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.

The optimal way to detect and treat cancer is to target cancer cells exclusively without affecting the surrounding tissue. One promising approach is to use radiolabelled molecules to target receptors that are overexpressed in cancer cells. Since the epidermal growth factor receptor (EGFR) family is overexpressed in many types of cancer, it is an attractive target for both diagnostic and therapeutic applications. This thesis can be divided into two parts. In part one (paper I), studies were conducted to modulate radionuclide uptake in tumour cells. The results showed that it was possible to modulate the cellular uptake of 125I delivered by trastuzumab (targeting HER2) by adding EGF (targeting EGFR). In part two (papers II-V) a high affinity EGFR-targeting affibody molecule (ZEGFR:955)2 was selected and analysed both in vitro and in vivo. In papers II, III and V, the results obtained when using (ZEGFR:955)2 were compared with those obtained with the two EGFR-binding molecules, EGF and cetuximab. These studies demonstrated that the affibody molecule bound specifically to EGFR (probably to subdomain III) with high affinity (~50 nM in biosensor analysis and ~1 nM in cellular studies) and produced intracellular signalling changes similar to those with cetuximab. In paper IV, in vivo studies were made, demonstrating that [111In](ZEGFR:955)2 gave a tumour-specific 111In uptake of 3.8±1.4% of injected dose per gram tumour tissue, 4 h post-injection. The tumours could be easily visualized with a gamma camera at this time-point. The results of these studies indicated that the affibody molecule (ZEGFR:955)2 is a possible candidate for radionuclide-based imaging of EGFR-expressing tumours. The biological effects of (ZEGFR:955)2 might be of interest for therapy applications.

Abnormal expression and signaling of the ErbB receptors is associated with the development and progression of several forms of cancer. In this thesis, new ErbB-targeting affibody molecules are evaluated regarding their cellular effects in vitro. Since ligand binding to an ErbB receptor might have an impact on the cell it is important to be aware of these effects as they may have consequences for the continued growth of the tumor when used in vivo. The affibody molecules are intended for tumor targeting with the prospect of clinical use in imaging or therapy. Three types of affibody molecules were studied, HER2-binding, EGFR-binding and bispecific binders that target both EGFR and HER2. The HER2-targeting (ZHER2:342)2 showed promising characteristics. It sensitized SKBR-3 cells to irradiation and decreased cell growth to the same extent as the clinically approved antibody Herceptin. The monomeric version, ZHER2:342, did not induce any large effects on intracellular signaling or biological outcome. This makes (ZHER2:342)2 interesting for therapy purposes, while ZHER2:342 may be better suited for imaging. The bispecific affibody molecules were all able to simultaneously bind to both EGFR and HER2, but none of the six constructs resulted in any large effects on cellular outcome. Interestingly, all three monovalent binders are more functional when positioned at the N-terminal part of the construct and the (S4G)3 linker renders higher affinity of the bispecific binders compared to (G4S)3. Tumors that co-express several ErbB receptors are often more aggressive and associated with a worse prognosis, suggesting that the total ErbB expression pattern might be more informative than the expression level of one receptor regarding cancer prognosis and prediction of response to targeted therapies. Bispecific ligands could thus be used as imaging agents with prognostic value. Another aspect of dual targeting is the possibility of increased tumor specificity since tumors are more likely than healthy tissue to express high amounts of two receptors.

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.

I Sverige drabbas närmare 8000 kvinnor av bröstcancer varje år. Bröstcancer är vanligast hos kvinnor över 40 år och utgör 30 % av alla cancersjukdomar hos kvinnor. Cirka 25 % av alla bröstcancerformer är HER2 (human epidermal growth factor receptor) -positiva. HER2-positiv bröstcancer resulterar i en aggressiv fenotyp och en väldigt dålig prognos.  Varje år får cirka 1300 – 1400 bröstcancerpatienter en spridning av sin sjukdom. Vid spridning bildas så kallade metastaser. Som behandling till HER2-positiv bröstcancer förskrivs målinriktat läkemedel tillsammans med cytostatika. Målinriktat läkemedel kan antingen vara antikroppar eller inhibitorer som hämmar HER2-medierad cellsignalering. Trastuzumab har sedan år 2007 använts som behandling vid spridd HER2-positiv bröstcancer. Pertuzumab är ett nyare tillskott i läkemedelsgruppen och används på samma indikation. Dessa två läkemedel är monoklonala antikroppar som binder till HER2. Skillnaden mellan dessa två läkemedel är att de binder till olika platser i HER2 och har olika verkningsmekanismer. Syftet med litteraturarbetet var att undersöka om pertuzumab som tillägg till trastuzumab och cytostatikumet docetaxel (s.k. TD-behandling) förlängde den progressionsfria perioden samt om den ökade livskvalitén hos patienter med spridd HER2-positiv bröstcancer. I bedömningen beaktas också om biverkningsmönstret förändras. Arbetet är en litteraturstudie omfattande fem vetenskapliga studier. De fem vetenskapliga artiklarna identifierades med databasen PubMed.  Arbetet baseras på två kliniska studier och tre kohortstudier. CLEOPATRA-studien är en stor studie som har undersökt effekten av pertuzumab som tillägg till TD-behandling. Utifrån studieresultatet ansågs det att pertuzumab som tillägg till TD-behandling förlängde den progressionsfria överlevnaden signifikant med 6,1 månader.  Studier som hade analyserat data från CLEOPATRA, har visat att tilläggsbehandlingen förlängde också tiden till sjukdomsförsämring och förbättrad livskvalité hos patienten. Behandlingen visade statistiskt signifikant förbättrad progressionsfri överlevnad hos alla åldersgrupper. Den visade inte heller att tilläggsbehandling med pertuzumab gav ökad risk för svåra biverkningar. Många av biverkningarna som registrerades var av grad 1 och 2.  Slutsatsen är att pertuzumab som tillägg till TD-behandling som förstahandsbehandling hos patienter med metastaserande HER2-positiv bröstcancer är fördelaktig. / In Sweden, about one in ten women develop breast cancer, which makes this the most common type of cancer disease in women. The risk of getting cancer increases with age. Almost 8000 women are diagnosed with breast cancer every year in Sweden. Human epidermal growth factor receptor 2 (HER2) – positive breast cancer, is an aggressive phenotype. About 25 % of the breast cancer tumors are overexpressing HER2 on the cell surface. HER2 is a subunit of the dimeric tyrosin kinase EFGR (HER) that stimulates cell proliferation. Every year, about 1300- 1400 patients advance in their illness and form metastases. Pertuzumab and trastuzumab, are both HER2 monoclonal antibodies. Pertuzumab inhibits the HER2 from heterodimerization with HER3, while trastuzumab inhibits HER2 homodimerization. Since 2007 trastuzumab and cytostatics were approved for treatment of metastatic HER2-positive breast cancer. The aim of this study was to evaluate if pertuzumab as an addition to trastuzumab and docetaxel (cytostatic) could prolong the time patients with metastatic HER2-positive breast cancer experienced free from progression of their illness in comparison with treatment with only trastuzumab and docetaxel. This study was based on scientific articles identified from the database PubMed. Five studies were selected, one of the studies was the CLEOPATRA-study. CLEOPATRA compared the efficiency and safety of the two selected treatments. Three studies further analyzed and followed up results of the CLEOPATRA-study. Study five investigated the effect of pertuzumab in patients with metastatic HER2-positive breast cancer, where disease progression had occurred during trastuzumab treatment. The primary endpoint was progression-free survival (PFS) and overall survival (OS). All articles showed that PFS increased in patients treated with pertuzumab as addition to trastuzumab and docetaxel. Overall survival was also improved during and after the follow-up. The conclusion of this study was that pertuzumab, trastuzumab and docetaxel as treatment for metastatic HER2-positive breast cancer is promising and should be used as first-line treatment of metastatic breast cancer with HER2 overexpression.

Notre travail s’est focalisé sur l’utilisation du trastuzumab‐immunoPET afin d’étudier et deguider une approche nouvelle visant à surmonter la résistance au médicament trastuzumab,causée par la surexpression de MUC4 dans le cancer du sein.Pour ce faire, nous avons préparé et utilisé du 89Zr‐trastuzumab dans le but de suivresa capacité de liaison au récepteur HER2 ainsi que son accumulation dans des cellulescancéreuses mammaires. Ensuite, nous avons formulé l’hypothèse que des agentsmucolytiques, tels que la N‐Acétylcystéine (NAC), en démêlant les réseaux formés par lesmucines, permettent l’amélioration de la captation du radiotraceur in vitro et in vivo. Eneffet, l’addition du NAC a occasionné une accumulation significative de 89Zr‐trastuzumab,sans altération ni changement de l’affinité de liaison au récepteur. Ceci semble égalementproduire une meilleure sensibilité des imageries PET dans le modèle animal choisi.Dans une seconde étape, nous avons évalué, dans un modèle murin de cancer du seinrésistant au trastuzumab et surexprimant la MUC4, si cette captation accrue se traduit parun bénéfice thérapeutique en utilisant le NAC combiné au trastuzumab. Nous avons obtenuun effet inhibiteur qui réduit de moitié la croissance tumorale, comparable à celui observépour la tumeur mammaire sensible au trastuzumab (implantée dans le même animal).En conclusion, notre étude démontre l’efficacité de l’utilisation de traceurs PETsurtout à visée théranostique, comme c’est le cas du 89Zr‐trastuzumab, pour étudier etévaluer la résistance aux médicaments ciblés apparentés au radiotraceur lui‐même. Ellepropose l’utilisation du NAC pour améliorer l’accessibilité du récepteur pour le radiotraceurainsi que pour le médicament « froid » ouvrant, de ce fait, une perspective vers uneutilisation clinique chez un sous‐type de patientes atteintes d’un cancer du sein. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

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京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12879号 / 論農博第2806号 / 新制||農||1028(附属図書館) / 学位論文||H26||N4878(農学部図書室) / 31597 / (主査)教授 植田 和光, 教授 植田 充美, 教授 矢﨑 一史 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM