Curcumin Enhances the Antitumoral Effect Induced by the Recombinant Vaccinia Neu Vaccine (rV-neuT) in Mice with Transplanted Salivary Gland Carcinoma Cells.

The survival rate for head and neck cancer patients has not substantially changed in the last two decades. We previously showed that two rV-neuT intratumoral injections induced an efficient antitumor response and rejection of transplanted Neu (rat ErbB2/neu oncogene-encoded protein)-overexpressing salivary gland tumor cells in BALB-neuT mice (BALB/c mice transgenic for the rat ErbB2/neu oncogene). However, reiterated poxviral vaccinations increase neutralizing antibodies to viral proteins in humans that prevent immune response against the recombinant antigen expressed by the virus. Curcumin (CUR) is a polyphenol with antineoplastic and immunomodulatory properties. The aim of this study was to employ CUR administration to boost the anti-Neu immune response and anticancer activity induced by one rV-neuT intratumoral vaccination in BALB-neuT mice. Here, we demonstrated that the combined rV-neuT+CUR treatment was more effective at reducing tumor growth and increasing mouse survival, anti-Neu humoral response, and IFN-γ/IL-2 T-cell release in vitro than the individual treatment. rV-neuT+CUR-treated mice showed an increased infiltration of CD4+/CD8+ T lymphocytes within the tumor as compared to those that received the individual treatment. Overall, CUR enhanced the antitumoral effect and immune response to Neu induced by the rV-neuT vaccine in mice. Thus, the combined treatment might represent a successful strategy to target ErbB2/Neu-overexpressing tumors.

Retargeting Lentiviruses via SpyCatcher-SpyTag Chemistry for Gene Delivery into Specific Cell Types.

We report a simple strategy for the creation of lentiviral vectors specific to any desired target cells. SpyTag is inserted into an engineered Sindbis virus envelope protein and displayed on the lentivirus surface to create Sindbis virus-SpyTag pseudoparticles (Sind-SpyTag-pp). The SpyTag serves as the covalent anchoring site for a target-cell-specific cell-binding protein (CBP) that is fused to a truncated SpyCatcher (SpyCatcherΔ). Target-cell-specific lentiviruses are created by mixing the Sind-SpyTag-pp and CBP-SpyCatcherΔ in vitro We first used a HER2-binding designed ankyrin repeat protein (DARPin.9.26) as the model CBP. The DARPin-conjugated lentivirus transduced HER2+ SKOV3 cells with an infectious titer of 5.2 × 106 IU/ml, >500-fold higher than the unfunctionalized "naked" virions (<104 IU/ml). The ability of the DARPin-conjugated lentivirus to transduce HER2+ cells correlated with the surface expression level of HER2. Furthermore, these lentiviruses preferentially transduced HER2+ cells in cocultures containing HER2+ and HER2- cells. To enable the use of commercially available monoclonal antibodies (MAbs) as the CBP, we developed a convenient click chemistry-based approach to conjugate MAb-derived Fab fragments to a variant SpyCatcherΔ protein containing a nonnatural amino acid, 4-azido-l-phenylalanine (AzF). Using the HER2-binding trastuzumab as a model cell-specific MAb, we created Fab-conjugated lentiviral vectors that transduced HER2+ SKOV3 cells with an infectious titer of 2.8 × 106 IU/ml, on par with the result achieved using the DARPin-SpyCatcherΔ fusion protein. The ability to create cell-specific lentiviral vectors through chemical conjugation of a CBP should make this approach generalizable to any antibody, giving it broad utility for a wide range of research and clinical applications.IMPORTANCE Lentiviral vectors hold great potential in gene therapy. However, it remains a major hurdle to robustly engineer cell-specific lentiviral vectors. This article reports a simple and effective strategy to functionalize lentiviral vectors with cell-binding proteins, thus retargeting these viruses to cells expressing the binding partner of the CBP. The CBP is genetically or chemically linked to the SpyCatcher. The SpyTag is displayed on the virion surface as a fusion to an engineered Sindbis virus envelope protein and is used as the anchorage site for SpyCatcher-linked CBP. Using this strategy, we created lentiviral vectors highly infectious toward HER2+ cancer cells. The ability to rapidly create cell-specific lentiviral vectors targeting a wide range of cell types should accelerate the development of custom lentiviral vectors for many research and clinical applications.

Oncogene addiction and immunity: clinical implications of tumour infiltrating lymphocytes in breast cancers overexpressing the HER2/neu oncogene.

PURPOSE OF REVIEW: To review the evidence that correlates tumour infiltrating lymphocytes, a surrogate biomarker of pre-existing host antitumour immunity, and survival in HER2-overexpressing breast cancers. This is of particular relevance to developing immune biomarkers and harnessing new immunotherapeutics in this breast cancer subtype. RECENT FINDINGS: Oncogene addiction, in which cancer cells become reliant on a single oncogenic pathway for tumour growth and progression, has traditionally been thought of as a cell intrinsic characteristic. However, increasing evidence from multiple studies exploring the relationship between markers of an antitumour immune response and clinical outcome in HER2-overexpressing breast cancer points to the importance of a permissive microenvironment in oncogene-addicted tumours. SUMMARY: Characterizing the immune microenvironment in HER2-overexpressing breast cancer has the potential to furnish predictive and prognostic biomarkers that may be useful in routine clinical decision-making. The host-tumour immune interface is emerging as a key aspect of breast cancer biology that is likely to yield novel therapies in the near future.

Simultaneous analysis of HER2 gene and protein on a single slide facilitates HER2 testing of breast and gastric carcinomas.

This study sought to evaluate a new combined gene and protein detection platform in the context of HER2 evaluation in breast and gastric carcinomas. HER2 immunohistochemistry (IHC) and dual color in situ hybridization (Dual ISH) were combined on a single slide. Results were compared with conventional HER2 IHC and fluorescence ISH. Results from the gene and protein assay were reliable and highly reproducible for both breast and gastric carcinomas. Concordance was found between conventional HER2 IHC and ISH testing and the gene and protein assay in the same laboratory (>95% for Dual ISH; lower for IHC because of different antibody clones), between IHC and Dual ISH performed on the same slide (>92%), and in the gene and protein assays between laboratories (>96%). This cost- and time-effective method provides fast and definitive results (IHC confirmed by means of Dual ISH) to aid in rapid treatment decisions. It can also be applied to other gene and protein combinations.

Erbb2 DNA vaccine combined with regulatory T cell deletion enhances antibody response and reveals latent low-avidity T cells: potential and limits of its therapeutic efficacy.

Rat (r)Erbb2 transgenic BALB-neuT mice genetically predestined to develop multiple invasive carcinomas allow an assessment of the potential of a vaccine against the stages of cancer progression. Because of rErbb2 expression in the thymus and its overexpression in the mammary gland, CD8(+) T cell clones reacting at high avidity with dominant rErbb2 epitopes are deleted in these mice. In BALB-neuT mice with diffuse and invasive in situ lesions and almost palpable carcinomas, a temporary regulatory T cells depletion combined with anti-rErbb2 vaccine markedly enhanced the anti-rErbb2 Ab response and allowed the expansion of latent pools of low-avidity CD8(+) T cells bearing TCRs repertoire reacting with the rErbb2 dominant peptide. This combination of a higher Ab response and activation of a low-avidity cytotoxic response persistently blocked tumor progression at stages in which the vaccine alone was ineffective. However, when diffuse and invasive microscopic cancers become almost palpable, this combination was no longer able to secure a significant extension of mice survival.

An adenoviral vaccine encoding full-length inactivated human Her2 exhibits potent immunogenicty and enhanced therapeutic efficacy without oncogenicity.

PURPOSE: Overexpression of the breast cancer oncogene HER2 correlates with poor survival. Current HER2-directed therapies confer limited clinical benefits and most patients experience progressive disease. Because refractory tumors remain strongly HER2+, vaccine approaches targeting HER2 have therapeutic potential, but wild type (wt) HER2 cannot safely be delivered in immunogenic viral vectors because it is a potent oncogene. We designed and tested several HER2 vaccines devoid of oncogenic activity to develop a safe vaccine for clinical use. EXPERIMENTAL DESIGN: We created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM), and full-length HER2 inactivated for kinase function (Ad-HER2-ki), and determined their immunogenicity and antitumor effect in wild type (WT) and HER2-tolerant mice. To assess their safety, we compared their effect on the cellular transcriptome, cell proliferation, anchorage-dependent growth, and transformation potential in vivo. RESULTS: Ad-HER2-ki was the most immunogenic vector in WT animals, retained immunogenicity in HER2-transgenic tolerant animals, and showed strong therapeutic efficacy in treatment models. Despite being highly expressed, HER2-ki protein was not phosphorylated and did not produce an oncogenic gene signature in primary human cells. Moreover, in contrast to HER2-wt, cells overexpressing HER2-ki were less proliferative, displayed less anchorage-independent growth, and were not transformed in vivo. CONCLUSIONS: Vaccination with mutationally inactivated, nononcogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translates into strong and effective antitumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials.

Role of immunohistochemistry in the diagnosis of lung cancer.

The role of immunohistochemistry is to recognize antigens and, consequently, to identify and classify specific cells within a cell population whose morphology is heterogenous or apparently homogenous. The visualization of the antigen-antibody complex is made possible through the addition of either a fluorochrome conjugate or an enzyme to the antibody, which is then viewed under microscopy. Immunohistochemistry can be used in the routine diagnosis of lung cancer, in order to identify biological markers (diagnostic and prognostic). The essential immunohistochemistry panels will be discussed in this review.

Tamoxifen combined to anti-HER-2/neu cell vaccine does not hamper cancer immunopreventive efficacy.

The possible interference of tamoxifen with anti-tumor vaccines was studied in a translational view of combined preventive approaches. Tamoxifen treatment of HER-2/neu transgenic mice combined to anti-HER-2/neu cell vaccine did not hamper the efficacy of cancer immunoprevention, and caused a significantly increased production of interferon-gamma. These data suggest that tamoxifen could even have a positive impact on the efficacy of cancer immunoprevention.

Recent advances relating to the clinical application of naked monoclonal antibodies in solid tumors.

This review focuses on the recent advances in clinical data regarding antibody-based therapy in the management of solid tumors. We also discuss perspectives on antibody-based therapy in the future. Thorough understanding of the complex interactions between components of the immunological response has led to interest in the concept of immune-mediated therapy for solid tumors. Over the last few years, several humanized and chimeric monoclonal antibodies (MAbs) targeting human epidermal receptor 2 (HER2), epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) have been employed in treating solid tumors, including breast, colorectal, lung, head and neck, and gynecologic cancers. Trastuzumab, bevacizumab, cetuximab, and panitumumab are MAbs that are most widely used in clinical practice with acceptable rates of adverse events. Combination of MAbs with small-molecule inhibitors of the same pathway could potentially increase the efficacy and specificity of antibody-based treatment. Immune-mediated effects may be further exploited with the use of bivalent molecules.

[HER2 status in breast cancer of Chinese women: a study of 1170 cases using fluorescence in-situ hybridization].

OBJECTIVE: To assess the prevalence of HER2 amplification according to HER2 and chromosome 17 copy numbers and HER2 FISH (fluorescence in-situ hybridization) ratio in breast cancer occurring in Chinese women. METHODS: Eleven hundreds and seventy cases of breast cancer occurring in Chinese women, who would be treated by trastuzumab and/or relevant chemotherapy based on HER2 status, were enrolled into the study. The formalin-fixed and paraffin-embedded tumor tissues were tested by FISH (PathVysion, Vysis). RESULTS: Among the 1170 cases of breast cancer studied, 408 cases (34.87%) were FISH-negative, whereas 762 cases (65.13%) were FISH-positive, including 87 cases (87/762, 11.42%) with highly amplified HER2 gene (signals arranged in aggregates). As for the remaining 675 FISH-positive cases, 159 cases (23.56%) showed low amplification (HER2/CEP17 ratio = 2 to 4), 422 cases (62.52%) showed moderate amplification (ratio = 4 to 10) and 94 cases (13.93%) showed high amplification (ratio > 10) for HER2 gene. Only 14 of the 1170 cases (1.20%) had indeterminate results (ratio between 1.8 and 2.2), including 1.23% (5/408) borderline FISH-negative (ratio between 1.8 and 2.0) and 1.18% (9/762) borderline FISH-positive (ratio between 2.0 and 2.2). Our data showed that 73.00% (854/1170) of cases were chromosome 17 aneusomy, including 22.65% (265/1170) hypodisomy (chromosome 17 copy number per cell < or = 1.75), 38.38% (449/1170) low polysomy (chromosome 17 copy number per cell 2.26 to 3.75) and 11.97% (140/1170) high polysomy (chromosome 17 copy number per cell > or = 3.76). The frequency of chromosome 17 polysomy was 50.34%. In the FISH-positive subgroup, 23.88% (182/762) was disomy (chromosome 17 copy number per cell between 1.76 and 2.25), 24.15% (184/762) hypodisomy, 39.37% (300/762) low polysomy and 12.60% (96/762) high polysomy. The frequency of chromosome 17 polysomy in the FISH-positive subgroup was 51.97%. In the FISH-negative subgroup, 32.84% (134/408) were disomy, 19.85% (81/408) hypodisomy, 36.52% (149/408) low polysomy and 10.78% (44/408) high polysomy. The frequency of chromosome 17 polysomy in the FISH-negative subgroup was 47.30%. On the other hand, HER2 monoallelic deletion (HER2/CEP17 < or = 0.7) was observed in 2.39% of cases. Chromosome 17 monosomy was detected in 5.00% (38/762) and 4.41% (18/408) of HER2-positive and HER2-negative groups, respectively. A HER2 ratio of < 1.5 was noted in 32.30% of all cases (including 92.65% of HER2-negative cases), compared with 9.23% (108/1170) with ratio between 1.5 and 2.2. CONCLUSIONS: The results show that a high amplification of HER2 gene is detected by FISH. Moderate amplification of HER2 gene and chromosome 17 polysomy are commonly seen in breast cancer patients in China Mainland. These findings may carry significant clinical and pathogenetic implication.

Therapeutic vaccination halts disease progression in BALB-neuT mice: the amplitude of elicited immune response is predictive of vaccine efficacy.

The aim of this study was to evaluate the efficacy of genetic vaccination with rat ErbB2 antigen in a therapeutic setting for the BALB-neuT mouse model of mammary carcinoma and to establish immunological correlates with vaccine efficacy. To define an early therapeutic setting we performed imaging studies of mouse mammary glands with a high-frequency ultrasound system that allowed the diagnosis of tumor lesions before they become palpable, starting from week 13 after mouse births. An intensive immunization protocol of vaccination was implemented at this stage, consisting of four weekly DNA injections with electroporation followed by two injections of adenovirus carrying the codon usage-optimized cDNA encoding the extracellular-transmembrane domain of rat ErbB2. Immunological parameters were monitored in each individual mouse by analyzing peripheral blood leukocytes. The appearance of the first palpable tumor in vaccinated mice was delayed and there was a statistically significant time gap before additional masses developed, indicating disease stabilization. As a result of the immunization, antibodies and CD8(+) T cells to rat ErbB2 were detected and the amplitude of elicited responses correlated with the efficacy of vaccination. Moreover, the vaccination regimen specifically halted the rise in circulating myeloid suppressor cells (MSCs). All three parameters, that is, CD8(+) T cells, antibodies to rat ErbB2, and circulating MSCs, measured at the end of vaccination could be used as predictive biomarkers for future tumor development. This study emphasizes the potential of genetic vaccines for the therapeutic treatment of malignancies and suggests possible predictive biomarkers to be further validated in the clinic for the follow-up of vaccinated cancer patients.

Vaccination of fiber-modified adenovirus-transfected dendritic cells to express HER-2/neu stimulates efficient HER-2/neu-specific humoral and CTL responses and reduces breast carcinogenesis in transgenic mice.

HER-2/neu transgene-modified dendritic cell (DC)-based vaccines are potent at eliciting HER-2/neu-specific antitumor immunity. In this study, we constructed a recombinant adenovirus (RGD)AdVneu with fiber gene modified by RGD insertion into the viral knob's H1 loop. We transfected DCs with (RGD)AdVneu, and assessed/compared HER-2/neu-specific humoral and cytotoxic T lymphocyte (CTL) responses and antitumor immunity derived from the original AdVneu-transfected DCs (DCneu1) and (RGD)AdVneu-transfected DCs (DCneu2). We demonstrated that DCneu2 displayed increased HER-2/neu expression by 8.3-fold compared to DCneu1. We also demonstrated that DCneu2 vaccination induced stronger HER-2/neu-specific humoral and CTL immune responses than DCneu1 vaccination. DCneu2 vaccination protected all the mice from HER-2/neu-expressing Tg1-1 tumor cell challenge in wild-type FVB/NJ mice, compared to a partial protection in DCneu1-immunized mice. In addition, DCneu2 vaccination also significantly delayed tumor growth than DCneu1 immunization (P<0.05) in Tg FVBneuN mice. Three immunizations of DCneu2 starting at the mouse age of 2 months also significantly delayed breast cancer development in Tg mice compared to DCneu2 vaccine (P<0.05). Importantly, DCneu2 vaccine reduced breast carcinogenesis by 9% in Tg mice with self HER-2/neu tolerance. Therefore, vaccination of fiber-modified adenovirus-transfected DCs to enhance expression of tumor antigens such as HER-2/neu is likely representative of a new direction in DC-based vaccine of breast cancer.

The opposing effects of lipopolysaccharide on the antitumor therapeutic efficacy of DNA vaccine.

DNA vaccine represents a novel method to elicit immunity against infectious disease. Lipopolysaccharide (LPS) copurified with plasmid DNA may affect therapeutic efficacy and immunological response. We aimed to study the effect of LPS on the therapeutic efficacy of HER-2/neu DNA vaccine in a mouse tumor animal model. Plasmid DNA purified from commercial EndoFree plasmid purification kits functioned as a better therapeutic DNA vaccine than that purified from Non-EndoFree purification kit, which contains >or=0.5 microg LPS per 100 mg DNA plasmid. To further investigate the effect of LPS on the therapeutic efficacy of DNA vaccine, increasing amount of LPS was added to endotoxin-free plasmid DNA, and inoculated on mice with established tumors. One mug of LPS significantly attenuated the therapeutic effect of neu DNA vaccine and increased Th2 immune responses bias with interleukin-4 cytokine production. In contrast, high amount (100 microg) of LPS enhanced the therapeutic efficacy of neu DNA vaccine with an increase of cytotoxic T lymphocyte response and Th1 immune response. The effect of LPS on DNA vaccine was diminished when the tumor was grown in toll-like receptor 4 (TLR4)-mutant C3H/HeJ mice. Our results indicate that variation in the LPS doses exerts opposing effects on the therapeutic efficacy of DNA vaccine, and the observed effect is TLR4 dependent.

Inflammation and breast cancer. Inflammatory component of mammary carcinogenesis in ErbB2 transgenic mice.

This review addresses genes differentially expressed in the mammary gland transcriptome during the progression of mammary carcinogenesis in BALB/c mice that are transgenic for the rat neu (ERBB2, or HER-2/neu) oncogene (BALB-neuT664V-E mice). The Ingenuity knowledge database was used to characterize four functional association networks whose hub genes are directly linked to inflammation (specifically, the genes encoding IL-1beta, tumour necrosis factor, interferon-gamma, and monocyte chemoattractant protein-1/CC chemokine ligand-2) and are increasingly expressed during such progression. In silico meta-analysis in a human breast cancer dataset suggests that proinflammatory activation in the mammary glands of these mice reflects a general pattern of human breast cancer.

Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs.

Various methods have been explored to enhance antibody-based cancer therapy. The use of multivalent antibodies or fragments against tumor antigens has generated a great deal of interest, as various cellular signals, including induction of apoptosis, inhibition of cell growth/survival, or internalization of the surface molecules, can be triggered or enhanced on extensive cross-linking of the target/antibody complex by the multivalent form of the antibody. The goal of the studies reported here was to develop multivalent antibody constructs via grafting of antibody molecules onto liposome membranes to enhance antibody activity. Using trastuzumab and rituximab as examples, up to a 25-fold increase in the antibody potency in cell viability assay was observed when the antibodies were presented in the multivalent liposome formulation. Key cell survival signaling molecules, such as phosphorylated Akt and phosphorylated p65 nuclear factor-kappaB, were down-regulated on treatment with multivalent liposomal trastuzumab and liposomal rituximab, respectively. Potent in vivo antitumor activity was shown for liposomal trastuzumab. The data presented here showed the potential of liposome technology to enhance the therapeutic effect of antibodies via a mechanism that modulates cell survival through clustering of the target/antibody complex.

c-erbB-2 oncoprotein over-expression in breast cancer and its relationship to histology and grade in a Ugandan population.

OBJECTIVE: To evaluate c-erbB-2 oncoprotein over expression in breast cancer patients of Kyadondo county and also relate this oncoprotein to histological type and grade of the tumour. DESIGN: Cross-sectional study. SETTING: Pathology Department, Faculty of Medicine, Makerere University. SUBJECTS: Seventy two breast cancer patients who were among the 174 entered in survival study had their paraffin breast tissue blocks retrieved from archives of Pathology Department and had their c-erbB-2 determined by peroxidase-labeled streptavidin-biotin immunohistochemical method. RESULTS: c-erbB-2 oncoprotein was over expressed in 33.3% of the tumours and correlated positively with histological grade (p = 0.007). TH oncoprotein over expression was not restricted to any particular histological type and there was no association with age of patient. CONCLUSION: The over expression of c-erbB-2 oncoprotein observed in one third of breast cancer patients of this African population could partly explain the observed poor survival rate reported in this community.

Modulation of HER2 expression by ferulic acid on human breast cancer MCF7 cells.

BACKGROUND: The molecular mechanisms underlying the mitogenic effect of ferulic acid (FA), an active compound derived from Angelica sinensis, have never been elucidated. It was the aim of this study to investigate the proliferative effect of FA on human breast cancer cell lines and to elucidate its modulation mechanism on HER2 expression in MCF7 line. MATERIALS AND METHODS: By using MCF7 (oestrogen receptor-positive; ER+, HER2-low), BT474 (ER+, HER2-high), MDAMB231 (ER-, HER2-low) and SKBR3 (ER-, HER2-high) human breast cancer cell lines as in vitro models, the mitogenic effects of FA were assessed by trypan blue dye exclusion assay and DNA flow cytometry. Ferulic acid-modulated cell signalling and HER2 gene expression were evaluated in MCF7 line by Western blot and real-time RT-PCR analysis. RESULTS: Ferulic acid ER-dependently stimulated cell proliferation on MCF7 cells in a concentration-dependent manner. The HER2 oncogene (one of the prognostic factors of breast cancer) and ESR1 gene (oestrogen receptor-alpha; ERalpha) transcription were markedly up-regulated by FA treatment. Besides, HER2 signalling and its downstream molecules such as AKT and ERK1/2 were involved in FA-modulated ERalpha and cyclin D1 synthesis. Addition of anti-HER2 antibody, trastuzumab, abrogated FA-enhanced proliferative effect on MCF7 cells, indicated a positive feedback control for the action of HER2 in this setting. The fact that the ER antagonist blocked most of the FA-up-regulated HER2 expression, and that trastuzumab down-regulated ERalpha gene expression, suggested a cross-talk between ERalpha and HER2 signalling on MCF7 cells. CONCLUSION: The authors' conclude that FA causes human breast cancer cell proliferation by up-regulation of HER2 and ERalpha expression.

Vaccination strategy determines the emergence and dominance of CD8+ T-cell epitopes in a FVB/N rat HER-2/neu mouse model of breast cancer.

The HER-2/neu oncogene has >25 HLA epitopes, yet only one FVB/N mouse CD8(+) T-cell epitope has been mapped to date. This epitope has been termed the immunodominant epitope for the FVB/N mouse, but we propose that the vaccination strategy determines the dominance of epitopes. Using a series of overlapping peptides, we have mapped another CD8(+) T-cell epitope that emerges in the FVB/N mouse following vaccination with Listeria monocytogenes-based vaccines that express fragments of HER-2/neu. Following the identification of this novel H-2K(q)-restricted epitope, we sought to compare the T-cell response to this epitope with the previously identified PDSLRDLSVF epitope. This newly identified epitope and the previously identified epitope lie within fragments contained in different vaccines, the PDSLRDLSVF epitope in Lm-LLO-EC2 and the newly identified PYNYLSTEV epitope in Lm-LLO-EC1; thus, it has been possible to compare the responses of these epitopes independent of any competing response between the epitopes. CTL analysis of individual peptide-pulsed target cells and intracellular cytokine stain for IFN-gamma produced by splenocytes from Lm-LLO-EC1 compared with Lm-LLO-EC2 vaccinated FVB/N mice shows that there is no difference between the responses generated to either of these epitopes. We also show that the avidity of the CD8(+) T cells for either of these epitopes is similar based on the concentration of peptide necessary to mediate similar levels of lysis of target cells. In addition, HER-2/neu DNA vaccination followed by CTL analysis further showed that both of these peptides can emerge as epitopes.

A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells.

Tumor-targeted vectors with controllable expression of therapeutic genes and specific antitumor antibodies are promising tools for the reduction of malignant tumors. Here we describe a new plasmid for the eukaryotic expression of an anti-HER2/neu mini-antibody-barnase fusion protein (4D5 scFv-barnase-His(5)) with an NH(2)-terminal leader peptide. The 4D5 scFv-barnase-His(5) gene was placed downstream of the tetracycline responsive-element minimal promoter in the vector using the Tet-Off gene-expression system. The Bacillus amyloliquefaciens ribonuclease barnase is toxic for the host cells. To overcome this problem, barstar gene under its own minimal cytomegalovirus promoter was used in designed vector. Barstar inhibits the background level of barnase in the cells in the presence of tetracycline in culture medium. The HEK 293T cells were transfected with the designed vector, and the 4D5 scFv-barnase-His(5) fusion protein was identified by anti-barnase antibodies in cell culture medium and after purification from cell lysates using metal-affinity chromatography. The overexpression of the anti-HER2/neu mini-antibody-barnase fusion protein decreased the intensity of fluorescence of HEK 293T cells co-transfected with the generated plasmid and a plasmid containing the gene of enhanced green fluorescent protein (pEGFP-N1), in comparison with the intensity of fluorescence of HEK 293T cells transfected with pEGFP-N1, in the absence of tetracycline in the medium. The effect of the 4D5 scFv-barnase-His(5) on EGFP fluorescence indicates that the introduced barnase functions as a ribonuclease inside the cells. The anti-HER2/neu mini-antibody could be used to deliver barnase to HER2/neu-positive cells and provide its penetration into the target cells, as HER2/neu is a ligand-internalizing receptor. This expression vector has potential applications to both gene and antibody therapies of cancer.