Preclinical development of cancer vaccines

Triplex cell vaccine is a cancer immunopreventive cell vaccine that can prevent almost completely mammary tumor onset in HER-2/neu transgenic mice. A future translation of cancer immunoprevention from preclinical to clinical studies should take into account several aspects. The work reported in this thesis deals with the study of three of these aspects: vaccine schedule, activity in a therapeutic set-up and second-generation DNA vaccines. An important element in determining human acceptance and compliance of a treatment protocol is the number of vaccinations. In order to improve the vaccination schedule a minimal protocol was searched, i.e. a schedule consisting of a lower number of administrations than standard protocol but with a similar efficacy. A candidate optimal protocol was identified by the use of an in silico model, SimTriplex simulator. The in vivo test of this schedule in HER-2/neu transgenic mice only partially confirmed in silico predictions. This result shows that in silico models have the potential ability to aid in searching of optimal treatment protocols, provided that they will be further tuned on experimental data. As a further result this preclinical study highlighted that kinetic of antibody response plays a major role in determining cancer prevention, leading to the hypothesis of a threshold that must be reached rapidly and maintained lifetime. Early clinical trials would be performed in a therapeutic, rather than preventive, setting. Thus, the activity of Triplex vaccine was investigated against experimental lung metastases in HER-2/neu transgenic mice in order to evaluate if the immunopreventive Triplex vaccine could be effective also against a pre-existing tumor mass. This preclinical model of aggressive metastatic development showed that the vaccine was an efficient treatment also 4 for the cure of micrometastases. However the immune mechanisms activated against tumor mass were not antibody dependent, i.e. different from those preventing the onset of primary mammary carcinoma. DNA vaccines could be more easily used than cellular ones. A second generation of Triplex vaccine based on DNA plasmids was evaluated in an aggressive preclinical model (BALBp53neu female mice) and compared with the preventive ability of cellular Triplex vaccine. It was observed that Triplex DNA vaccine was as effective as Triplex cell vaccine, exploiting a more restricted immune stimulation.

Emodin, a tyrosine kinase inhibitor, and human cancer

Many human diseases, including cancers, result from aberrations of signal transduction pathways. The recent understanding of the molecular biochemistry of signal transduction in normal and transformed cells enable us to have a better insight about cancer and design new drugs to target this abnormal signaling in the cancer cells. Tyrosine kinase pathway plays a very important role in normal and cancer cells. Enhanced activity of tyrosine kinases has been associated with many human cancer types. Therefore, identifying the type of tyrosine kinases involved in a particular cancer type and blocking these tyrosine kinase pathways may provide a way to treat cancer. Receptor tyrosine kinase expression, namely epidermal growth factor receptor (EGFR) family, was examined in the oral squamous cell carcinoma patients. The expression levels of different members of the EGFR family were found to be significantly associated with shorter patients' survival. Combining EGFR, HER-2/neu, and HER-3 expression can significantly improve the predicting power. The effect of emodin, a tyrosine kinase inhibitor, on these receptors in head and neck squamous cell carcinoma cell lines was examined. Emodin was found to suppress the tyrosine phosphorylation of HER-2/neu and EGF-induced tyrosine phosphorylation of EGFR. Emodin also induced apoptosis and downregulated the expression of anti-apoptotic protein bcl-2 in oral squamous cell carcinoma cells. It is known that tyrosine kinase pathways are involved in estrogen receptor signaling pathway. Therefore, the effects of inhibiting the tyrosine kinase pathway in estrogen receptor-positive breast cancers was studied. Emodin was found to act similarly to antiestrogens, capable of inhibiting estrogen-stimulated growth and DNA synthesis, and the phosphorylation of Rb protein. Interestingly, emodin, and other tyrosine kinase inhibitors, such as RG 13022 and genistein, depleted cellular levels of estrogen receptor protein. Emodin-induced depletion of estrogen receptor was mediated by the proteasome degradation pathway. In summary, we have demonstrated that tyrosine kinase pathways play an important role in oral squamous cell carcinoma and estrogen receptor-positive breast cancer. Targeting the tyrosine kinases by inhibitors, such as emodin, may provide a potential way to treat the cancer patients. ^

A model-based approach for assessing in vivo combination therapy interactions

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach compares in vivo tumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.

T Cell Receptor-induced Activation and Apoptosis In Cycling Human T Cells Occur throughout the Cell Cycle

Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0–G1, S, and G2–M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca2+ flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle.

Transformation studies of a nontumorigenic rat prostatic epithelial cell line with activated {\it myc\/} and {\it neu\/} oncogenes

A cloned nontumorigenic prostatic epithelial cell line, NbE-1.4, isolated from Noble (nbl/crx) rat ventral prostate, was used to examine the potential role of activated myc and neu oncogenes in prostate carcinogenesis. Transfection of SV40 promoter/enhancer driven constructs containing either v-myc, truncated c-myc, or neu-T (activated neu) oncogenes was accomplished using calcium phosphate-mediated DNA transfer. Cells were cotransfected, as necessary, with pSV2neo, allowing for selection of positive clones using the antibiotic geneticin (G418). G418 resistant colonies were pooled in some cases or limiting dilution exclusion cloned in others as described. Transfection of NbE-1.4 cells with activated myc oncogenes resulted only in the partial transformation. These cells display an altered morphology and decreased dependence on serum factors in vitro; however, saturation density, soft agar colony formation and growth assay in male athymic nude mice were all negative. Transfection and overexpression of NbE-1.4 cells with an activated neu oncogene alone resulted in tumorigenic conversion. Cell transformation was evident following an examination of the altered cellular morphology, an increased soft agar colony formation, and an acquisition of a tumorigenic potential when injected s.c. into male athymic nude mice. neu-transformed NbE-1.4 cells displayed elevated activity of the neu receptor tyrosine kinase. Furthermore, qualitative changes in tyrosine phosphorylated proteins were found in neu transformed cell clones. These changes were associated with elevated expression of mRNAs for laminin $\beta$1, $\beta$2, and procollagen type IV. The expression of fibronectin and E-cadherin, which are often lost during tumorigenesis, did not correlate with the tumorigenic phenotype. Therefore, it appears that neu oncogene overexpression has been found to be associated with the transformation of rat prostatic epithelial cells, presumably through alterations in gene expression that regulate extracellular matrix. The possible interrelationship and functional significance between neu oncogene expression and the elevated extracellular matrix gene expression is discussed. ^

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.

Ubiquitin- and ubiquitin-like proteins-conjugating enzymes (E2s) in breast cancer.

Breast cancer is the most common malignancy in women and a significant cause of morbidity and mortality. Sub-types of breast cancer defined by the expression of steroid hormones and Her2/Neu oncogene have distinct prognosis and undergo different therapies. Besides differing in their phenotype, sub-types of breast cancer display various molecular lesions that participate in their pathogenesis. BRCA1 is one of the common hereditary cancer predisposition genes and encodes for an ubiquitin ligase. Ubiquitin ligases or E3 enzymes participate together with ubiquitin activating enzyme and ubiquitin conjugating enzymes in the attachment of ubiquitin (ubiquitination) in target proteins. Ubiquitination is a post-translational modification regulating multiple cell functions. It also plays important roles in carcinogenesis in general and in breast carcinogenesis in particular. Ubiquitin conjugating enzymes are a central component of the ubiquitination machinery and are often perturbed in breast cancer. This paper will discuss ubiquitin and ubiquitin-like proteins conjugating enzymes participating in breast cancer pathogenesis, their relationships with other proteins of the ubiquitination machinery and their role in phenotype of breast cancer sub-types.

Selection of peptides and synthesis of pentameric peptabody molecules reacting specifically with ErbB-2 receptor.

The HER-2/ErbB-2 oncoprotein is overexpressed in human breast and ovarian adenocarcinomas and is clearly associated with the malignant phenotype. Although no specific ligand for this receptor has been positively identified, ErbB-2 was shown to play a central role in a network of interactions with the related ErbB-1, ErbB-3 and ErbB-4 receptors. We have selected new peptides binding to ErbB-2 extracellular domain protein (ECD) by screening 2 newly developed constrained and unconstrained random hexapeptide phage libraries. Out of 37 phage clones, which bound specifically to ErbB-2 ECD, we found 6 constrained and 10 linear different hexapeptide sequences. Among the latter, 5 consensus motifs, all with a common methionine and a positively charged residue at positions 1 and 3, respectively, were identified. Furthermore, 3 representative hexapeptides were fused to a coiled-coil pentameric recombinant protein to form the so-called peptabodies recently developed in our laboratory. The 3 peptabodies bound specifically to the ErbB-2 ECD, as determined by enzyme-linked immunosorbent assay and BIAcore analysis and to tumor cells overexpressing ErbB-2, as shown by flow cytometry. Interestingly, one of the free selected linear peptides and all 3 peptabodies inhibited the proliferation of tumor cells overexpressing ErbB-2. In conclusion, a novel type of ErbB-2-specific ligand is described that might complement presently available monoclonal antibodies.

Cox-2 e sua associação com fatores prognósticos e resposta à quimioterapia primária em pacientes com câncer de mama

OBJETIVO: Avaliar em pacientes com câncer de mama a expressão imunoistoquímica da cox-2 antes da quimioterapia primária com 5-fluorouracil, epirrubicina e ciclofosfamida (FEC) e a associação desta com tamanho inicial do tumor, estado linfonodal, receptores hormonais, expressão da Her-2 e com a resposta clínica e anatomopatológica. MÉTODOS: Estudo retrospectivo com 41 mulheres portadoras do diagnóstico histopatológico de carcinoma ductal de mama. Foram submetidas à quimioterapia primária com esquema FEC (5-fluorouracil, epirrubicina e ciclofosfamida) na dosagem de 500mg/m2, 75mg/m2 e 500 mg/m2, respectivamente. Os critérios de inclusão foram intervalo etário entre 30 e 70 anos, estadiamento II a IIIA, após comprovação da ausência de metástase, tumor primário de mama, único e unilateral, tipo histológico ductal invasivo e ausência de cardiopatia e gestação. Para avaliação da expressão da proteína Her 2 neuutilizaram-se anticorpos monoclonais de coelho. Para visibilizar a expressão da proteína cox-2 utilizaram-se anticorpos policlonais obtidos do soro de cabras. A avaliação da resposta clínica ao tratamento foi realizada por exame físico mensurando-se o maior eixo tumoral por paquímetro. As medidas foram realizadas à admissão e após os ciclos de quimioterapia primária. Após três sessões quimioterápicas com intervalos de 21 dias realizou-se o procedimento cirúrgico. Adotaram-se os critérios do RECIST. Após a operação foi avaliada a resposta anatomopatológica local, sendo considerada completa quando da ausência de neoplasia invasiva e do componente in situ. Na avaliação imumoistoquímica para os receptores de estrogênio utilizaram-se estrogen receptor NCL-ER6F11 e para progesterona, progesterone receptor, NCL-PGR-312 considerando positiva quando da coloração em 10% ou mais das células tumorais. RESULTADOS: A distribuição segundo estadiamento clínico UICC verificaram-se seis no estádio IIA (14,6%), 22 no estádio IIB (53,6%) e 13 estádio IIIA (31,8%). A avaliação clínica inicial do maior eixo tumoral variou de 2,5 a 15 cm e mediana de 5 cm. Foram identificadas 14 pacientes (34,1%) com estado linfonodal negativo e 27 positivo (65,9%). Observou-se que 19 (46,3%) apresentavam-se no menacme e 22 (53,6%) na menopausa. CONCLUSÃO: Houve associação da expressão da cox-2 à fatores de pior prognóstico no câncer de mama como estado linfonodal positivo, receptores hormonais negativos e expressão da Her-2.

PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

The transcriptional regulation of the {\it neu\/} gene: Examples of activation, repression, and cell-specific expression

The neu oncogene encodes a growth factor receptor-like protein, p185, with an intrinsic tyrosine kinase activity. A single point mutation, an A to T transversion resulting in an amino acid substitution from valine to glutamic acid, in the transmembrane domain of the rat neu gene was found to be responsible for the transforming and tumorigenic phenotype of the cells that carry it. In contrast, the human proto-neu oncogene is frequently amplified in tumors and cell lines derived from tumors and the human neu gene overexpression/amplification in breast and ovarian cancers is known to correlate with poor patient prognosis. Examples of the human neu gene overexpression in the absence of gene amplification have been observed, which may suggest the significant role of the transcriptional and/or post-transcriptional control of the neu gene in the oncogenic process. However, little is known about the transcriptional mechanisms which regulate the neu gene expression. In this study, three examples are presented to demonstrate the positive and negative control of the neu gene expression.^ First, by using band shift assays and methylation interference analyses, I have identified a specific protein-binding sequence, AAGATAAAACC ($-$466 to $-$456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.^ Second, it has been reported that the E3 region of adenovirus 5 induces down-regulation of epidermal growth factor (EGF) receptor through endocytosis. I found that the human neu gene product, p185, (an EGF receptor-related protein) is also down-regulated by adenovirus 5, but via a different mechanism. I demonstrate that the adenovirus E1a gene is responsible for the repression of the human neu gene at the transcriptional level.^ Third, a differential expression of the neu gene has been found in two cell model systems: between the mouse fibroblast Swiss-Webster 3T3 (SW3T3) and its variant NR-6 cells; and between the mouse liver tumor cell line, Hep1-a, and the mouse pancreas tumor cell line, 266-6. Both NR-6 and 266-6 cell lines are not able to express the neu gene product, p185. I demonstrate that, in both cases, the transcriptional repression of the neu gene may account for the lack of the p185 expression in these two cell lines. ^

Regulation of Bcl-2 expression by the product of the prostate apoptosis response gene -4, Par-4

Elevated expression levels of the bcl-2 proto-oncogene have been correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t (14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer, there being no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2 expressing, androgen-independent prostate cancer cell lines, by all trans-retinoic acid treatment but this did not occur in the androgen-dependent cell lines expressing low levels of Bcl-2. Changes in localization of Par-4, and an induction in the expression of WT1 protein accompanied the decrease in the Bcl-2 protein and transcript following all trans-retinoic acid treatment, in the androgen-independent prostate cancer cell line. In stable clones expressing ectopic Par-4 we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1 binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1 binding site on the bcl-2 promoter. ^

Element concentration and GDGT fractional abundances of sediment core GeoB3910-2

In dieser Arbeit wurde das Zeitintervall zwischen 20 und 10 ka vor heute einschließlich des Heinrichevent 1 und der Younger Dryas am Kern GeoB 3910-2 neu untersucht. An organischen Parametern, basierend auf der Verteilung von bakteriellen GDGTs, und Elementkonzentrationen wurde eine Rekonstruktion der klimatischen Bedingungen und Veränderungen im Hinterland von NO Brasilien durchgeführt. Es zeigt sich, dass sich die durchschnittliche Landtemperatur gleich der Oberflächenwassertemperatur verhält und im Gegensatz zu den Phasen von H6 bis H2 dem antarktischen Erwärmungstrend ab 17 ka vor heute folgt. Weiterhin konnte gezeigt werden, dass durch die südwärts Verlagerung der ITCZ während H1 und der YD die Niederschläge in NO Brasilen intensiviert wurden, was eine Ausbreitung der Flüsse und Änderung der Erosionsgebiete zur Folge hatte.

Suppression and promotion of tumor growth by monoclonal antibodies to ErbB-2 differentially correlate with cellular uptake.

Amplification and overexpression of the erbB-2/neu protooncogene are frequently associated with aggressive clinical course of certain human adenocarcinomas, and therefore the encoded surface glycoprotein is considered a candidate target for immunotherapy. We previously generated a series of anti-ErbB-2 monoclonal antibodies (mAbs) that either accelerate or inhibit the tumorigenic growth of erbB-2-transformed murine fibroblasts. The present study extended this observation to a human tumor cell line grown as xenografts in athymic mice and addressed the biochemical differences between the two classes of mAbs. We show that the inhibitory effect is dominant in an antibody mixture, and it depends on antibody bivalency. By using radiolabeled mAbs we found that all of three tumor-inhibitory mAbs became rapidly inaccessible to acid treatment when incubated with tumor cells. However, a tumor-stimulatory mAb remained accessible to extracellular treatments, indicating that it did not undergo endocytosis. In addition, intracellular fragments of the inhibitory mAbs, but not of the stimulatory mAb, were observed. Electron microscopy of colloidal gold-antibody conjugates confirmed the absence of endocytosis of the stimulatory mAb but detected endocytic vesicles containing an inhibitory mAb. We conclude that acceleration of cell growth by ErbB-2 correlates with cell surface localization, whereas inhibition of tumor growth is associated with an intrinsic ability of anti-ErbB-2 mAbs to induce endocytosis. These conclusions are relevant to the selection of optimal mAbs for immunotherapy and may have implications for the mechanism of cellular transformation by an overexpressed erbB-2 gene.

[the New Classification Of Breast Cancers: Finding The Luminal A].

To compare the distributions of patients with clinical-pathological subtypes of luminal B-like breast cancer according to the 2011 and 2013 St. Gallen International Breast Cancer Conference Expert Panel. We studied 142 women with breast cancer who were positive to estrogen receptor and had been treated in São Paulo state, southeast Brazil. The expression of the following receptors was assessed by immunohistochemistry: estrogen, progesterone (PR) and Ki-67. The expression of HER-2 was measured by fluorescent in situ hybridization analysis in tissue microarray. There were 29 cases of luminal A breast cancers according to the 2011 St. Gallen International Breast Cancer Conference Expert Panel that were classified as luminal B-like in the 2013 version. Among the 65 luminal B-like breast cancer cases, 29 (45%) were previous luminal A tumors, 15 cases (20%) had a Ki-67 >14% and were at least 20% PR positive and 21 cases (35%) had Ki-67 >14% and more than 20% were PR positive. The 2013 St. Gallen consensus updated the definition of intrinsic molecular subtypes and increased the number of patients classified as having luminal B-like breast cancer in our series, for whom the use of cytotoxic drugs will probably be proposed with additional treatment cost.