京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20799号 / 医博第4299号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 武藤 学, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les nanotechnologies appliquées à la médecine, et plus particulièrement à l’oncologie, ont permis le développement d’une nouvelle classe d’entités, appelées communément nanomédicaments ou médicaments vectorisés. Ce projet de recherche a pour objectif d’encapsuler du docétaxel dans un vecteur lipidique unilamellaire furtif, puis de greffer en surface le trastuzumab afin d’en améliorer le profil pharmacocinétique, notamment en optimisant la spécificité de la phase de distribution. Les résultats obtenus montrent qu’il est possible de développer un immunoliposome furtif de 140 nm encapsulant 90 % de docétaxel avec un taux de greffage de trastuzumab de 30 %. L’approche en cytométrie de flux que nous avons développée et appliquée a permis une quantification absolue du nombre d’anticorps présents en surface. In vitro, un double screening en culture 2D et en sphéroïde 3D a démontré la supériorité antiproliférative de l’immunoliposome comparativement à tous les autres traitements, indépendamment du statut Her2 des lignées étudiées. Les études in vivo ont confirmé cette supériorité, y compris comparativement au T-DM1, l’antibody-drug conjugate de référence dans la pathologie. Les études de biodistribution ont montré que l’accumulation de notre forme vectorielle dépendait de la taille et du degré de vascularisation des tumeurs, plus que statut Her2 tumoral. En conclusion, nous avons démontré l’intérêt thérapeutique de développer des formes vectorielles dans la prise en charge du cancer du sein, comparativement aux traitements standard. Une optimisation de la phase de distribution explique la supériorité antiproliférative obtenue avec l’immunoliposome. / The application of nanotechnology in medicine, especially oncology, has allowed for the development of a new class of entities, commonly called nanomedicine or vectorized medicine.This research project aims to encapsulate docetaxel in a stealthy, unilamellar, lipidic vector, then graft trastuzumab onto its surface to improve its pharmacokinetic profile, specifically by optimizing the specificity of the distribution phase.The results show that it is possible to develop a stealthy immunoliposome of 140 nm encapsulating 90% docetaxel and a trastuzumab grafting rate of 30 %. The flow cytometry approach that we developed and applied allowed for an absolute quantification of the number of antibodies present on the surface. In vitro, a double screening in 2D culture and in 3D spheroid showed the antiproliferative superiority of the immunoliposome compared to all the other treatments, regardless of the Her2 status in the cells studied. In vivo studies have confirmed said superiority compared to T-DM1; the benchmark antibody-drug conjugate for this pathology. Biodistribution studies have shown that the accumulation of our vector depends moreover on the size and degree of tumor vascularization than its Her2 status. In conclusion, we have demonstrated the therapeutic value of developing vector forms in the management of breast cancer therapy compared to standard treatments. The optimization of the distribution phase explains the antiproliferative superiority obtained by using the immunoliposome.

Le trastuzumab est un anticorps monoclonal déjà utilisé dans le traitement du cancer du sein sur-exprimant la protéine HER2. Malgré son efficacité, la résistance est souvent apparue. Ici, nous avons étudié l'impact des cellules adipocytaires sur la cytotoxicité cellulaire dépendante de l'anticorps (ADCC), une des mécanismes d'action principaux du trastuzumab. Nous avons trouvé que les adipocytes, ainsi que les pré- adipocytes, inhibent l'ADCC médié par le trastuzumab. Nous avons montré que les factors dérivées d'adipocytes, comme des protéines ou des exosomes, causent l'inhibition d'ADCC. Aucune séquestration ou dégradation de l'anticorps a été observée. Des analyses phénotypiques n'ont pas révélé de modification des récepteurs des cellules NK, ni du niveau de HER2 sur les cellules cancéreuses en présence d'adipocytes. La pré-incubation des cellules cancéreuses avec le surnageant des adipocytes réduit la sensitivité tumorale à l'ADCC. Nous avons trouvé que le factor de croissance et de différenciation GDFI5 est rapidement induit dans les cellules cancéreuses exposées au surnageant d'adipocytes. Une diminution de l'expression de GDFI5 par les siRNA réverse l'inhibition d'ADCC induite par les adipocytes. En conclusion, nous avons démontré que les adipocytes jouent un rôle dans la résistance des cellules de cancer du sein à l'ADCC médié par le trastuzumab, et nous suggérons que cibler GDFI5 ou l'interaction entre les adipocytes et les cellules cancéreuses pourrait sensibiliser les cellules cancéreuses au traitement par l'anticorps monoclonal / Trastuzumab is a monoclonal antibody already approved in the treatment of HER2-expressing breast cancer. Despite its efficacy, resistance often occurs. Here, we investigated the impact of adipocytes on antibody dependent cellular cytotoxicity (ADCC), one of the main mechanisms of action of trastuzumab. We found that adipocytes, as well as preadipocytes, inhibited trastuzumab-mediated ADCC. We showed that adipocyte-derived factors, likely proteins or exosomes, mediated the inhibition of ADCC. No titration or degradation of the antibody was detected. Analysis of cell phenotype did not reveal any modification of NK cell receptors, nor of HER2 levels on cancer cells in the presence of adipocytes. Pre-incubation of cancer cells with adipocyte-conditioned medium reduced sensitivity of cancer cells to ADCC. We found that growth differentiation factor l5 (GDFl5) was rapidly induced in cancer cells exposed to adipocyte-conditioned medium. Down-regulation of GDFl5 by siRNA reversed the adipocyte-induced inhibition of ADCC. In conclusion, we demonstrated that adipocytes play a role in the resistance of breast cancer to trastuzumab-mediated ADCC, and suggested that targeting GDFl5 or the crosstalk between adipocytes and cancer cells may sensitize cancer cells to monoclonal antibody treatment

Monoclonal antibodies (mAbs) conjugated to nuclear localization sequences (NLS) and labeled with Auger electron-emitters have great potential for targeted radiotherapy of cancer. This approach may be especially appropriate for the 25-30% of patients with breast cancer whose tumors display overexpression of HER2. Trastuzumab (Herceptin) is a humanized anti-HER2 mAb approved for immunotherapy of HER2-amplified breast cancer. The goal of this research was to radiolabel trastuzumab with [111]In, and to modify it with peptides harboring the NLS (CGYGPKKKRKVGG) of the simian virus 40 large-T antigen for targeted radiotherapy of breast cancer. It was hypothesized that the NLS-peptides would mediate the translocation of covalently linked [111]In-trastuzumab molecules into the nuclei of HER2-overexpressing breast cancer cells where subcellular-range Auger electrons are most damaging to DNA and lethal to cells. Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate for reaction with NLS-peptides and labeled with [111]In using diethylenetriaminepentaacetic acid. The dissociation constant for binding of [111]In-NLS-trastuzumab to HER2-overexpressing SK-BR-3 human breast cancer cells was reduced < 3-fold compared to [111]In-trastuzumab, demonstrating relatively preserved receptor-binding affinity. The NLS-peptides did not affect the biodistribution of [111]In-trastuzumab, but promoted its nuclear uptake in HER2-overexpressing MDA-MB-361 xenografts. The cytotoxicity of [111]In-NLS-trastuzumab on breast cancer cells correlated with their HER2 expression. Moreover, [111]In-NLS-trastuzumab was 2-fold and 5-fold more potent at killing MDA-MB-361 and SK-BR-3 cells compared to [111]In-trastuzumab, and nearly 3-fold and 6-fold more effective than unlabeled trastuzumab, respectively. Methotrexate is a known radiosensitizer that can amplify the lethal effects of ionizing radiation on tumor cells. Non-cytotoxic, but radiosensitizing doses of methotrexate were therefore combined with [111]In-NLS-trastuzumab; this enhanced the sensitivity of HER2-overexpressing breast cancer cells to [111]In-NLS-trastuzumab. The blood t1/2 of [111]In-NLS-trastuzumab in non-tumor bearing BALB/c mice was 23-34 h when administered intravenously or intraperitoneally. The maximum tolerated dose was 9.2-18.5 MBq; doses >18.5 MBq caused decreased leukocyte and platelet counts. [111]In-NLS-trastuzumab exhibited strong anti-tumor effects against HER2-overexpressing MDA-MB-361 xenografts, reducing their growth rate 2-fold and 3-fold compared to mice administered [111]In-trastuzumab or unlabeled trastuzumab, respectively. These promising results suggest that [111]In-NLS-trastuzumab may be a useful Auger electron radioimmunotherapeutic agent for HER2-positive breast cancer in humans.

The human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% of breast cancers (BCs) and confers an aggressive tumour phenotype with a poor prognosis. Trastuzumab (Herceptin®) is a humanized IgG1 monoclonal antibody (mAb) approved for treatment of HER2-positive breast cancer (BC), however many eligible patients do not respond. The hypothesis was that molecular imaging strategies that probe: i) the expression of HER2; ii) one of the mechanisms of action of trastuzumab or iii) evaluate the viability of tumour cells by their glucose utilization would be useful in predicting and monitoring the response of BC to treatment with trastuzumab. The relationship between tumour HER2 density, uptake of 111In-DTPA-trastuzumab and response to trastuzumab was evaluated by gamma camera imaging, biodistribution studies and monitoring tumour growth in mice implanted with BC xenografts. There was a non-linear relationship between HER2 expression and uptake of this radiopharmaceutical when tumour uptake was corrected for non-specific IgG accumulation and/or circulating blood radioactivity (r2=0.87-0.99). Tumour response corresponded better with the uncorrected tumour uptake of 111In-DTPA-trastuzumab. HER2 downregulation, a putative mechanism of action of trastuzumab, was noted as decreased tumour uptake on microSPECT/CT of mice bearing MDA-MB-361 xenografts administered 111In-DTPA-pertuzumab. Tumour uptake of 111In-DTPA-pertuzumab was reduced by 53% in mice treated for 3 days with trastuzumab (P<0.05) associated with an early molecular response to the drug. Furthermore, tumour uptake of 111In-DTPA-pertuzumab was reduced by 78% (P<0.001) in mice treated for 3 weeks, which corresponded with a reduction in HER2-positive tumour cells, indicating a therapeutic response. The relationship between changes in tumour uptake of 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) and response to trastuzumab was examined in mice bearing MDA-MB-361 and MDA-MB-231 BC xenografts, with high or very low HER2 expression, treated with trastuzumab. MicroPET imaging and biodistribution studies detected a 43-60% (P<0.03) reduction in tumour uptake of 18F-FDG in mice with MDA-MB-361 xenografts, treated with trastuzumab compared to PBS-treated controls. In contrast, there was no change in 18F-FDG uptake in MDA-MB-231 xenografts, that did not respond to trastuzumab. I conclude that molecular imaging is a promising tool for monitoring response of BC to treatment with trastuzumab.

Monoclonal antibodies (mAbs) conjugated to nuclear localization sequences (NLS) and labeled with Auger electron-emitters have great potential for targeted radiotherapy of cancer. This approach may be especially appropriate for the 25-30% of patients with breast cancer whose tumors display overexpression of HER2. Trastuzumab (Herceptin) is a humanized anti-HER2 mAb approved for immunotherapy of HER2-amplified breast cancer. The goal of this research was to radiolabel trastuzumab with [111]In, and to modify it with peptides harboring the NLS (CGYGPKKKRKVGG) of the simian virus 40 large-T antigen for targeted radiotherapy of breast cancer. It was hypothesized that the NLS-peptides would mediate the translocation of covalently linked [111]In-trastuzumab molecules into the nuclei of HER2-overexpressing breast cancer cells where subcellular-range Auger electrons are most damaging to DNA and lethal to cells. Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate for reaction with NLS-peptides and labeled with [111]In using diethylenetriaminepentaacetic acid. The dissociation constant for binding of [111]In-NLS-trastuzumab to HER2-overexpressing SK-BR-3 human breast cancer cells was reduced < 3-fold compared to [111]In-trastuzumab, demonstrating relatively preserved receptor-binding affinity. The NLS-peptides did not affect the biodistribution of [111]In-trastuzumab, but promoted its nuclear uptake in HER2-overexpressing MDA-MB-361 xenografts. The cytotoxicity of [111]In-NLS-trastuzumab on breast cancer cells correlated with their HER2 expression. Moreover, [111]In-NLS-trastuzumab was 2-fold and 5-fold more potent at killing MDA-MB-361 and SK-BR-3 cells compared to [111]In-trastuzumab, and nearly 3-fold and 6-fold more effective than unlabeled trastuzumab, respectively. Methotrexate is a known radiosensitizer that can amplify the lethal effects of ionizing radiation on tumor cells. Non-cytotoxic, but radiosensitizing doses of methotrexate were therefore combined with [111]In-NLS-trastuzumab; this enhanced the sensitivity of HER2-overexpressing breast cancer cells to [111]In-NLS-trastuzumab. The blood t1/2 of [111]In-NLS-trastuzumab in non-tumor bearing BALB/c mice was 23-34 h when administered intravenously or intraperitoneally. The maximum tolerated dose was 9.2-18.5 MBq; doses >18.5 MBq caused decreased leukocyte and platelet counts. [111]In-NLS-trastuzumab exhibited strong anti-tumor effects against HER2-overexpressing MDA-MB-361 xenografts, reducing their growth rate 2-fold and 3-fold compared to mice administered [111]In-trastuzumab or unlabeled trastuzumab, respectively. These promising results suggest that [111]In-NLS-trastuzumab may be a useful Auger electron radioimmunotherapeutic agent for HER2-positive breast cancer in humans.

The human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% of breast cancers (BCs) and confers an aggressive tumour phenotype with a poor prognosis. Trastuzumab (Herceptin®) is a humanized IgG1 monoclonal antibody (mAb) approved for treatment of HER2-positive breast cancer (BC), however many eligible patients do not respond. The hypothesis was that molecular imaging strategies that probe: i) the expression of HER2; ii) one of the mechanisms of action of trastuzumab or iii) evaluate the viability of tumour cells by their glucose utilization would be useful in predicting and monitoring the response of BC to treatment with trastuzumab. The relationship between tumour HER2 density, uptake of 111In-DTPA-trastuzumab and response to trastuzumab was evaluated by gamma camera imaging, biodistribution studies and monitoring tumour growth in mice implanted with BC xenografts. There was a non-linear relationship between HER2 expression and uptake of this radiopharmaceutical when tumour uptake was corrected for non-specific IgG accumulation and/or circulating blood radioactivity (r2=0.87-0.99). Tumour response corresponded better with the uncorrected tumour uptake of 111In-DTPA-trastuzumab. HER2 downregulation, a putative mechanism of action of trastuzumab, was noted as decreased tumour uptake on microSPECT/CT of mice bearing MDA-MB-361 xenografts administered 111In-DTPA-pertuzumab. Tumour uptake of 111In-DTPA-pertuzumab was reduced by 53% in mice treated for 3 days with trastuzumab (P<0.05) associated with an early molecular response to the drug. Furthermore, tumour uptake of 111In-DTPA-pertuzumab was reduced by 78% (P<0.001) in mice treated for 3 weeks, which corresponded with a reduction in HER2-positive tumour cells, indicating a therapeutic response. The relationship between changes in tumour uptake of 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) and response to trastuzumab was examined in mice bearing MDA-MB-361 and MDA-MB-231 BC xenografts, with high or very low HER2 expression, treated with trastuzumab. MicroPET imaging and biodistribution studies detected a 43-60% (P<0.03) reduction in tumour uptake of 18F-FDG in mice with MDA-MB-361 xenografts, treated with trastuzumab compared to PBS-treated controls. In contrast, there was no change in 18F-FDG uptake in MDA-MB-231 xenografts, that did not respond to trastuzumab. I conclude that molecular imaging is a promising tool for monitoring response of BC to treatment with trastuzumab.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references (p. 167-181).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 273-291).

Bröstcancer är en av de vanligaste cancerformerna bland kvinnor i Sverige idag med över 8000 patienter diagnostiserade varje år. Sedan 1960 har den 10-åriga överlevnaden ökat med mer än 30 % och fortfarande ses en stadig ökning. En anledning till denna stegring är den ökade förståelsen för sjukdomen och utvecklingen av nya mer avancerade analys- och behandlingsmetoder. En av de nyare analysmetoderna som används vid diagnostisering av bröstcancer är klassificering av intrinsic subtype, där uttrycket av biomarkörer på tumörcellernas yta undersöks. En sådan biomarkör är Human Epidermal Growth Factor Receptor 2 (HER2), en tyrosinkinasreceptor som är överuttryckt i 20-30 % av alla fall av bröstcancer. Denna receptor är normalt en viktig del av cellens intracellulära signaleringsvägar som stimulerar tillväxt och celldelning. Vid överuttryck på tumörcellens yta är denna receptor dock associerad med en mer aggressiv cancerform och en ökad återfallsrisk. Identifieringen av HER2 som en riskfaktor för en aggressiv typ av cancer har tillåtit utvecklingen av trastuzumab, en monoklonal antikropp riktad specifikt mot denna receptor. Trastuzumab verkar genom att binda in till receptorn och hämma dess tillväxtstimulerande effekter och på så sätt hämma sjukdomsförloppet. Trastuzumab har i flera år använts för behandling av HER2-positiv bröstcancer men den optimala behandlingsregimen är till stor del fortfarande okänd. Syftet med detta litteraturarbete var därför att undersöka om trastuzumab bör vara förstahandspreparat vid behandling av HER2-positiv bröstcancer och i sådana fall med vilka behandlingsrekommendationer. Litteraturstudien baserades på analys av fem kliniska prövningar där effekt, säkerhet och behandlingslängd undersökts. Resultaten som presenteras i denna studie tyder på att trastuzumab är ett effektivt alternativ för behandling av HER2-positiv bröstcancer. Resultaten stöttar även de nuvarande behandlingsrekommendationerna på 12 månaders adjuvant behandling med trastuzumab. Säkerhetsanalysen visade att trastuzumab generellt är ett säkert läkemedel och att allvarliga biverkningar är ovanliga. Behandlingen är dock inte utan risk och noggrann monitorering av patienterna krävs för att minska risken för allvarliga biverkningar. / Breast cancer is one of the most common types of cancer in Sweden, with over 8000 patients diagnosed every year. Since 1960 the survival rate for breast cancer has climbed over 30 % and is still rising. There could be several reasons for this development. One reason could be the more effective screening programs implemented into the Swedish health care system, which allows the cancer to be found and treated at an early stage of the disease. Early treatment is one of the key factors in treatment success. Another reason is the development of more efficient targeted cancer therapies that are now used to treat breast cancer. One of the new diagnostic tools that are used in the diagnosing of breast cancer is classification of intrinsic subtype. This analysis serves to discover specific proteins and receptors that are overexpressed in the tumour cells, and that could act as potential drug targets. One such receptor is the Human Epidermal Growth Factor Receptor 2 (HER2). The HER2 is a tyrosin kinase receptor which upon activation plays a major role in cell growth and cell division. In approximately 20-30 % of all breast cancers the expression of this receptor on the cell surface and/or the gene coding for the receptor is drastically amplified. This amplification is one of the factors that allow the tumour cells to grow and divide almost unlimitedly. The extreme potency of this receptor makes the HER2-positive breast cancer one of the most aggressive breast cancer types there is. By identifying this receptor as a major cause of the cancer research has allowed the development of a very specific targeted therapy in the form of the monoclonal antibody trastuzumab. Trastuzumab binds to the HER2-receptor and inhibits its growth stimulating effects. For several years it has been used to treat patients with HER2-positive breast cancer. However, to some extent the optimal treatment regimen is still unknown. The aim of this study was therefore to evaluate the current treatment regimen of HER2-positive breast cancer with trastuzumab. The evaluation will be based on analysis of the treatments’ efficacy, safety and optimal treatment duration.   This is a literature study based on the analysis of five clinical trials that were collected using the medical an bioscientific database PubMed. The search was limited to only include studies that analysed the use of trastuzumab as adjuvant treatment for early HER2-positive breast cancer. Studies that analysed the effect in metastatic breast cancer were excluded to enable a more specific analysis. The studies that were selected analysed efficacy and/or treatment duration as primary end points and safety as a secondary end point or as a part of the primary endpoint. Based on the results presented in these studies trastuzumab is an effective and safe treatment of choice for early HER-2 positive breast cancer. The studies also showed evidence to support the current recommended treatment duration of 12 months. The safety analysis showed that trastuzumab is a generally well tolerated treatment; however as with any cancer treatment there are certain risks, cardiac toxicity being the most serious. In conclusion, this literature study supports the current treatment regimen with trastuzumab for HER2-positive breast cancer.