Daniela Peruzzi

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.

Telomerase and HER-2/neu as targets of genetic cancer vaccines in dogs.

Pet dogs represent a valuable pre-clinical model to assess the efficacy of oncology drugs. Additionally, 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. The telomerase reverse transcriptase (TERT) is reactivated in most of human and dog tumors. Similarly, HER-2/neu oncoprotein is overexpressed in a proportion of canine breast cancers. Therefore, TERT and HER-2/neu can constitute valid tumor associated antigens (TAA), suitable targets for translational cancer immunotherapy in dogs. In this study, we have evaluated the ability of DNA electroporation (DNA-EP) and Adenovirus serotype 6 (Ad6) to induce immune responses against dog TERT (dTERT) and HER-2/neu in healthy dogs. Vaccination was effective in all treated animals and the adaptive immune response remained detectable and long-lasting in the absence of autoimmunity or other side-effects. Our results show that DNA-EP/Ad6-based cancer vaccine induces adaptive immune responses against TAA in canine subjects and support further evaluation of this approach in cancer dog patients.

Engineered adenovirus serotypes for overcoming anti-vector immunity.

Adenovirus (Ad)-based gene transfer has been successfully utilised in gene therapy and vaccine applications. To date, an increasing number of human clinical trials utilise recombinant Ad-based vectors as a gene transfer platform. In particular, progress has been made recently in utilising Ad-based vectors as a vaccine platform in HIV, cancer immunotherapy approaches and in vaccination for other infections. Despite these successes, the scientific and bio-industrial communities have recently recognised that innate and pre-existing immunity against Ad vectors can constitute a serious obstacle to the development and application of this technology. It is essential to overcome vector-mediated immune responses, such as production of inflammatory cytokines and pre-existing immunity to Ad, because the induction of these responses not only shortens the period of gene expression but also leads to serious side effects. This review focuses on the biology of Ad infection and the approaches that are being adopted to overcome immunity against the Ad-based vectors.

Coadministration of telomerase genetic vaccine and a novel TLR9 agonist in nonhuman primates.

The human telomerase reverse transcriptase (hTERT) is an attractive target for human cancer vaccination because its expression is reactivated in most human tumors. We have evaluated the ability of DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to induce immune responses against hTERT in nonhuman primates (NHPs) (Macaca mulatta). Vaccination was effective in all treated animals, and the adaptive immune response remained detectable and long lasting without side effects. To further enhance the efficacy of the hTERT vaccine, we evaluated the combination of hTERT vaccine and a novel TLR9 agonist, referred to as immunomodulatory oligonucleotide (IMO). Monkeys were dosed weekly with IMO concurrently with the vaccine regimen and showed increases in cytokine secretion and activation of natural killer (NK) cells compared with the group that received vaccine alone. Using a peptide array, a specific profile of B-cell reactive epitopes was identified when hTERT vaccine was combined with IMO. The combination of IMO with hTERT genetic vaccine did not impact vaccine-induced TERT-specific cell-mediated immunity. Our results show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against hTERT with IMO induces multiple effects on innate and adaptive immune responses in NHPs.

A TLR9 agonist enhances therapeutic effects of telomerase genetic vaccine

The telomerase reverse transcriptase (TERT) is an attractive target for cancer vaccination because its expression is reactivated in most tumors. In this study, we have evaluated the ability of a genetic vaccine targeting murine TERT (mTERT) based on DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to exert therapeutic effects in combination with a novel TLR9 agonist, referred to as immune modulatory oligonucleotide (IMO), as an adjuvant. IMO was administered to mice at the same time as vaccine. IMO induced dose-dependent cytokine secretion and activation of NK cells. Most importantly, vaccination of mice with IMO in combination with mTERT vaccine conferred therapeutic benefit in tumor bearing animals and this effect was associated with increased NK, DC and T cell tumor infiltration. These data show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against TERT with IMO induces multiple effects on innate and adaptive immune responses resulting in a significant antitumor efficacy.

A therapeutic cancer vaccine targeting carcinoembryonic antigen in intestinal carcinomas.

A genetic vaccine platform based on DNA electroporation (DNA-EP) and adenovirus (Ad) was used to generate immune response against human carcinoembryonic antigen (CEA) and antitumor effects in murine models with spontaneous tumors arising in an orthotopic location. CEA transgenic (CEA.Tg) mice treated with the carcinogen 1,2-dimethylhydrazine developed CEA-overexpressing tumors that resembled human sporadic colorectal cancer. APC1638N/CEA hybrid mice, generated by crossing mice carrying the adenomatous polyposis coli (Apc1638N) gene mutation with CEA.Tg mice, are representative of human familial polyposis and develop polyps that overexpress the antigen. In both models, the DNA-EP/Ad vaccine succeeded in breaking immune tolerance and achieved significant antitumor effects in therapeutic settings. Our data suggest that genetic vaccines targeting CEA may be feasible strategies against gut tumors that overexpress the antigen. In addition, these models are powerful systems for evaluating antigen-specific tumor immunity and assessing therapeutic vaccine strategies for human colorectal cancer.

An efficient T-cell epitope discovery strategy using in silico prediction and the iTopia assay platform

Functional T-cell epitope discovery is a key process for the development of novel immunotherapies, particularly for cancer immunology. In silico epitope prediction is a common strategy to try to achieve this objective. However, this approach suffers from a significant rate of false-negative results and epitope ranking lists that often are not validated by practical experience. A high-throughput platform for the identification and prioritization of potential T-cell epitopes is the iTopiaTM Epitope Discovery SystemTM, which allows measuring binding and stability of selected peptides to MHC Class I molecules. So far, the value of iTopia combined with in silico epitope prediction has not been investigated systematically. In this study, we have developed a novel in silico selection strategy based on three criteria: (1) predicted binding to one out of five common MHC Class I alleles; (2) uniqueness to the antigen of interest; and (3) increased likelihood of natural processing. We predicted in silico and characterized by iTopia 225 candidate T-cell epitopes and fixed-anchor analogs from three human tumor-associated antigens: CEA, HER2 and TERT. HLA-A2-restricted fragments were further screened for their ability to induce cell-mediated responses in HLA-A2 transgenic mice. The iTopia binding assay was only marginally informative while the stability assay proved to be a valuable experimental screening method complementary to in silico prediction. Thirteen novel T-cell epitopes and analogs were characterized and additional potential epitopes identified, providing the basis for novel anticancer immunotherapies. In conclusion, we show that combination of in silico prediction and an iTopia-based assay may be an accurate and efficient method for MHC Class I epitope discovery among tumor-associated antigens.

Immunogenicity and Therapeutic Efficacy of a Dual-Component Genetic Cancer Vaccine Cotargeting Carcinoembryonic Antigen and HER2/neu in Preclinical Models

Several cancer vaccine efforts have been directed to simultaneously cotarget multiple tumor antigens, with the intent to achieve broader immune responses and more effective control of cancer growth. Genetic cancer vaccines based on in vivo muscle electro-gene-transfer of plasmid DNA (DNA-EGT) and adenoviral vectors represent promising modalities to elicit powerful immune responses against tumor-associated antigens (TAAs) such as carcinoembryonic antigen (CEA) and human epidermal growth factor receptor-2 (HER2)/neu. Combinations of these modalities of immunization (heterologous prime-boost) can induce superior immune reactions as compared with single-modality vaccines. We have generated a dual component-dual target genetic cancer vaccine consisting of a DNA moiety containing equal amounts of two plasmids, one encoding the extracellular and transmembrane domains of HER2 (ECD.TM) and the other encoding CEA fused to the B subunit of Escherichia coli heat-labile toxin (LTB), and of an adenoviral subtype 6 dicistronic vector carrying the same two tumor antigens gene constructs. The CEA/HER2 vaccine was tested in two different CEA/HER2 double-transgenic mouse models and in NOD/scid-DR1 mice engrafted with the human immune system. The immune response was measured by enzyme-linked immunospot assay, flow cytometry, and ELISA. The CEA/HER2 vaccine was able to break immune tolerance against both antigens. Induction of a T cell and antibody immune response was detected in immune-tolerant mice. Most importantly, the vaccine was able to slow the growth of HER2/neu⁺ and CEA⁺ tumors. A significant T cell response was measured in NOD/scid-DR1 mice engrafted with human cord blood cells. In conclusion, the CEA/HER2 genetic vaccine was immunogenic and able to confer significant therapeutic effects. These data warrant the evaluation of this vaccination strategy in human clinical trials.

Abstract 291: Targeting the stroma to hit the tumor: MMP11 as a novel target for cancer immunotherapy

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that mediate a number of physiological and pathological processes, such as matrix degradation, tissue remodeling, inflammation and tumor metastasis. The objective of this study was to develop and characterize a vaccine targeting stromal antigens expressed by cancer associated fibroblasts (CAFs). We focused on MMP11 (or stromelysin 3, ST3), that has been detected primarily in CAFs and its expression correlates with aggressive clinical behavior and invasiveness of different types of carcinoma. We demonstrated that intramuscular injection of genetic vectors such as Adenovirus (Ad) and plasmid DNA encoding MMP11 engineered variants followed by in vivo electroporation (DNA-EP) resulted in breakage of immune tolerance and induction of both cell mediated and humoral immune response. Importantly, MMP11 vaccine was able to confer significant antitumoral protection in a chemically induced, MMP11 overexpressing colon cancer model both in prophylactic and therapeutic settings. To determine the mechanism of action of MMP11 vaccine, we have utilized IFNgamma- and μmicroMT-knock out mice, devoid of T- and B-cell immune response, respectively. Our results show that both arms of the immune response are important to confer the therapeutic effect. Moreover, MMP11 stromal vaccine showed synergic effects when combined with genetic vaccines targeting classic tumor associated antigens, such as telomerase reverse transcriptase (TERT) and carcinoembryonic antigen (CEA). Finally, to assess the immunogenicity and the safety of MMP11 vaccine in a large animal model, nonhuman primates have been vaccinated with Ad and DNA-EP. A strong immune response was measured with no detectable side effects. In addition, MMP11 detection and spontaneous immune responses in the blood of breast and prostate cancer patients further corroborate MMP11 as a valid target for immune intervention. Taken together, these data support the use of MMP11 as a potential candidate for cancer immunotherapy in human clinical trials. Citation Format: Laura Luberto, Rita Mancini, Arianna Di Napoli, Daniela Peruzzi, Federica Mori, Giuseppe Roscilli, Emanuele Marra, Manuela Cappelletti, Gennaro Ciliberto, Luigi Aurisicchio. Targeting the stroma to hit the tumor: MMP11 as a novel target for cancer immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 291. doi:10.1158/1538-7445.AM2015-291

Safety and efficacy of a genetic vaccine targeting Telomerase plus chemotherapy for the therapy of canine B-cell lymphoma.

Abstract Client-owned pet dogs represent exceptional translational models for advancement of cancer research because they reflect the complex heterogeneity observed in human cancer. We have recently shown that a genetic vaccine targeting dog telomerase reverse transcriptase (dTERT) and based on adenovirus DNA electro-gene-transfer (Ad/DNA-EGT) technology can induce strong cell-mediated immune responses against this tumor antigen and increase overall survival of dogs affected by B-cell lymphosarcoma (LSA) in comparison with historical controls when combined with a cyclophosphamide, vincristine, and prednisone (COP) chemotherapy regimen. Here, we have conducted a double-arm clinical trial with an extended number of LSA patients, measured the antigen-specific immune response, and evaluated potential toxic effects of the immunotherapy along with a follow-up of patients survival for 3.5 years. The immune response was measured by enzyme-linked immunospot assay. The expression of dTERT was quantified by quantita...