ErbB2 genetic cancer vaccine in nonhuman primates: relevance of single nucleotide polymorphisms.

Aberrant Her2/neu expression is associated with the development of epithelial-derived human carcinomas and for this reason it is considered a good target for immunologic intervention. To define methods to circumvent immunologic tolerance and to elicit immunity against the Her2/neu tumor-associated antigen in a suitable animal model, we have isolated the cDNA encoding the rhesus monkey homolog of human Her2/neu (RhErbB2) to construct DNA plasmids and adenoviral vectors for the development of a cancer vaccine against this protein. To further increase the immunogenic potency of these vectors, a synthetic codon-optimized RhErbB2 cDNA (RhErbB2OPT) was constructed and characterized. Genetic vaccination of rhesus monkeys was effective in inducing a response against RhErbB2 in immunized animals; importantly, the elicited immunity was associated with natural RhErbB2 polymorphisms, thus distinguishing responses against "self " and "nonself " epitopes. In particular, the postpriming response recognized mainly nonself epitopes whereas the boosted response cross-reacted with self epitopes. Our findings are particularly relevant in the investigation of the impact of TAA polymorphisms on the efficacy of a cancer vaccine strategy.

Differential screening of phage-ab libraries by oligonucleotide microarray technology.

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.

Immunogenicity and safety of a DNA prime/adenovirus boost vaccine against rhesus CEA in nonhuman primates.

Scaling up experimental protocols from rodents to humans is often not a straightforward procedure, and this particularly applies to cancer vaccines, where vaccination technology must be especially effective to overcome a variety of immune suppressive mechanisms. DNA electroporation (DNA-EP) and adenoviral vectors (Ad) have shown high potency and therapeutic efficacy for different antigens in several pre-clinical models. To evaluate the ability of DNA-EP and Ad to break tolerance to a self-antigen in large animals, we have cloned the CEA homologue (rhCEA) from rhesus monkeys (Macaca mulatta) colon tissue samples. rhCEA is a 705 aa protein and shares 78.9% homology to human CEA protein. Immunogenicity of rhCEA expressing vectors was tested in mice and subsequently in rhesus monkeys. To further increase the immunogenic potency of these vectors, a synthetic codon optimized rhCEA cDNA (rhCEAopt) was constructed. Genetic vaccination of rhesus monkeys was effective in breaking immune tolerance to rhCEA in all immunized animals, maintaining over time the elicited immune response, and most importantly, neither autoimmunity nor other side-effects were observed upon treatment. Our data confirm the efficacy of genetic cancer vaccines in large animals such as nonhuman primates and show that development of modified expression cassettes that result in increased potency of plasmid DNA and adenovirus may have a significant impact on vaccine development against malignancies expressing tumor associated antigens in patients.

Adenovirus vaccination against neu oncogene exerts long-term protection from tumorigenesis in BALB/neuT transgenic mice.

The transforming rat HER2/neu oncogene (neu), when embedded in the genome of transgenic BALB/c (neuT) mice, provokes the development of an invasive carcinoma in each of their 10 mammary glands. We used the neuT mice model system to evaluate the immunization efficiency and the protective effect of intramuscular injection of adenovirus (Ad) and/or of DNA with electrostimulation (DNA+ES), both expressing the rat p185(neu) protein. A neu cDNA sequence, which exclusively contains codons preferred by highly expressed mammalian genes, was used in this study. This "optimized" cDNA displayed higher expression in cultured cells and greater cell-mediated response than the original gene when injected as DNA+ES. Ad expressing the optimized sequence (Ad5-neu.opt) induced a higher immune response, as measured by the frequency of IFN-gamma-secreting spleen cells and antibody titers. Different Ad/DNA combinations and immunization schedules confirmed the superiority of Ad5-neu.opt in inducing a strong Th1-skewed humoral and CD8(+) cell-mediated response. Two Ad5-neu.opt injections of 10(9) viral particles at week 10 and 12 were sufficient to induce the highest response, which persisted at detectable levels up to 33 weeks of age. Anti-Ad5 antibodies elicited by previous injections neutralized the effect of an additional Ad5-neu.opt immunization at week 19. A group, which received 3 injections of DNA+ES at week 23, 27 and 31, in addition to the 3 Ad injections at week 10, 12 and 19 showed an increased frequency of IFN-gamma(+), CD8(+) PBMC at week 25, which persisted at detectable levels till week 38. Ad5-neu.opt administration at 10 and 12 weeks of age had a significant impact on tumor progression. At 44 weeks, 40% of the mice were completely protected from tumors with a mean tumor of 3.8. In contrast, control mice developed 10 tumors and died by week 27. Vaccination blocked the tumor development at the atypical hyperplasia stage present at the time of treatment. Tumors developing at later times express reduced levels of rat p185(neu) protein.

Xenogeneic immunization in mice using HER2 DNA delivered by an adenoviral vector.

The protective efficacy of xenogeneic vaccination with DNA encoding the HER2 oncogene was evaluated in BALB/c mice transgenic for the transforming form of the neu oncogene, which spontaneously develops carcinomas in all mammary glands. Intramuscular injection of either plasmid DNA followed by electrical stimulation (pVIJ-HER2 with ES) or an adenoviral vector (Ad5-HER2), both expressing the HER2 oncogene, was tested. Immunization using pVIJ-HER2 with ES elicited a cell-mediated response that was much lower than that elicited by the immunization with Ad5-HER2, as measured by the frequency of IFN-gamma-secreting spleen cells. The dominant T-cell epitope of the HER2 protein product (p185) in the BALB/c (H-2(d)) genetic background was identified. While the T-cell response elicited was only partially crossreactive with the corresponding rat epitopes because of sequence variations (89% similarity), a cytotoxic T-lymphocyte activity against the rat immunodominant epitope was also evident. The Ad5-HER2 vaccination induced also antibodies against p185, which crossreacted with the rat protein homolog. Both T- and B-cell responses slowly declined with time. Vaccination with Ad5-HER2 at 6 and 9 weeks of age delayed incidence and reduced multiplicity of tumors in neu transgenic mice.

General strategy for broadening adenovirus tropism.

In spite of its broad host range, adenovirus type 5 (Ad5) transduces a number of clinically relevant tissues and cell types inefficiently, mostly because of low expression of the coxsackievirus-adenovirus receptor (CAR). To improve gene transfer to such cells, we modified the Ad5 fiber knob to recognize novel receptors. We expressed a functional Ad5 fiber knob domain on the capsid of phage lambda and employed this display system to construct a large collection of ligands in the HI loop of the Ad5 knob. Panning this library on the CAR-negative mouse fibroblast cell line NIH 3T3 resulted in the identification of three clones with increased binding to these cells. Adenoviruses incorporating these ligands in the fiber gene transduced NIH 3T3 cells 2 or 3 orders of magnitude better than the parent vector. The same nonnative tropism was revealed in other cell types, independently of CAR expression. These Ad5 derivatives proved capable of transducing mouse and human primary immature dendritic cells with up to 100-fold increased efficiency.