapy of solid tumors.