Genetic immunization against neu /erbB2 transgenic breast cancer

erbB2/neu, an overexpressed oncogene product, has been proposed as a human cancer vaccine target. In the present study, transgenic (rat neuNT oncogene) FVB/neu mice, developing metastasizable mammary carcinoma, were immunized with a plasmid DNA encoding are not tolerant to the self antigen and sequences. We report that transgenic tumour-bearing mice, like some breast cancer patients erbB2+X, develop anti-neu autoimmune responses, which can be boosted and skewed to a Th1 phenotype by DNA immunization. Intramuscular injections of neuNT plasmid drastically reduced (or even prevented in a small number of treated mice) the outgrowth of mammary neoplasms as well as their metastatic penetrance. Furthermore, DNA immunization caused haemorrhagic necrosis of established cancer nests, leaving a greatly reduced portion of the tumour burden for the host to cope with. The antitumour activities we obtained, in this very challenging model for cancer immunotherapy, lay the foundation for DNA-based immunization to control erbB2/neu-overexpressing neoplasms. Received: 19 April 1998 / Accepted: 20 August 1998     

Humoral and cellular responses raised against the human HER2 oncoprotein are cross-reactive with the homologous product of the neu proto-oncogene,but do not protect rats against B104 tumors expressing mutated neu

 The neu proto-oncogene encodes a plasma membrane protein belonging to the epidermal growth factor receptor family. The cell line B104, derived from a BDIX rat neuroblastoma, carries a point mutation in neu, and forms a tumor when injected into these rats. The human homologue of the neu oncogene (here called HER2) is overexpressed in certain types of cancer. Rats were immunized with HER2 protein (HER2) to investigate a possible cross-reaction between the homologous proteins which could protect them against subsequent inoculation with B104. Specific antibody in the serum was measured by cell-based enzyme-linked immunosorbent assay and fluorescence immunocytochemistry, and delayed-type hypersensitivity by an ear assay. Sera from animals immunized with the HER2 extracellular domain (HER2-ECD) reacted with both HER2- and neu-expressing cells. In the ear assay, a significant cellular response to both HER2-ECD (P <0.05) and neu protein (P <0.001) was observed in HER2-ECD-immunized rats. However, the growth of B104 tumors in rats was not affected by preimmunization with HER2-ECD. The results indicate that an autoreactive immune response to neu was induced by immunization with HER2-ECD, but was too weak to affect the growth of the neu-bearing tumor. Received: 9 November 1995 / Accepted: 2 February 1996     

Autoantibody to p185 erbB2/neu oncoprotein by vaccination with xenogenic DNA

 The passive transfer of antibodies and vaccination procedures against p185, the erbB2/neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4⁺ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year. Received: 29 August 1996 / Accepted: 31 October 1996     

A murine B cell lymphoma expressing human HER2/neu undergoes spontaneous tumor regression and elicits antitumor immunity

 In the present study we describe a novel murine tumor model in which the highly malignant murine B cell lymphoma 38C13 has been transduced with the cDNA encoding human tumor-associated antigen HER2/neu. This new cell line (38C13-HER2/neu) showed stable surface expression but not secretion of human HER2/neu. It also maintained expression of the idiotype (Id) of the surface immunoglobulin of 38C13, which serves as another tumor-associated antigen. Surprisingly, spontaneous tumor regression was observed following s.c. but not i.v. injection of 38C13-HER2/neu cells in immunocompetent syngeneic mice. Regression was more frequently observed with larger tumor cell challenges and was mediated through immunological mechanisms because it was not observed in syngeneic immunodeficient mice. Mice that showed complete tumor regression were immune to challenge with the parental cell line 38C13 and V1, a variant of 38C13 that does not express the Id. Immunity could be transferred with sera, suggesting that an antibody response mediated rejection and immunity. Continuously growing s.c. tumors as well as metastatic tumors obtained after the i.v. injection of 38C13-HER2/neu maintained expression of human HER2/neu, which can serve as a target for active immunotherapy. As spontaneous tumor regression has not been observed in other human murine models expressing human HER2/neu, our results illustrate the enormous differences that can exist among different murine tumors expressing the same antigen. The present model provides a useful tool for the study of the mechanisms of protective immunity to B cell lymphoma and for the evaluation of different therapeutic approaches based on the stimulation or suppression of the immune response. Received: 2 August 2000 / Accepted: 20 September 2000     

Local delivery of recombinant vaccinia virus encoding for neu counteracts growth of mammary tumors more efficiently than systemic delivery in neu transgenic mice

Recombinant vaccinia virus has been widely employed as a cancer vaccine in several clinical trials. In this study we explored, employing BALB/c mice transgenic for the rat neu oncogene, the ability of the recombinant vaccinia virus neu (rV-neuT) vaccine to inhibit growth of neu+ mammary carcinomas and whether the efficacy of vaccination was dependent on: (a) carcinogenesis stage at which the vaccination was initiated; (b) number of vaccinations and (c) route of delivery (systemic vs. local). BALB-neuT mice were vaccinated one, two and three times by subcutaneous (s.c.) and intramammary gland (im.g.) injection with rV-neuT or V-wt (wild-type vaccinia virus) starting at the stage in which mouse mammary gland displays atypical hyperplasia, carcinoma in situ or invasive carcinoma. We demonstrated that vaccination using rV-neuT was more effective when started at an earlier stage of mammary carcinogenesis and after three vaccinations. The im.g. vaccination was more effective than the s.c. vaccination in inhibiting mammary carcinogenesis, eliciting anti-Neu antibodies, increasing anti-Neu IgG2a/G3 isotypes and inducing antibodies able to trigger mammary tumor cells apoptosis and antibody-dependent cellular cytotoxicity. The better protective ability of rV-neuT im.g. vaccination was associated with its capacity to induce a superior degree of in vivo mammary cancer cells apoptosis. Our research suggests that intratumoral vaccination using recombinant vaccinia virus could be employed to increase the activity of a genetic cancer vaccine. This study may have important implications for the design of cancer vaccine protocols for the treatment of breast cancer and of accessible tumors using recombinant vaccinia virus.     

Dendritic cells pulsed with an anti-idiotype antibody mimicking Her-2/neu induced protective antitumor immunity in two lines of Her-2/neu transgenic mice

Her-2/neu proto-oncogene is overexpressed in 20-30% of human breast cancers and is associated with high recurrence risk. To test the efficacy of immune-based strategies in eliciting an antitumor response, we have evaluated the vaccine potential of an anti-idiotype (Id) antibody, 6D12 in tolerant hosts. Immunization of human Her-2/neu transgenic mice with 6D12-pulsed dendritic cells (DC) could reverse Her-2/neu unresponsiveness and result in the induction of Her-2/neu-specific humoral and cellular immune responses and protection against tumors expressing Her-2/neu. Furthermore, the tumor rejection in 6D12-pulsed DC immunized mice was associated with development of memory response. Vaccination of transgenic female FVB-neuN mice that carry the rat Her-2/neu oncogene, markedly delayed tumor onset and developed significantly fewer spontaneous mammary tumors compared with mice treated with control vaccine. Tumor growth inhibition was associated with the induction of Her-2/neu-specific immune responses. These data suggest the potential use of anti-Id antibody 6D12 as a vaccine for immunotherapy of Her-2/neu-positive human cancer.     

Identification and characterization of a HER-2/neu epitope as a potential target for cancer immunotherapy

Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828–836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu ⁺ tumor cell lines. HER-2/neu(828-836), [HER-2(9₈₂₈)], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9₈₂₈) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8⁺ T-lymphocytes specifically recognizing HER-2(9₈₂₈) in 8 out of 20 HLA-A*0201⁺ HER-2/neu ⁺ breast cancer patients. Moreover, HER-2(9₈₂₈)-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9₈₂₈) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9₈₂₈) as a promising candidate for peptide-based cancer vaccines.     

Dmp1α Inhibits HER2/neu-Induced Mammary Tumorigenesis

Our recent study shows a pivotal role of Dmp1 in quenching hyperproliferative signals from HER2 to the Arf-p53 pathway as a safety mechanism to prevent breast carcinogenesis. To directly demonstrate the role of Dmp1 in preventing HER2/neu-driven oncogenic transformation, we established Flag-Dmp1α transgenic mice (MDTG) under the control of the mouse mammary tumor virus (MMTV) promoter. The mice were viable but exhibited poorly developed mammary glands with markedly reduced milk production; thus more than half of parous females were unable to support the lives of new born pups. The mammary glands of the MDTG mice had very low Ki-67 expression but high levels of Arf, Ink4a, p53, and p21^Cip1, markers of senescence and accelerated aging. In all strains of generated MDTG;neu mice, tumor development was significantly delayed with decreased tumor weight. Tumors from MDTG;neu mice expressed Flag-Dmp1α and Ki-67 in a mutually exclusive fashion indicating that transgenic Dmp1α prevented tumor growth in vivo. Genomic DNA analyses showed that the Dmp1α transgene was partially lost in half of the MDTG;neu tumors, and Western blot analyses showed Dmp1α protein downregulation in 80% of the cases. Our data demonstrate critical roles of Dmp1 in preventing mammary tumorigenesis and raise the possibility of treating breast cancer by restoring Dmp1α expression.     

HER-2/neu Cancer Vaccines: Present Status and Future Prospects

Immunotherapeutic approaches to cancer should focus on novel undertakings that modulate immune responses by synergistic enhancement of anti-tumor immunological parameters. Cancer vaccines should preferably be composed of multiple defined tumor antigen specific B- and T-cell epitopes. The main focus of this article is to briefly review the present status of Her-2/neu vaccine strategies and to describe the innovative strategies developed in my laboratory for a vaccine against HER-2/neu (ErbB-2) with emphasis on the humoral arm of the immune response. Elucidating the underlining mechanisms of anti-tumor effects elicited by peptide vaccines against a self-protein is a requirement for developing an immunotherapeutic strategy that might be effective in human cancer vaccines. Our approach entails the identification of biologically relevant epitopes, establishing relevant in vitro assays for monitoring vaccine efficacy, devising strategies to engineer conformationally dependent sequences, developing highly immunogenic vaccines for an outbred population and delivering the immunogen/vaccine in a safe and efficacious vehicle, utilizing transgenic animal models for assessing tumor development, and developing challenge models using transplantable tumors to study efficacy of vaccine constructs. We have developed a multi-HER-2/neu B-cell epitope approach and shown in preclinical studies that immunization with a combination of two B-cell epitope was more effective in preventing mammary tumors than a single epitope. We have translated that work to the clinic (OSU 0105) in an FDA approved, NCI sponsored “Phase 1 Active Immunotherapy trial with Chimeric and Multi-epitope based peptide vaccine targeting HER-2 oncoprotein and nor-MDP adjuvant in patients with metastatic and/or recurrent solid tumors” at the James Cancer Hospital at the Ohio State University. The correlation between overexpression of HER-2/neu and up-regulation of VEGF has been demonstrated in breast cancer patients. Thus, blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. The hypothesis that combination of anti-angiogenic therapy and tumor immunotherapy of cancer may be synergistic is an important future goal. In this review, I will discuss insights into our preclinical studies that might aid in the design of the next generation of cancer vaccines and become an integrated component of prophylactic/preventive and therapeutic approach.     

Antibody-induced activation of p185HER2 in the human lung adenocarcinoma cell line Calu-3 requires bivalency

In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185^HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by theHER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185^HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amount of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185^HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185^HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185^HER2 receptor.     

Long-term immunity elicited by antibody–cytokine fusion proteins protects against sequential challenge with murine mammary and colon malignancies

We have previously reported that the antibody fusion proteins anti-HER2/neu IgG3 fused to IL-12 [(IL-12)-IgG3] or GM-CSF [IgG3-(GM-CSF)] independently or in combination are effective anti-tumor agents against D2F2/E2 murine mammary cancer cells expressing human HER2/neu in the peritoneum. Importantly, the long-term survivors were immune to the subcutaneous challenge with D2F2/E2 and the parental D2F2 not expressing HER2/neu. We now show that these long-term survivors also exhibit significant protection against subsequent subcutaneous challenge with the murine colon carcinoma CT26-HER2/neu, and later against subcutaneous challenge with the parental CT26. These results suggest that the long-term systemic protection against mammary cancer elicited by treatment with antibody–cytokine fusion proteins can be extended to prevent the growth of a tumor from different origin expressing HER2/neu, and that this protection is not limited to this antigen alone, since it also prevented the growth of the parental tumor cells.     

Biological studies and potential therapeutic applications of monoclonal antibodies and small molecules reactive with theneu/c-erbB-2 protein

The overexpression of the growth factor receptor p185 ^neu/c-erbB-2 has been observed in a number of human adenocarcinomas and is mechanistically linked to neoplastic growth. Monoclonal antibodies raised against extracellular domains of the p185 ^neu/c-erbB-2 receptor oncoprotein have been utilized to inhibit the pathway ofneu-induced tumor development. Our laboratory has demonstrated a direct effect of anti-p185 ^neu/c-erbB2 antibodies which requires receptor ligation. This induced aggregation causes the downmodulation of cell-surface expression and eventual degradation of p185 ^neu/c-erbB-2 protein. In cells transformed by theneu oncogene, the result of antibody-induced p185 ^neu/c-erbB-2 receptor modulation is the reversion of the malignant pheno-type. We are exploiting the direct efficacy of this monoclonal antibody by developing small molecules (peptides and organic mimietics) based on anti-p185 ^neu/c-erbB-2 antibody structure that can mediate similar receptor binding and biological effects.     

Promoting effect of Antrodia camphorata as an immunomodulating adjuvant on the antitumor efficacy of HER-2/neu DNA vaccine

It is well known that DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. Antrodia camphorata (AC) is a unique basidiomycete fungus of the Polyporaceae family that only grows on the aromatic tree Cinnamomum kanehirai Hayata (Lauraceae) endemic to Taiwan. Importantly, AC has been shown to be highly beneficial in the treatment and prevention of cancer. The goal of this study was to investigate whether AC is able to augment the antitumor immune properties of a HER-2/neu DNA vaccine in a mouse model in which p185neu is overexpressed in MBT-2 tumor cells. Compared with the mice that received the HER-2/neu DNA vaccine alone, co-treatment with AC suppressed tumor growth and extended the survival rate. This increase in the antitumor efficacy was attributed to the enhancement of the Th1-like cellular immune response by the HER-2/neu DNA vaccine–AC combination. Evidence for this came from the marked increase in the IFN-γ mRNA expression in CD4⁺ T cells in the draining inguinal lymph nodes, an increase in the number of functional HER-2/neu-specific CTLs, and the increased tumor infiltration of both CD4⁺ and CD8⁺ T cells, depletion of which abolishes the antitumor effect of the HER-2/neu DNA vaccine–AC therapy. Our results further indicate that the treatment of mice with AC enhanced DC activation and production of Th1-activating cytokines (e.g. IL-12, and IFN-α) in the draining lymph nodes, which were sufficient to directly stimulate T cell proliferation and higher IFN-γ production in response to ErbB2. Overall, these results clearly demonstrate that AC represents a promising immunomodulatory adjuvant that could enhance the therapeutic potency of HER-2/neu DNA vaccines in cancer therapy.     

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of the neu oncogene-encoded p185 protein

Theneu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in theneu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658–667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an -helix with a sharp bend (CD* conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all -helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect. These results suggest there may be a normal and a transforming conformation for theneu-encoded p185 proteins which may explain their differences in transforming activity.     

Assessment of immunologic response and recurrence patterns among patients with clinical recurrence after vaccination with a preventive HER2/neu peptide vaccine: from US Military Cancer Institute Clinical Trials Group Study I-01 and I-02

Background  E75, a HER2/neu immunogenic peptide, is expressed in breast cancer (BCa). We have performed clinical trials of E75 + GM-CSF vaccine in disease-free, node-positive and node-negative BCa patients at high recurrence risk and recurrences were noted in both control and vaccine groups. Methods  Among the 186 BCa patients enrolled, 177 completed the study. Patients were HLA typed; the HLA-A2⁺/A3⁺ patients were vaccinated; HLA-A2⁻/A3⁻ patients were followed as controls. Standard clinicopathological factors, immunologic response to the vaccine, and recurrences were collected and assessed. Results  The control group recurrence rate was 14.8 and 8.3% in the vaccinated group (P = 0.17). Comparing the 8 vaccinated recurrences (V-R) to the 88 vaccinated nonrecurrent patients (V-NR), the V-R group had higher nodal stage (≥N2: 75 vs. 5%, P = 0.0001) and higher grade tumors (%grade 3: 88 vs. 31%, P = 0.003). The V-R group did not fail to respond immunologically as noted by equivalent dimer responses and post-DTH responses. Compared to control recurrent patients (C-R), V-R patients trended toward higher-grade tumors and hormone-receptor negativity. C-R patients had 50% bone-only recurrences, compared to V-R patients with no bone-only recurrences (P = 0.05). Lastly, V-R mortality rate was 12.5% compared with 41.7% for the C-R group (P = 0.3). Conclusions  The vaccinated patients who recurred had more aggressive disease compared to V-NR patients. V-R patients had no difference in immune response to the vaccine either in vitro or in vivo. V-R patients, when compared to C-R patients, trended towards more aggressive disease, decreased recurrence rates, decreased mortality, and no bone-only recurrences. Supported by the United States Military Cancer Institute and the Department of Clinical Investigation at Walter Reed Army Medical Center. Funded primarily by the Clinical Breast Care Project. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army, the Department of the Navy, or the Department of Defense. This work represents original research that has not been submitted elsewhere for publication.     

CDK inhibitor p57Kip2 is downregulated by Akt during HER2-mediated tumorigenicity

HER2/neu oncogene is frequently deregulated in cancers, and the (PI3K)-Akt signaling is one of the major pathways in mediating HER2/neu oncogenic signal. p57^Kip2, an inhibitor of cyclin-depependent kinases, is pivotal in regulating cell cycle progression, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to p57^Kip2 regulation, and that Akt is a negative regulator of p57^Kip2. Ectopic expression of Akt can decrease the expression of p57^Kip2, while Akt inhibition leads to p57^Kip2 stabilization. Mechanistic studies show that Akt interacts with p57^Kip2 and causes cytoplasmic localization of p57^Kip2. Akt phosphorylates p57 on Ser 282 or Thr310. Akt activity results in destabilization of p57 by accelerating turnover rate of p57 and enhancing p57 ubiquitination. Importantly, the negative impact of HER2/Akt on p57 stability contributes to HER2-mediated cell proliferation, transformational activity and tumorigenicity. p57 restoration can attenuate these defects caused by HER2. Significantly, Kaplan-Meier analysis of tumor samples demonstrate that in tumors where HER2 expression was observed, high expression levels of p57^Kip2 were associated with better overall survival. These data suggest that HER2/Akt is an important negative regulator of p57^Kip2, and that p57 restoration in HER2-overexpressing cells can reduce breast tumor growth. Our findings indicate the applicability of employing p57 regulation as a therapeutic intervention in HER2-overexpressing cancers.     

Immunological responses induced by <Emphasis Type="Italic">asd</Emphasis> and <Emphasis Type="Italic">wzy/asd</Emphasis> mutant strains of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium in BALB/c mice

Attenuated bacteria have long been developed as vaccine candidates but can have some disadvantages, such as the potential for damage to immune organs due to insufficient clearance. To minimize these disadvantages, we generated Salmonella enterica serovar Typhimurium mutants SHJ2104 (asd::cm) and HTSaYA (wzy::km, asd::cm). The wzy gene codes for the O-antigen polymerase, which is involved in lipopolysaccharide (LPS) biosynthesis, and asd codes for aspartate β-semialdehyde dehydrogenase, which participates in cell wall formation. The strains synthesized LPS with a short-chain length, and showed lower cytotoxicity and reduced intracellular proliferation in animal cells compared to wild-type bacteria. After oral infection, the mutants were cleared in immune tissues, including the Peyer’s patch, mesenteric lymph node, and spleen, within 5 days. The LD50 of the mutants in Balb/c mice was estimated to be 10⁶ higher than wild-type bacteria when administered either via an oral or i.p. route, indicating that the two strains are highly attenuated. To compare the immune response to and protective effects of the mutants against wild-type bacterial infection, we inoculated the mutants into mice via an oral (1×10¹⁰CFU) or i.p. (1×10⁷ CFU) route once or twice at a two week interval. All immune responses, such as serum IgG and secretory IgA levels, cytokine production, and delayed hypersensitivity, were highly induced by two rounds of immunization. HTSaYA and SHJ2104 induced similar immune responses, and mice immunized with HTSaYA or SHJ2104 via an i.p. route were protected against wild-type Salmonella infection even at 100-fold of the LD₅₀ (5×10⁶ CFU). Taken together, these data indicate that HTSaYA and SHJ2104 could be developed as live attenuated Salmonella vaccine candidates.     

Measuring the Pharmacodynamic Effects of a Novel Hsp90 Inhibitor on HER2/neu Expression in Mice Using 89Zr-DFO-Trastuzumab

Background

The positron-emitting radionuclide ⁸⁹Zr (t _1/2 = 3.17 days) was used to prepare ⁸⁹Zr-radiolabeled trastuzumab for use as a radiotracer for characterizing HER2/neu-positive breast tumors. In addition, pharmacodynamic studies on HER2/neu expression levels in response to therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted.

Methodology/Principal Findings

Trastuzumab was functionalized with desferrioxamine B (DFO) and radiolabeled with [⁸⁹Zr]Zr-oxalate at room temperature using modified literature methods. ImmunoPET and biodistribution experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) tumor xenografts were conducted. The change in ⁸⁹Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 was evaluated by biodistribution studies, Western blot analysis and immunoPET. ⁸⁹Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity 104.3±2.1 MBq/mg (2.82±0.05 mCi/mg of mAb). In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87±0.07. In vivo biodistribution experiments revealed high specific BT-474 uptake after 24, 48 and 72 h (64.68±13.06%ID/g; 71.71±10.35%ID/g and 85.18±11.10%ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by biodistribution studies and immunoPET of ⁸⁹Zr-DFO-trastuzumab. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75±4.43%ID/g and 41.42±3.64%ID/g, respectively). By 72 h radiotracer uptake (73.64±12.17%ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels.

Conclusions/Significance

The results indicate that ⁸⁹Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors, and has potential to be used to measure the efficacy of long-term treatment with Hsp90 inhibitors, like PU-H71, which display extended pharmacodynamic profiles.     

The Phosphoenolpyruvate Carboxykinase of Mycobacterium Tuberculosis Induces Strong Cell-Mediated Immune Responses in Mice

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes guanosine or adenosine mononucleotide-dependent reversible conversion of oxaloacetate (OAA) and phosphoenolpyruvate (PEP). Mycobacterium (M) tuberculosis possesses a putative GTP-dependent PEPCK. To analyze the immune responses caused by PEPCK, the effects of PEPCK on the induction of CD4⁺ T cells and cytokines such as IFN-γ, IL-12 and TNF-α were evaluated in mice. It was found that the number of CD4⁺ T cells was increased in the PEPCK immunized mice although the change of the number of CD8⁺ T cells was not significant. The cytokines IFN-γ, IL-12 and TNF-α were increased significantly in the mice immunized with PEPCK than those of incomplete adjuvant. These characteristics were further demonstrated in the mice infected by pckA mutated BCG strain. The results indicate that PEPCK can effectively induce cell-mediated immune response by increasing activity of cytokines and PEPCK may be a promising new subunit vaccine candidate for tuberculosis.