Luigi Aurisicchio

Baculovirus Vectors Elicit Antigen-Specific Immune Responses in Mice

ABSTRACT To characterize the induction of antigen-specific immune response mediated by baculovirus, vectors expressing the E2 glycoprotein of hepatitis C virus or the carcinoembryonic antigen (CEA) under the control of the cytomegalovirus immediate-early promoter-enhancer were constructed. Additionally, a baculovirus vector encoding the E2 glycoprotein (Bac-G-E2) and expressing vesicular stomatitis virus glycoprotein (VSV-G) in the viral envelope was generated by inserting the VSV-G coding sequence downstream of the polyhedrin promoter. Mice were subjected to intramuscular, intranasal, or subcutaneous inoculations with Bac-E2 and the cellular immune response was monitored by ELISPOT and intracellular staining. Additionally, humoral response was monitored by titrating anti-E2 antibodies. Induction of a measurable anti-E2 T-cell response was observed only after intramuscular injection and was predominantly CD8+ specific. The immunogenic properties of baculovirus as vaccine vector were not restricted to E2 because a CEA-specific CD4+ T-cell response was observed upon intramuscular injection of Bac-CEA. Interestingly, the Bac-G-E2 vector was shown to be a more efficient immunogen than Bac-E2, in view of the 10-fold difference in the minimal dose required to elicit a measurable T-cell response upon intramuscular injection. Induction of inflammatory cytokines such as gamma interferon, tumor necrosis factor alpha, and interleukin-6 was detected as early as 6 h postinjection of Bac-G-E2. Most importantly, both vectors elicited CD8+ T cells with effector function capable of lysing target cells loaded with a hepatitis C virus-specific epitope. Additionally, enhanced NK cytolytic activity was detected in immunized mice. Thus, these results further demonstrate that baculovirus may be considered a useful vector for gene therapy.

Quantitative Optical Imaging of Primary Tumor Organoid Metabolism Predicts Drug Response in Breast Cancer

There is a need for technologies to predict the efficacy of cancer treatment in individual patients. Here, we show that optical metabolic imaging of organoids derived from primary tumors can predict the therapeutic response of xenografts and measure antitumor drug responses in human tumor-derived organoids. Optical metabolic imaging quantifies the fluorescence intensity and lifetime of NADH and FAD, coenzymes of metabolism. As early as 24 hours after treatment with clinically relevant anticancer drugs, the optical metabolic imaging index of responsive organoids decreased (P < 0.001) and was further reduced when effective therapies were combined (P < 5 × 10(-6)), with no change in drug-resistant organoids. Drug response in xenograft-derived organoids was validated with tumor growth measurements in vivo and staining for proliferation and apoptosis. Heterogeneous cellular responses to drug treatment were also resolved in organoids. Optical metabolic imaging shows potential as a high-throughput screen to test the efficacy of a panel of drugs to select optimal drug combinations. Cancer Res; 74(18); 5184-94. ©2014 AACR.

Efficient induction of T‐cell responses to carcinoembryonic antigen by a heterologous prime‐boost regimen using DNA and adenovirus vectors carrying a codon usage optimized cDNA

The immunogenic properties of plasmid DNA and recombinant adenovirus (Ad) encoding the carcinoembryonic antigen (CEA) were examined in mice by measuring both the amplitude and type of immune response, and the immunogenicity of codon usage optimized cDNA encoding CEA (CEAopt) was assessed both in C57Bl/6 and CEA transgenic mice. Vectors were injected into quadriceps muscle either alone or in combination, and plasmid DNA was electroporated to enhance gene expression efficiency and immunogenicity. Injection of plasmid pVIJ/CEA followed by Ad‐CEA boost elicited the highest amplitude of both CD4+ and CD8+ T‐cell response to the target antigen, measured by both IFNγ‐ELIspot assay and intracellular staining. Vectors carrying cDNA of CEAopt expressed a greater amount of the CEA protein than their wild‐type counterparts, and this enhanced expression was associated with greater immunogenicity. Both CD4+ and CD8+ T‐cell epitopes were mapped in the C‐terminal portion of the protein. In CEA transgenic mice, only immunization based on repeated injections of pVIJ/CEAopt followed by Ad‐CEAopt was able to elicit a CEA‐specific CD8+ T‐cell response, whereas the wild‐type vectors did not break tolerance to this target antigen. MC38‐CEA tumor cells injected s.c. in CEA transgenic mice vaccinated with CEAopt vectors exhibited delayed growth kinetics. These studies demonstrate that this type of genetic vaccine is highly immunogenic and can break tolerance to CEA tumor antigen in CEA transgenic mice.

Liver-Specific Alpha 2 Interferon Gene Expression Results in Protection from Induced Hepatitis

The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-alpha). However, systemic delivery of r-hIFN-alpha is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-alpha antiviral efficacy, we have explored the therapeutic potential of murine IFN-alpha2 (mIFNalpha2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-alpha2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-alpha2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-alpha2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-alpha2.

Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells

In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

Phase 1 studies of the safety and immunogenicity of electroporated HER2/CEA DNA vaccine followed by adenoviral boost immunization in patients with solid tumors

BackgroundDNA electroporation has been demonstrated in preclinical models to be a promising strategy to improve cancer immunity, especially when combined with other genetic vaccines in heterologous prime-boost protocols. We report the results of 2 multicenter phase 1 trials involving adult cancer patients (n=33) with stage II-IV disease.MethodsPatients were vaccinated with V930 alone, a DNA vaccine containing equal amounts of plasmids expressing the extracellular and trans-membrane domains of human HER2, and a plasmid expressing CEA fused to the B subunit of Escherichia coli heat labile toxin (Study 1), or a heterologous prime-boost vaccination approach with V930 followed by V932, a dicistronic adenovirus subtype-6 viral vector vaccine coding for the same antigens (Study 2).ResultsThe use of the V930 vaccination with electroporation alone or in combination with V932 was well-tolerated without any serious adverse events. In both studies, the most common vaccine-related side effects were injection site reactions and arthralgias. No measurable cell-mediated immune response (CMI) to CEA or HER2 was detected in patients by ELISPOT; however, a significant increase of both cell-mediated immunity and antibody titer against the bacterial heat labile toxin were observed upon vaccination.ConclusionV930 vaccination alone or in combination with V932 was well tolerated without any vaccine-related serious adverse effects, and was able to induce measurable immune responses against bacterial antigen. However, the prime-boost strategy did not appear to augment any detectable CMI responses against either CEA or HER2.Trial registrationStudy 1 – ClinicalTrials.gov, NCT00250419; Study 2 – ClinicalTrials.gov, NCT00647114.

MMP11: A Novel Target Antigen for Cancer Immunotherapy

Purpose: Matrix metalloproteinases (MMP) are zinc-dependent endopeptidases that mediate numerous physiologic and pathologic processes, including matrix degradation, tissue remodeling, inflammation, and tumor metastasis. To develop a vaccine targeting stromal antigens expressed by cancer-associated fibroblasts, we focused on MMP11 (or stromelysin 3). MMP11 expression correlates with aggressive profile and invasiveness of different types of carcinoma. Experimental Design: To show the efficacy of a vaccine targeting MMP11, we constructed a series of plasmid DNA vectors expressing murine MMP11. Mice were vaccinated by i.m. injection followed by in vivo DNA electroporation. A chemically induced, MMP11-overexpressing colon cancer model was established and characterized. Antibody and T-cell responses were determined, and immunoreactive epitopes were characterized. To analyze the possible use of MMP11 as tumor-associated antigen in cancer patients, HLA-A2.1 transgenic mice (HHD) were used to identify reactive epitopes as tools to assess immunogenicity in humans. Results: Using microarray, we confirmed the overexpression of MMP11 mRNA in a large panel of human tumor samples. MMP11 vaccine induced cell mediated and antibody immune response and exerted significant antitumoral protection in mice with colon cancer in prophylactic and therapeutic settings. HHD transgenic mice were vaccinated with a plasmid encoding human MMP11, and a HLA-A2.1–restricted epitope (hMMP237) was identified. hMMP237 was shown to be immunogenic in human peripheral blood mononuclear cells (PBMC) by in vitro priming. Conclusion: Our study describes the identification of MMP11 as a novel broadly expressed tumor associated antigen as target candidate for cancer immunotherapy.

A Vaccine Targeting Telomerase Enhances Survival of Dogs Affected by B-cell Lymphoma

Canine cancers occur with an incidence similar to that of humans and share many features with human malignancies including histological appearance, tumor genetics, biological behavior, and response to conventional therapies. As observed in humans, the telomerase reverse transcriptase (TERT) activity is largely confined to tumor tissues and absent in the majority of normal dog tissues. Therefore, dog TERT (dTERT) can constitute a valid target for translational cancer immunotherapy. We have evaluated the ability of adenovirus serotype 6 (Ad6) and DNA electroporation (DNA-EP) to induce immune responses against dTERT in dogs affected by malignant lymphoma (ML). The vaccine was combined with standard chemotherapy regimen [cyclophosphamide, vincristine, prednisone (COP)]. dTERT-specific immune response was induced in 13 out of 14 treated animals (93%) and remained detectable and long-lasting with the absence of autoimmunity or other side effects. Most interestingly, the survival time of vaccine/Chemo-treated dogs was significantly increased over historic controls of Chemo-treated animals (>97.8 versus 37 weeks, respectively, P = 0.001). Our results show that Ad6/DNA-EP-based cancer vaccine against dTERT overcomes host immune tolerance, should be combined with chemotherapy, induces long-lasting immune responses, and significantly prolongs the survival of ML canine patients. These data support further evaluation of this approach in human clinical trials.

Spheres Derived from Lung Adenocarcinoma Pleural Effusions: Molecular Characterization and Tumor Engraftment

Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.

Treatment of Mammary Carcinomas in HER-2 Transgenic Mice through Combination of Genetic Vaccine and an Agonist of Toll-Like Receptor 9

Purpose: Oligodeoxynucleotides containing unmethylated CpG dinucleotides induce innate and adaptive immunity through Toll-like receptor 9 (TLR9). In the present study, we have examined the ability of a novel agonist of TLR9, called immunomodulatory oligonucleotide (IMO), to enhance effects of a HER-2/neu plasmid DNA electroporation/adenovirus (DNA-EP/Ad) vaccine. Experimental Design: BALB/NeuT mice were treated with DNA-EP vaccine alone, IMO alone, or the combination of two agents starting at week 13, when all mice showed mammary neoplasia. Tumor growth and survival were documented. Antibody and CD8+ T-cell responses were determined. Peptide microarray analysis of sera was carried out to identify immunoreactive epitopes. Additionally, microCT and microPET imaging was carried out in an advanced-stage tumor model starting treatment at week 17 in BALB/NeuT mice. Results: The combination of DNA-EP and IMO resulted in significant tumor regression or delay to tumor progression. 2-Deoxy-2-[18F]fluoro-d-glucose microPET and microCT imaging of mice showed reduced tumor size in the DNA-EP/IMO combination treatment group. Mice treated with the combination produced greater antibody titers with IgG2a isotype switch and antibody-dependent cellular cytotoxicity activity than did mice treated with DNA-EP vaccine. An immunogenic B-cell linear epitope, r70, within the HER-2 dimerization domain was identified through microarray analysis. Heterologous DNA-EP/Ad vaccination combined with IMO increased mice survival. Conclusion: The combination of HER-2/neu genetic vaccine and novel agonist of TLR9 had potent antitumor activity associated with antibody isotype switch and antibody-dependent cellular cytotoxicity activities. These results support possible clinical trials of the combination of DNA-EP/Ad-based cancer vaccines and IMO.