Natural CD8<Superscript>+</Superscript> T-cell responses against MHC class I epitopes of the HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein in patients with epithelial tumors

HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (⁺) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2–binding nona-peptide 369-377 (HER-2(9₃₆₉)). In the present study, we examined patients with HER-2/neu⁺ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu–derived and HLA-A2–restricted "cytotoxic" peptides and to a novel one spanning amino acids 777–785 also with HLA-A2–binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon (IFN-) ELISpot assay detecting T cells at the single cell level secreting IFN-. CTLp were defined as peptide-specific precursors per 10⁶ peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/neu-negative (^-) tumors and healthy individuals. Of the HER-2/neu⁺ patients examined, 31% had increased CTLp to HER-2(9₉₅₂), 19% to HER-2(9₆₆₅), 16% to HER-2(9₆₈₉), and 12.5% HER-2(9₄₃₅), whereas only 2 of 32 patients (6%) responded to HER-2(9₇₇₇). The CTLp recognizing HER-2(9₉₅₂) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu^- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN- production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9₄₃₅), HER-2(9₉₅₂), HER-2(9₆₈₉), HER-2(9₆₆₅), and HER-2(9₇₇₇) is present in patients with HER-2/neu⁺ tumors of distinct histology, (2) HER-2(9₇₇₇) is a naturally processed peptide expressed on the surface of HER-2/neu⁺ tumors, as are the other four peptides, and (3) HER-2/neu⁺ prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy.     

AE37 peptide vaccination in prostate cancer: identification of biomarkers in the context of prognosis and prediction

A fundamental challenge in administering immunotherapies for cancer is the establishment of biomarkers that can predict patients’ responsiveness to treatment. In this study, our aim was to predict the immunologic and clinical responses of vaccination therapy with an Ii-key-modified HER-2/neu peptide (Ii-key/HER-2(776–790) or AE37), applied in our recent phase I study in patients with prostate cancer. To this end, we retrospectively analyzed our data derived from immunologic determinations before, during and after primary series of vaccinations with AE37, as well as after one AE37 booster injection. Using the obtained data, we then observed the relationship between the immunologic parameters and clinical outcome of patients. We found that preexisting levels of transforming growth factor beta (TGF-β) had an inverse correlation with in vivo and in vitro immunologic responses to the AE37 vaccine which were measured as delayed-type hypersensitivity (DTH) and interferon gamma (IFN-γ) production in response to the native HER-2(776–790) (or AE36) peptide, respectively. Patients with preexistent IFN-γ immunity to AE36 developed positive DTH reactions after primary vaccinations and booster. Moreover, we could detect a direct correlation between IFN-γ production and DTH reactions in response to AE36 challenge in our vaccinated patients. DTH reactions were a stronger indicator for patients’ overall survival (OS) than preexistent or vaccine-induced IFN-γ immunity. In contrast, we found that preexisting TGF-β levels were correlated with shorter patients’ OS. These retrospective analyses suggest that the above biomarkers at the time-points measured offer promise for evaluating immunologic and clinical responses to AE37-based vaccinations.     

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.     

Immunobiology of HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein and its potential application in cancer immunotherapy

HER-2/neu (also known as HER2 or c-erb-B2) is a 185-kDa protein receptor with tyrosine kinase activity and extensive homology to the epidermal growth factor (EGF) receptor. HER-2/neu is expressed in many epithelial tumors and known to be overexpressed in approximately 20–25% of all ovarian and breast cancers, 35–45% of all pancreatic adenocarcinomas, and up to 90% of colorectal carcinomas. HER-2/neu overexpression represents a marker of poor prognosis. HER-2/neu-positive tumor cells are potentially good targets for tumor-reactive cytotoxic T lymphocytes which have been utilized in immunotherapeutic trials. In addition, the humanized monoclonal antibody Herceptin has been tested in several clinical trials and proved to be an effective adjuvant therapy for HER-2/neu-positive breast and ovarian cancers. Vaccinations aiming at generating T-cell responses are being examined in both experimental and clinical trials. Natural immunity at the level of T and B cells has been observed in patients with HER-2/neu-positive tumors confirming the immunogenicity of HER-2/neu and encouraging vaccination trials with HER-2 protein–derived subunits or synthetic peptides. This review summarizes recent data from patients with various types of HER-2/neu–overexpressing cancers carrying different HLA alleles and exhibiting preexistent immunity to HER-2/neu–derived synthetic peptides. It also discusses potential advantages of the various vaccination approaches to immunotherapy targeting the HER-2/neu molecule.Keywords ImmunobiologyHER-2/neu OncoproteinCancer ImmunologyThis work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Paclitaxel enhances early dendritic cell maturation and function through TLR4 signaling in mice

Subclinical doses of Paclitaxel (PTX) given 1 day prior to a HER-2/neu (neu)-targeted, granulocyte-macrophage colony stimulating factor (GM-CSF)-secreting whole-cell vaccine enhances neu-specific T cell responses and slows neu⁺ tumor growth in tolerized HER-2/neu (neu-N) mice. We demonstrate that co-administration of PTX and Cyclophosphamide (CY) synergizes to slow tumor growth, and that in vitro, DC precursors exposed to PTX before LPS maturation results in greater co-stimulatory molecule expression, IL-12 production, and the ability to induce CD8⁺ T cells with enhanced lytic activity against neu⁺ tumors. PTX treatment also enhances maturation marker expression on CD11c⁺ DCs isolated from vaccine-draining lymph nodes. Ex vivo, these DCs activate CD8⁺ T cells with greater lytic capability than DC’s from vaccine alone-treated neu-N mice. Finally, PTX treatment results in enhanced antigen-specific, IFN-γ-secreting CD8⁺ T cells in vivo. Thus, administration of PTX with a tumor vaccine improves T cell priming through enhanced maturation of DC.     

Generation of human tumor-specific CTLs in HLA-A2.1–transgenic mice using unfractionated peptides from eluates of human primary breast and ovarian tumors

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with malignant transformation and aggressive disease. Due to its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. Peptide extracts derived from primary HLA-A*0201-positive (+) HER-2/neu⁺ human tumors by acid elution (acid cell extracts (ACEs)) were tested for their capacity to elicit in HLA-A*0201 transgenic mice, cytotoxic T lymphocytes (CTLs) lysing HLA-A*0201⁺ HER-2/neu⁺ tumor cells. Injections of ACE in transgenic mice induced CTLs capable of specifically lysing HER-2/neu⁺ tumor cell lines (also including the original HER-2/neu⁺ primary tumor cells from which the ACEs were derived) in an HLA-A*0201–restricted fashion. Adoptive transfer of ACE-induced CTLs was sufficient to significantly prolong survival of SCID mice inoculated with HLA-A*0201⁺ HER-2/neu⁺ human tumor cell lines. Cytotoxicity of such ACE-induced CTL lines was directed, at least as detected herein, also against the HER-2/neu peptides HER-2 (9₃₆₉) and HER-2 (9₄₃₅) demonstrating the immunodominance of these epitopes. HER-2 peptide–specific CTLs generated in the HLA-A*0201–transgenic mice, upon peptide immunization, lysed in vitro HER-2/neu⁺ human tumor cell lines in an HLA-A*0201–restricted manner and, when adoptively transferred, conferred sufficient protection in SCID mice inoculated with the same human tumor cell lines as above. However, CTLs induced by ACEs displayed enhanced efficacy in the therapy of xenografted SCID mice compared with the HER-2 peptide–specific CTLs (i.e., HER-2 [9₃₆₉] or HER-2 [9₄₃₅]). Even by administering mixtures of CTLs specific for each of these peptides, the prolongation of survival achieved was still inferior compared with that obtained with ACE-induced CTLs. This suggested that additional epitopes may contribute to the immunogenicity of such tumor-derived ACEs. Thus, immunization with ACEs from HER-2/neu⁺ primary tumor cells appears to be an effective approach to generate multiple and potent CTL-mediated immune responses against HER-2/neu⁺ tumors expressing the appropriate HLA allele(s). By screening ACE-induced CTL lines with synthetic peptides encompassing the HER-2/neu sequence, it is feasible to identify immunodominant epitopes which may be used in mixtures as vaccines with enhanced efficacy in both the prevention and therapy of HER-2/neu⁺ malignancies.This work was supported by grants from the Regional Operational Program Attika (No. 20, MIS code 59605GR) to M.P., and from the GSRT Program (No. PENED 01ED55) to C.N.B.     

3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

Herceptin failure is a major clinical problem in breast cancer. A subset of breast cancer patients with high HER-2/neu levels eventually experience metastatic disease progression when treated with Herceptin as a single agent. Mechanistic details of development of this aggressive disease are not clear. Therefore, there is a dire need to better understand the mechanisms by which drug resistance develops and to design new combined treatments that benefit patients with aggressive breast cancer and have minimal toxicity. We hypothesized that 3, 3′-diindolylmethane (DIM), a non-toxic agent can be combined with Herceptin to treat breast cancers with high levels of HER-2/neu. Here, we evaluated the effects of Herceptin alone and in combination with DIM on cell viability, apoptosis and clonogenic assays in SKBR3 (HER-2/neu-expressing) and MDA-MB-468 (HER-2/neu negative) breast cancer cells. We found that DIM could enhance the effectiveness of Herceptin by significantly reducing cell viability, which was associated with apoptosis-induction and significant inhibition of colony formation, compared with single agent treatment. These results were consistent with the down-regulation of Akt and NF-kB p65. Mechanistic investigations revealed a significant upregulation of miR-200 and reduction of FoxM1 expression in DIM and Herceptin-treated breast cancer cells. We, therefore, transfected cells with pre-miR-200 or silenced FoxM1 in these cells for understanding the molecular mechanism involved. These results provide experimental evidence, for the first time, that DIM plus Herceptin therapy could be translated to the clinic as a therapeutic modality to improve treatment outcome of patients with breast cancer, particularly for the patients whose tumors express high levels of HER-2/neu.     

Silencing of the PP2A catalytic subunit causes HER-2/neu positive breast cancer cells to undergo apoptosis

Protein phosphatase 2A (PP2A), in its activated form as a phosphatase, is a tumour suppressor. However, when PP2A is phosphorylated at the tyrosine residue (pY307), it loses its phosphatase activity and becomes inactivated. In our previous study, we found a higher expression of pY307-PP2A in HER-2/neu positive breast tumour samples and significantly correlated to tumour progression, and in this context, it could function as a proto-oncogene. The above and subsequent findings led us to postulate that the critical role of PP2A in maintaining the balance between cell survival and cell death may be linked to its phosphorylation status at its Y307 residue. Hence, we further investigated the effects of knocking down the PP2A catalytic subunit which contains the Y307 amino acid residue in two HER-2/neu positive breast cancer cell lines, BT474 and SKBR3. We showed that this causes the silenced HER-2/neu breast cancer cells to undergo apoptosis and furthermore, that such apoptosis is mediated by p38 MAPK-caspase 3/PARP activation. Understanding the role of PP2A in HER2/neu positive cells might thus provide insight into new targets for breast cancer therapy.     

CD4+ and CD8+ T Cells Can Act Separately in Tumour Rejection after Immunization with Murine Pneumotropic Virus Chimeric Her2/neu Virus-Like Particles

Background

Immunization with murine pneumotropic virus virus-like particles carrying Her2/neu (Her2MPtVLPs) prevents tumour outgrowth in mice when given prophylactically, and therapeutically if combined with the adjuvant CpG. We investigated which components of the immune system are involved in tumour rejection, and whether long-term immunological memory can be obtained.

Methodology and Results

During the effector phase in BALB/c mice, only depletion of CD4⁺ and CD8⁺ in combination, with or without NK cells, completely abrogated tumour protection. Depletion of single CD4⁺, CD8⁺ or NK cell populations only had minor effects. During the immunization/induction phase, combined depletion of CD4⁺ and CD8⁺ cells abolished protection, while depletion of each individual subset had no or negligible effect. When tumour rejection was studied in knock-out mice with a C57Bl/6 background, protection was lost in CD4^−/−CD8^−/− and CD4^−/−, but not in CD8^−/− mice. In contrast, when normal C57Bl/6 mice were depleted of different cell types, protection was lost irrespective of whether only CD4⁺, only CD8⁺, or CD4⁺ and CD8⁺ cells in combination were eradicated. No anti-Her2/neu antibodies were detected but a Her2/neu-specific IFNγ response was seen. Studies of long-term memory showed that BALB/c mice could be protected against tumour development when immunized together with CpG as long as ten weeks before challenge.

Conclusion

Her2MPtVLP immunization is efficient in stimulating several compartments of the immune system, and induces an efficient immune response including long-term memory. In addition, when depleting mice of isolated cellular compartments, tumour protection is not as efficiently abolished as when depleting several immune compartments together.     

Effect of the tyrosine kinase inhibitor lapatinib on CUB-domain containing protein (CDCP1)-mediated breast cancer cell survival and migration

The surface receptor CUB domain-containing protein 1 (CDCP1) is highly expressed in several adenocarcinomas and speculated to participate in anchorage-independent cell survival and cell motility. Tyrosine kinase phosphorylation seems to be crucial for intracellular signaling of CDCP1. Lapatinib, a tyrosine kinase inhibitor (TKI), is approved for treatment of HER-2/neu overexpressing metastatic breast cancer and functions by preventing autophosphorylation following HER-2/neu receptor activation. This study aimed to investigate the effect of CDCP1 expression on anchorage-independent growth and cell motility of breast cancer cells. Moreover, studies were performed to examine if lapatinib provided any beneficial effect on HER-2/neu^(+)/−/CDCP1⁺ breast cancer cell lines. In our studies, we affirmed that CDCP1 prevents cells from undergoing apoptosis when cultured in the absence of cell–substratum anchorage and that migratory and invasive properties of these cells were decreased when CDCP1 was down-regulated. However, only HER-2/neu⁺, but not HER-2/neu^(+)/− cells showed decreased proliferation and invasion and an enhanced level of apoptosis towards loss of anchorage when treated with lapatinib. Therefore, we conclude that CDCP1 might be involved in regulating adhesion and motility of breast cancer cells but that lapatinib has no effect on tyrosine kinases regulating CDCP1. Nonetheless, other TKIs might offer therapeutic approaches for CDCP1-targeted breast cancer therapy and should be studied considering this aspect.     

Trafficking of high avidity HER-2/neu-specific T cells into HER-2/neu-expressing tumors after depletion of effector/memory-like regulatory T cells

Background

Cancer vaccines are designed to activate and enhance cancer-antigen-targeted T cells that are suppressed through multiple mechanisms of immune tolerance in cancer-bearing hosts. T regulatory cell (Treg) suppression of tumor-specific T cells is one barrier to effective immunization. A second mechanism is the deletion of high avidity tumor-specific T cells, which leaves a less effective low avidity tumor specific T cell repertoire available for activation by vaccines. Treg depleting agents including low dose cyclophosphamide (Cy) and antibodies that deplete CD25-expressing Tregs have been used with limited success to enhance the potency of tumor-specific vaccines. In addition, few studies have evaluated mechanisms that activate low avidity cancer antigen-specific T cells. Therefore, we developed high and low avidity HER-2/neu-specific TCR transgenic mouse colonies specific for the same HER-2/neu epitope to define the tolerance mechanisms that specifically affect high versus low avidity tumor-specific T cells.

Methodology/Principal Findings

High and low avidity CD8⁺ T cell receptor (TCR) transgenic mice specific for the breast cancer antigen HER-2/neu (neu) were developed to provide a purified source of naïve, tumor-specific T cells that can be used to study tolerance mechanisms. Adoptive transfer studies into tolerant FVB/N-derived HER-2/neu transgenic (neu-N) mice demonstrated that high avidity, but not low avidity, neu-specific T cells are inhibited by Tregs as the dominant tolerizing mechanism. High avidity T cells persisted, produced IFNγ, trafficked into tumors, and lysed tumors after adoptive transfer into mice treated with a neu-specific vaccine and low dose Cy to deplete Tregs. Analysis of Treg subsets revealed a Cy-sensitive CD4⁺Foxp3⁺CD25^low tumor-seeking migratory phenotype, characteristic of effector/memory Tregs, and capable of high avidity T cell suppression.

Conclusion/Significance

Depletion of CD25^low Tregs allows activation of tumor-clearing high avidity T cells. Thus, the development of agents that specifically deplete Treg subsets should translate into more effective immunotherapies while avoiding autoimmunity.     

Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer

The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.     

Unconventional cytokine profiles and development of T cell memory in long-term survivors after cancer vaccination

Cancer vaccine trials frequently report on immunological responses, without any clinical benefit. This paradox may reflect the challenge of discriminating between effective and pointless immune responses and sparse knowledge on their long-term development. Here, we have analyzed T cell responses in long-term survivors after peptide vaccination. There were three main study aims: (1) to characterize the immune response in patients with a possible clinical benefit. (2) To analyze the long-term development of responses and effects of booster vaccination. (3) To investigate whether the Th1/Th2-delineation applies to cancer vaccine responses. T cell clones were generated from all nine patients studied. We find that surviving patients harbor durable tumor-specific responses against vaccine antigens from telomerase, RAS or TGFβ receptor II. Analyses of consecutive samples suggest that booster vaccination is required to induce robust T cell memory. The responses exhibit several features of possible clinical advantage, including combined T-helper and cytotoxic functionality, recognition of naturally processed antigens and diverse HLA-restriction and fine-specificity. CD4⁻CD8⁻ T cell clones display unconventional cytotoxicity and specifically kill tumor cells expressing mutated TGFβ receptor II. Cytokine profiling on the long-term survivors demonstrates high IFNγ/IL10-ratios, favoring immunity over tolerance, and secretion of multiple chemokines likely to mobilize the innate and adaptive immune system. Interestingly, these pro-inflammatory cytokine profiles do not follow a Th1/Th2-delineation. Most IFNγ^high/IL4^low/IL10^low cultures include high concentrations of hallmark Th2-cytokines IL-5 and IL-13. This does not reflect a mixture of Th1- and Th2-clones, but applies to 19/20 T cell clones confirmed to be monoclonal through TCR clonotype mapping. The present study identifies several factors that may promote clinical efficacy and suggests that cytokine profiling should not rely on the Th1/Th2-paradigm, but assess the overall inflammatory milieu and the balance between key cytokines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis

Background

In spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB.

Methods/Principal Findings

In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin- a key latency antigen of M. tuberculosis to boost the BCG induced immunity. ‘BCG prime – DNA boost’ regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log₁₀ and 1.96 log₁₀ fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3⁺) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2.

Conclusions/Significance

These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3⁺ Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.     

Ontak reduces the immunosuppressive tumor environment and enhances successful therapeutic vaccination in HER-2/<Emphasis Type="Italic">neu</Emphasis>-tolerant mice

Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT ⁺ (neuT ⁺) triple transgenic mice represent an improvement over neuT ⁺ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85–94) (p85) CTL and HER-2(776–790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT ⁺ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8⁺ CTLs and CD4⁺ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4⁺ and CD8⁺ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4⁺ and CD8⁺ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.     

In the FVB/N HER-2/<Emphasis Type="Italic">neu</Emphasis> transgenic mouse both peripheral and central tolerance limit the immune response targeting HER-2/neu induced by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>-based vaccines

Listeria monocytogenes-based vaccines for HER-2/neu are capable of breaking tolerance in FVB/N rat HER-2/neu transgenic mice. The growth of implanted NT-2 tumors, derived from a spontaneously occurring tumor in the FVB/N HER-2/neu transgenic mouse, was significantly slower in these mice following vaccination with a series of L. monocytogenes-based vaccines for HER-2/neu. Mechanisms of T cell tolerance that exist in these transgenic mice include the absence of functional high avidity anti-HER-2/neu CD8⁺ T cells and the presence of CD4⁺CD25⁺ regulatory T cells. The in vivo depletion of these regulatory T cells resulted in the slowing in growth of tumors even without the treatment of mice with an anti-HER-2/neu vaccine. The average avidities of responsive CD8⁺ T cells to six of the nine epitopes in HER-2/neu we examined, four of which were identified in this study, are shown here to be of a lower average avidity in the transgenic mice versus wild type FVB/N mice. In contrast, the average avidity of CD8⁺ T cells to three epitopes that showed the lowest avidity in the wild-type mice did not differ between wild type and transgenic mice. This study demonstrates the ability of L. monocytogenes-based vaccines to impact upon tolerance to HER-2/neu in FVB/N HER-2/neu transgenic mice and further defines some of the aspects of tolerance in these mice.     

Loss of Multi-Epitope Specificity in Memory CD4+ T Cell Responses to B. Pertussis with Age

Pertussis is still occurring in highly vaccinated populations, affecting individuals of all ages. Long-lived Th1 CD4⁺ T cells are essential for protective immunity against pertussis. For better understanding of the limited immunological memory to Bordetella pertussis, we used a panel of Pertactin and Pertussis toxin specific peptides to interrogate CD4⁺ T cell responses at the epitope level in a unique cohort of symptomatic pertussis patients of different ages, at various time intervals after infection. Our study showed that pertussis epitope-specific T cell responses contained Th1 and Th2 components irrespective of the epitope studied, time after infection, or age. In contrast, the breadth of the pertussis-directed CD4⁺ T cell response seemed dependent on age and closeness to infection. Multi-epitope specificity long-term after infection was lost in older age groups. Detailed knowledge on pertussis specific immune mechanisms and their insufficiencies is important for understanding resurgence of pertussis in highly vaccinated populations.     

Anti-tumor immunity and autoimmunity: a balancing act of regulatory T cells

Regulatory T (Treg) cell activity has been observed in anti-tumor and autoimmunity since the 1970s. Functional and molecular characterization of Treg cells has been made possible by the recent association of cell markers, such as CD25, CTLA-4, GITR, and Foxp3 gene product, with immunoregulatory activity. Here the influence of Treg cells in both anti-tumor immunity and autoimmunity was measured in BALB/c mice. Depletion of CD4⁺CD25⁺ Treg cells with CD25 mAb resulted in mammary tumor regression and increased susceptibility to thyroiditis. This in vivo priming to both tumor-associated antigens and self-thyroglobulin attests to the presence of otherwise undetectable immune effectors which are under negative regulation. Modulation of Treg cells is a powerful strategy in cancer therapy, but may potentiate autoimmune complications. Murine models exhibiting breakable tolerance to tumor-associated antigens, such as ErbB-2 (HER-2/neu), and increased susceptibility to autoimmunity following Treg-cell depletion are being established to test new vaccination or therapeutic strategies involving Treg-cell modulation.This article forms part of the Symposium in Writing Inhibitors of immunosurveillance and anti-tumor immunity, published in Vol. 53.