Federica Moschella

Cyclophosphamide Enhances the Antitumor Efficacy of Adoptively Transferred Immune Cells through the Induction of Cytokine Expression, B-Cell and T-Cell Homeostatic Proliferation, and Specific Tumor Infiltration

Purpose: Immunotherapy is a promising antitumor strategy, which can be successfully combined with current anticancer treatments, as suggested by recent studies showing the paradoxical chemotherapy-induced enhancement of the immune response. The purpose of the present work is to dissect the biological events induced by chemotherapy that cooperate with immunotherapy in the success of the combined treatment against cancer. In particular, we focused on the following: (a) cyclophosphamide-induced modulation of several cytokines, (b) homeostatic proliferation of adoptively transferred lymphocytes, and (c) homing of transferred lymphocytes to secondary lymphoid organs and tumor mass. Experimental Design: Here, we used the adoptive transfer of tumor-immune cells after cyclophosphamide treatment of tumor-bearing mice as a model to elucidate the mechanisms by which cyclophosphamide can render the immune lymphocytes competent to induce tumor rejection. Results: The transfer of antitumor immunity was found to be dependent on CD4+ T cells and on the cooperation of adoptively transferred cells with the host immune system. Of note, tumor-immune lymphocytes migrated specifically to the tumor only in mice pretreated with cyclophosphamide. Cyclophosphamide treatment also promoted homeostatic proliferation/activation of transferred B and T lymphocytes. Optimal therapeutic responses to the transfer of immune cells were associated with the cyclophosphamide-mediated induction of a “cytokine storm” [including granulocyte macrophage colony-stimulating factor, interleukin (IL)-1β, IL-7, IL-15, IL-2, IL-21, and IFN-γ], occurring during the “rebound phase” after drug-induced lymphodepletion. Conclusions: The ensemble of these data provides a new rationale for combining immunotherapy and chemotherapy to induce an effective antitumor response in cancer patients.

Chemotherapy enhances vaccine-induced antitumor immunity in melanoma patients.

Combination of chemotherapy with cancer vaccines is currently regarded as a potentially valuable therapeutic approach for the treatment of some metastatic tumors, but optimal modalities remain unknown. We designed a phase I/II pilot study for evaluating the effects of dacarbazine (DTIC) on the immune response in HLA‐A2+ disease‐free melanoma patients who received anticancer vaccination 1 day following chemotherapy (800 mg/mq i.v.). The vaccine, consisting of a combination of HLA‐A2 restricted melanoma antigen A (Melan‐A/MART‐1) and gp100 analog peptides (250 μg each, i.d.), was administered in combination or not with DTIC to 2 patient groups. The combined treatment is nontoxic. The comparative immune monitoring demonstrates that patients receiving DTIC 1 day before the vaccination have a significantly improved long‐lasting memory CD8+ T cell response. Of relevance, these CD8+ T cells recognize and lyse HLA‐A2+/Melan‐A+ tumor cell lines. Global transcriptional analysis of peripheral blood mononuclear cells (PBMC) revealed a DTIC‐induced activation of genes involved in cytokine production, leukocyte activation, immune response and cell motility that can favorably condition tumor antigen‐specific CD8+ T cell responses. This study represents a proof in humans of a chemotherapy‐induced enhancement of CD8+ memory T cell response to cancer vaccines, which opens new opportunities to design novel effective combined therapies improving cancer vaccination effectiveness.

Combination strategies for enhancing the efficacy of immunotherapy in cancer patients

A major challenge in cancer immunotherapy is the identification of effective strategies for enhancing its clinical efficacy. One approach is based on adjuvants capable of breaking tolerance against tumor‐associated antigens. Interferon‐α(IFN‐α), an antiviral cytokine with a long record of clinical use, has recently been shown to act as an effective adjuvant in cancer patients. Notably, a special interest is currently focused on the use of dendritic cells (DC) generated in the presence of IFN‐α (IFN‐DC) for the preparation of anticancer vaccines. An additional approach for enhancing the response to immunotherapy relies on its combination with chemotherapy. In fact, an ensemble of results from both studies in animal models and pilot clinical trials suggest that certain chemotherapeutic agents can act, under defined conditions, as strong adjuvants for enhancing the efficacy of immunotherapy. These results open new opportunities for designing mechanism‐based combination therapies involving both chemotherapy and new‐generation cancer vaccines, including IFN‐DC‐based vaccines.

Exploitation of the propulsive force of chemotherapy for improving the response to cancer immunotherapy

Since the early clinical studies of cancer immunotherapy, the question arose as to whether it was possible to combine it with standard cancer treatments, mostly chemotherapy. The answer, now, is past history. The combined use of immunotherapy and chemotherapy is not only possible but, in certain cases, can be advantageous, depending on the drug, the dose and the combination modalities. In order to find the best synergisms between the two treatments and to turn weak immunotherapeutic interventions into potent anticancer instruments, it is mandatory to understand the complex mechanisms responsible for the positive interactions between chemotherapy and immunotherapy. In this article, we review the current knowledge on mechanisms involved in the immunostimulating activity of chemotherapy and summarize the main studies in both mouse models and patients aimed at exploiting such mechanisms for enhancing the response to cancer immunotherapy.

Exploiting dendritic cells in the development of cancer vaccines

Due to their central role in priming and modulating the immune response, dendritic cells (DCs) represent an ideal instrument for the design of effective immunotherapeutic strategies for cancer patients. Recent advancement on the knowledge of the numerous DC subtypes, their functions and T-cell polarizing abilities has led to the development of several protocols for the ex vivo differentiation of autologous DCs and their loading with tumor-associated antigens. Moreover, novel strategies for the in vivo targeting of tumor antigens and adjuvants to natural DC subsets have been developed. Despite the large number of clinical studies carried out in cancer patients, a consensus on the optimal treatment modalities has not been reached yet. In this review, we summarize our current knowledge on DC biology and on DC use in clinical trials. Special attention is given to the many open issues regarding DC-based vaccination to sensitize researchers in the field to the compelling need of conducting comparative studies systematically addressing the still unresolved problems.

The Janus face of cyclophosphamide A sterile inflammatory response that potentiates cancer immunotherapy

Cyclophosphamide, within well-defined therapeutic regimens, increases the antineoplastic effects of immunotherapy. We have recently identified multiple factors and mechanisms that underlie the paradoxical synergy between these two treatment modalities. In particular, we found that cyclophosphamide stimulates anticancer immune responses upon the perception by the immune system of inflammatory danger signals associated with the death of leukocytes, via p53 and type I interferon-related mechanisms.

Twenty-five years of type I interferon-based treatment: A critical analysis of its therapeutic use

Abstract The clinical exploitation of type I interferon (IFN) as an antiviral and antineoplastic agent is based on the properties originally attributed to this cytokine family, with schedules reflecting only their antiviral and antiproliferative activities. Nevertheless, type I IFN has emerged as a central activator of the innate immunity. As current schedules of treatment for chronic hepatitis C and for hematological and solid tumors, based on the continuous administration of recombinant type I IFN or pegylated formulations, disregard viral resistance, host genetic variants predicting treatment outcome and mechanisms of refractoriness, new administration schedules, the combination of type I IFN with new drugs and the increased monitoring of patients’ susceptibility to type I IFN are expected to provide a new life to this valuable cytokine.

The added value of type i interferons to cytotoxic treatments of cancer

Type I interferons (IFNs) exert anti-proliferative, antiviral and immunomodulatory activities. They are also involved in cell differentiation and anti-tumor defense processes. A growing body of literature indicates that the success of conventional chemotherapeutics, epigenetic drugs, targeted anticancer agents and radiotherapy (RT) relies, at least in part, on the induction of type I IFN signaling in malignant cells, tumor-infiltrating antigen presenting cells or other immune cells within lymphoid organs or blood. The mechanisms underlying type I IFN induction and the clinical consequences of these observations are only beginning to be elucidated. In the present manuscript, we reviewed the recent advances in the field and provided our personal view on the role of type I IFNs induced in the context of cytotoxic anticancer treatments and on its possible exploitation as a complement in cancer therapy.

MHV-68 producing mIFNα1 is severely attenuated in vivo and effectively protects mice against challenge with wt MHV-68

Human gammaherpesviruses such as Epstein-Barr virus (EBV) cause lifelong infections and associated diseases, by virtue of their ability to establish latent infection. Many studies performed in the past years in murine herpesvirus 68 (MHV-68) model of infection suggested that the limited immunity generated against isolated viral components by subunit vaccines cannot counteract the multiple immune evasion strategies operated by gammaherpesviruses. Indeed, a significant inhibition of long-term latency establishment could be observed in mice vaccinated with strains of genetically modified MHV-68 defective in reactivation or establishment of latency. In this study, we focused on the effects of interferon-α (IFN-α) on both the lytic and latent phase of MHV-68 infection, as exerted by the constitutive release of IFN-α1 by a clone of MHV-68 genetically modified to produce this cytokine (MHV-68mIFNα1). Although the MHV-68mIFNα1 recombinant virus exhibited in vitro replication features indistinguishable from those of the wild type MHV-68, its pathological properties were severely attenuated in vivo in immunocompetent mice and not in mice rendered genetically unresponsive to type I IFN, suggesting that a stronger immune response was primed in the presence of the cytokine. Notably, MHV-68mIFNα1 attenuation did not result in a reduced level of long-term spleen latency establishment. These results prompted us to evaluate the efficacy of MHV-68mIFNα1 in a prophylactic vaccination regimen aimed at inhibiting the symptoms of acute virus infection and the establishment of long-term latency after MHV-68 challenge. Our results show that mice vaccinated with MHV-68mIFNα1, administered as a live-attenuated or partially inactivated (by Psoralen and UV treatment) vaccine, were protected against the challenge with wt MHV-68 from all phases of infection. The ability of MHV-68mIFNα1 to produce IFN-α at the site of the infection, thus efficiently stimulating the immune system in case of virus reactivation from latency, makes this recombinant virus a safer live-attenuated vaccine as compared to the previously reported latency-deficient clones.

Disruption of IFN-I signaling promotes HER2/neu tumor progression and breast cancer stem cells

Type I interferon (IFN-I) inhibits tumor growth and activates antitumor responses. HER2-driven tumors in mice lacking IFN-I receptor were more aggressive, more vascularized, and were enriched in stem cells, suggesting that IFN-I exerts key control over tumor progression. Type I interferon (IFN-I) is a class of antiviral immunomodulatory cytokines involved in many stages of tumor initiation and progression. IFN-I acts directly on tumor cells to inhibit cell growth and indirectly by activating immune cells to mount antitumor responses. To understand the role of endogenous IFN-I in spontaneous, oncogene-driven carcinogenesis, we characterized tumors arising in HER2/neu transgenic (neuT) mice carrying a nonfunctional mutation in the IFNI receptor (IFNAR1). Such mice are unresponsive to this family of cytokines. Compared with parental neu+/− mice (neuT mice), IFNAR1−/− neu+/− mice (IFNAR-neuT mice) showed earlier onset and increased tumor multiplicity with marked vascularization. IFNAR-neuT tumors exhibited deregulation of genes having adverse prognostic value in breast cancer patients, including the breast cancer stem cell (BCSC) marker aldehyde dehydrogenase-1A1 (ALDH1A1). An increased number of BCSCs were observed in IFNAR-neuT tumors, as assessed by ALDH1A1 enzymatic activity, clonogenic assay, and tumorigenic capacity. In vitro exposure of neuT+ mammospheres and cell lines to antibodies to IFN-I resulted in increased frequency of ALDH+ cells, suggesting that IFN-I controls stemness in tumor cells. Altogether, these results reveal a role of IFN-I in neuT-driven spontaneous carcinogenesis through intrinsic control of BCSCs. Cancer Immunol Res; 6(6); 658–70. ©2018 AACR.