<p>HER2 Gene-Protein Assay (GPA) је посебно погодан за истовремено процењивање експресије HER2 протеина и статуса амплификације HER2 гена на нивоу појединачних ћелија и њихово повезивање са ћелијском морфологијом. Циљ истраживања био је испитати да ли су постојећи критеријуми препоручени од стране ASCO/CAP довољни за дијагностиковање HER2 позитивности код пацијенткиња које показују интратуморску хетерогеност, како у примарним туморима тако и у метастазама у регионалне лимфне чворове, учесталост HER2 хетерогености у макрометастазама лоцираним у лимфним чворовима, те да ли постоји јасна корелација између хетерогености нађене у примарном тумору дојке и припадајућим метастазама у лимфним чворовима. Испитивање је обухватило 41 од планиране 51 пацијенткиње које су испуниле све критеријуме укључивања. Репрезентативни парафински блокови метастатских лимфних чворова одабрани су из архивираног материјала, обојени GPA методом и процењени у складу са критеријумима ASCO/CAP 2013. Анализирано је 120 ћелија у хистолошком резу сваког метастатског лимфног чвора. Статус HER2 се разликовао између примарног тумора и његових метастаза у 13,2% (5/38) случајева. Један случај HER2 позитивног примарног тумора имао је HER2 негативне метастазе, два додатна случаја са HER2 позитивним примарним тумором су имала метастазе са статусом граничне амплификације без прекомерне експресије HER2 протеина и два случаја са HER2 негативним примарним тумором су имала метастазе са статусом граничне амплификације без прекомерне експресије HER2 протеина. У 17.4% (4/23) случајева са HER2 не-амплификованим примарним тумором метастазе су постале граничне у статусу генске амплификације. Једна од четири метастазе HER2 негативног примарног тумора показала је мали фокус HER2 позитивних туморских ћелија (&lt;3% тумора). Микрохетерогеност је анализирана у 108 лимфних чворова код 38 пацијенткиња и уочена у 22 лимфна чвора, тј. код четири пацијенткиње у свим анализираним лимфним чворовима, док је код једне пацијенткиње од 4 анализирана лимфна чвора микрохетерогеност потврђена у једном лимфном чвору. На основу добијених резултата може се закључити да постојећи критеријуми препоручени од стране ASCO/CAP применом прихваћених метода нису довољни за дијагностиковање HER2 позитивности код пацијенткиња које показују интратуморску и интертуморску хетерогеност како у примарним туморима тако и у метастазама, те да постоји статистички високо сигнификантан број макрометастаза лоцираних у лимфним чворовима које показују HER2 хетерогеност и позитивна корелација између хетерогености нађене у примарним туморима и припадајућим метастазама у лимфним чворовима.</p> / <p>HER2 Gene-Protein Assay (GPA) je posebno pogodan za istovremeno procenjivanje ekspresije HER2 proteina i statusa amplifikacije HER2 gena na nivou pojedinačnih ćelija i njihovo povezivanje sa ćelijskom morfologijom. Cilj istraživanja bio je ispitati da li su postojeći kriterijumi preporučeni od strane ASCO/CAP dovoljni za dijagnostikovanje HER2 pozitivnosti kod pacijentkinja koje pokazuju intratumorsku heterogenost, kako u primarnim tumorima tako i u metastazama u regionalne limfne čvorove, učestalost HER2 heterogenosti u makrometastazama lociranim u limfnim čvorovima, te da li postoji jasna korelacija između heterogenosti nađene u primarnom tumoru dojke i pripadajućim metastazama u limfnim čvorovima. Ispitivanje je obuhvatilo 41 od planirane 51 pacijentkinje koje su ispunile sve kriterijume uključivanja. Reprezentativni parafinski blokovi metastatskih limfnih čvorova odabrani su iz arhiviranog materijala, obojeni GPA metodom i procenjeni u skladu sa kriterijumima ASCO/CAP 2013. Analizirano je 120 ćelija u histološkom rezu svakog metastatskog limfnog čvora. Status HER2 se razlikovao između primarnog tumora i njegovih metastaza u 13,2% (5/38) slučajeva. Jedan slučaj HER2 pozitivnog primarnog tumora imao je HER2 negativne metastaze, dva dodatna slučaja sa HER2 pozitivnim primarnim tumorom su imala metastaze sa statusom granične amplifikacije bez prekomerne ekspresije HER2 proteina i dva slučaja sa HER2 negativnim primarnim tumorom su imala metastaze sa statusom granične amplifikacije bez prekomerne ekspresije HER2 proteina. U 17.4% (4/23) slučajeva sa HER2 ne-amplifikovanim primarnim tumorom metastaze su postale granične u statusu genske amplifikacije. Jedna od četiri metastaze HER2 negativnog primarnog tumora pokazala je mali fokus HER2 pozitivnih tumorskih ćelija (&lt;3% tumora). Mikroheterogenost je analizirana u 108 limfnih čvorova kod 38 pacijentkinja i uočena u 22 limfna čvora, tj. kod četiri pacijentkinje u svim analiziranim limfnim čvorovima, dok je kod jedne pacijentkinje od 4 analizirana limfna čvora mikroheterogenost potvrđena u jednom limfnom čvoru. Na osnovu dobijenih rezultata može se zaključiti da postojeći kriterijumi preporučeni od strane ASCO/CAP primenom prihvaćenih metoda nisu dovoljni za dijagnostikovanje HER2 pozitivnosti kod pacijentkinja koje pokazuju intratumorsku i intertumorsku heterogenost kako u primarnim tumorima tako i u metastazama, te da postoji statistički visoko signifikantan broj makrometastaza lociranih u limfnim čvorovima koje pokazuju HER2 heterogenost i pozitivna korelacija između heterogenosti nađene u primarnim tumorima i pripadajućim metastazama u limfnim čvorovima.</p> / <p><!--[if gte mso 9]><xml> <o:DocumentProperties> <o:Author>ilija vogel</o:Author> <o:Version>16.00</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings></xml><![endif]--><!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:TrackMoves/> <w:TrackFormatting/> <w:PunctuationKerning/> <w:ValidateAgainstSchemas/> 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The oncogenic isoform of HER2, HER2Δ16, is expressed with HER2 in nearly 50% of HER2 positive breast tumors where HER2Δ16 drives metastasis and resistance to multiple therapeutic interventions including tamoxifen and trastuzumab. The research carried out in this dissertation investigated the molecular mechanisms underlying HER2Δ16 activity contributing to primary trastuzumab resistance. In recent years microRNAs have been shown to influence multiple aspects of tumorigenesis and tumor cell response to therapy. Accordingly, the HER2Δ16 oncogene alters microRNA expression to promote endocrine resistance. With the goal of identifying microRNA suppressors of HER2Δ16 oncogenic activity, we investigated the contribution of altered microRNA expression to HER2Δ16-mediated tumorigenesis and trastuzumab resistance. Using a gene array strategy to compare the microRNA expression profiles of MCF-7 to MCF-7/HER2Δ16 cells, we found that HER2Δ16 suppresses expression of the miR-7 tumor suppressor. Reestablishing miR-7 expression significantly inhibits HER2Δ16-mediated tumor cell proliferation and migration, as well as sensitizes HER2Δ16-expressing cells to trastuzumab treatment. We propose that miR-7 regulated pathways, including EGFR and Src kinase, represent targets for the therapeutic intervention of refractory and metastatic HER2Δ16-driven breast cancer. Research in the past decade in HER2-positive breast cancer has focused on elucidating the molecular basis of primary and acquired trastuzumab resistance. Our laboratory has shown that critical and clinically important resistance pathways may be deregulated and only revealed during drug treatment. To identify potential resistance pathways deregulated during trastuzumab treatment, we used a phosphoproteomic approach to profile a subset of phosphorylation events after HER2Δ16-overexpressing cells were treated with trastuzumab. We discovered trastuzumab treatment significantly induced activation of ribosomal p70S6 kinase 1 (p70S6K) and aberrant signaling activity of this kinase is implicated in several disease models due to its role in regulating protein synthesis, cell proliferation and survival. Our data indicates that trastuzumab activates p70S6K to promote prosurvival signaling in breast cancer cells with inherent resistance. We propose that p70S6K can be evaluated in HER2-positive breast cancer patients undergoing trastuzumab treatment as a biomarker to predict therapeutic response. Overall, our research establishes that HER2Δ16 expression is an important genetic event in HER2 tumorigenesis and drives trastuzumab refractory breast cancer. / acase@tulane.edu

<p>Breast cancer mortality in Sweden is now in decline, thanks to early detection and the wide use of adjuvant endocrine therapy and chemotherapy. </p><p>While hormone receptor status is predictive of response to endocrine treatment, there is no clinically useful predictive marker of a patient’s response to chemotherapy. Consequently, patients receive chemotherapy with considerable toxicity but minimal benefit. The aim of this thesis was to investigate a number of methods with the potential to predict response to chemotherapy and thus enhance treatment efficacy in breast cancer patients.</p><p>We found that topo IIα, the key target enzyme of topo II inhibitors, is significantly expressed in nonproliferating breast cancer cells. This finding may explain why topo II inhibitors are effective in patients with slow growing tumors and a low proliferation rate.</p><p>Topo IIα gene amplification was suggestive of increased response to anthracyclines in advanced breast cancer, whereas the oncogene HER2 had no predictive value by itself. These findings are in accordance with current knowledge.</p><p>Cyclin A, a marker of cell proliferation, showed good prognostic value but did not predict response to chemotherapy in advanced breast cancer.</p><p>In vitro chemosensitivity testing with FMCA predicted tumor response in patients with advanced breast cancer with a sensitivity of 89% and a specificity of 53%. Our results are consistent with the results from similar assays, which predict drug resistance with good accuracy while clinical drug sensitivity is less reliably predicted. The use of FMCA and similar assays is not yet recommended outside clinical trials; their main utility is in preclinical testing of new anti-cancer drugs, including targeted therapies.</p><p>The combination of epirubicin, capecitabine, and cisplatin (EXC) demonstrated high clinical response rate (74%) and pathological complete response rate (22%) in locally advanced breast cancer, but with cumbersome toxicity. The fluoropyrimidine biomarkers TS, TP, and DPD did not predict response to the EXC regimen.</p>

Breast cancer mortality in Sweden is now in decline, thanks to early detection and the wide use of adjuvant endocrine therapy and chemotherapy. While hormone receptor status is predictive of response to endocrine treatment, there is no clinically useful predictive marker of a patient’s response to chemotherapy. Consequently, patients receive chemotherapy with considerable toxicity but minimal benefit. The aim of this thesis was to investigate a number of methods with the potential to predict response to chemotherapy and thus enhance treatment efficacy in breast cancer patients. We found that topo IIα, the key target enzyme of topo II inhibitors, is significantly expressed in nonproliferating breast cancer cells. This finding may explain why topo II inhibitors are effective in patients with slow growing tumors and a low proliferation rate. Topo IIα gene amplification was suggestive of increased response to anthracyclines in advanced breast cancer, whereas the oncogene HER2 had no predictive value by itself. These findings are in accordance with current knowledge. Cyclin A, a marker of cell proliferation, showed good prognostic value but did not predict response to chemotherapy in advanced breast cancer. In vitro chemosensitivity testing with FMCA predicted tumor response in patients with advanced breast cancer with a sensitivity of 89% and a specificity of 53%. Our results are consistent with the results from similar assays, which predict drug resistance with good accuracy while clinical drug sensitivity is less reliably predicted. The use of FMCA and similar assays is not yet recommended outside clinical trials; their main utility is in preclinical testing of new anti-cancer drugs, including targeted therapies. The combination of epirubicin, capecitabine, and cisplatin (EXC) demonstrated high clinical response rate (74%) and pathological complete response rate (22%) in locally advanced breast cancer, but with cumbersome toxicity. The fluoropyrimidine biomarkers TS, TP, and DPD did not predict response to the EXC regimen.

Dysregulation of protein activity, caused by alterations in protein sequence, expression, or localization, is associated with numerous diseases. In order to control the activity of harmful protein entities, affinity ligands such as proteins, oligonucleotides or small molecules can be engineered to specifically interact with them to modulate their function. In this thesis, non-immunoglobulin based affinity proteins known as affibody molecules are used to functionally inhibit proteins important for signaling through pathways that are overactive in different cancers.   In Paper I and Paper II, affibody molecules with high affinity for the receptor tyrosine kinases HER2 or EGFR are expressed in the secretory compartments of model cancer cell lines SKOV3 or A431 using a retrovirus-based gene delivery system. Equipping the affinity proteins with an ER retention tag, the affibody molecules together with their target protein are retained in the secretory compartments as shown by confocal fluorescence imaging. Flow cytometric analysis showed a 60 % or 80 % downregulation of surface located HER2 or EGFR in these cell lines, respectively. A significant decreased in proliferation rate of the cells was also observed, which for EGFR retention could be correlated with inhibition of phosphorylation in the kinase domain. In Paper III, novel affibody molecules interacting with the hormone binding site of the insulin growth factor-1 receptor were generated. One variant had high (1.2 nM) affinity for the receptor and could be used for immunofluorescence analysis and for receptor pull-out from cell lysates. Addition of this affibody molecule to MCF-7 cells had a dose dependent growth inhibitory effect on the cells. In Paper IV, novel affibody molecules against the intracellular oncoproteins H-Ras and Raf-1 were selected and characterized, and they proved to be specific for their target proteins. Mapping experiments showed that the affibody molecules selected against H-Ras interacted at over-lapping epitopes not affecting the interaction between Ras and Raf. In contrast, the predominant variant isolated during selection against Raf-1 could completely inhibit the Ras/Raf interaction in a real-time biospecific interaction analysis.   Taken together, the affibody molecules presented here and the strategies by which they are used to interfere with cancer related proteins and pathways may be valuable tools for further investigation of these systems and may possibly also be used to generate molecules suitable for cancer therapy. / QC 20100818

Recent advances in biotechnology have generated possibilities to investigate and measure parts of life previously left for believers to explain. Utilizing the same book of recipes, the genome, our cells produce selections of proteins at a time and thereby niche into a multitude of specialized cell types, tissues and organs comprising our body. Knowledge of the precise protein composition in a given organ at normal and disease condition would be of invaluable importance, both for identification of disease causes and the design of new pharmaceuticals, as well as for a deeper understanding of the processes of life. This doctoral thesis describes the start and progress of a visionary project (HPR) to localize all human proteins in our body, with emphasis on the generation and characterization of antibodies used as protein targeting missiles. To facilitate the identification of one human protein in a complex environment like our body, it is of significant importance to have precise and specific means of detection. The first two papers (I-II), describe software developed for generation of monospecific antibodies satisfying such needs, using a set of rules for antigen optimization. Five years after project start a large amount of antibodies with documented characteristics have been generated. The third paper (III), illustrates an attempt to sieve these antibody characteristics to develop a tool, for further improvement of antigen selection, based on the correlation between antigen sequence and amount of specific antibody generated.Having a panel of protein-specific antibodies is a possession of a great value, not only for localization studies, but also as possible target-directed pharmaceuticals. In such cases, knowledge of the precise epitope recognized by the antibody on its target protein, is an important aid, both for understanding its effect as well as unwanted cross-reactivity. Paper (IV) describes the development of a high-resolution method for epitope mapping of antibodies using staphylococcal display. An application of the method is described in the last paper (V) where it is used to map an anti-HER2 monospecific antibody with growth-inhibiting effects on breast cancer cells. The monospecific antibody was fractionated into separate populations and five novel epitopes related to cancer cell growth-inhibition was determined.Altogether these methods are valuable tools for generation and characterization of monospecific antibodies. / QC 20100907

Affibody molecules are small (7 kDa) affinity proteins of non-immunoglobulin origin that have been generated to specifically interact with a large number of clinically important molecular targets. In this thesis, Affibody molecules have been employed as tracers for radionuclide molecular imaging of HER2- and IGF-1R-expressing tumors, paper I-IV, and for surface knock-down of EGFR, paper V. In paper I, a tag with the amino acid sequence HEHEHE was fused to the N-terminus of a HER2-specific Affibody molecule, (ZHER2), and was shown to enable facile IMAC purification and efficient tri-carbonyl 99mTc-labeling. In vivo evaluation of radioactivity uptake in different organs showed an improved biodistribution, including a 10-fold lower radioactivity uptake in liver, compared to the same construct with a H6-tag. In paper II, it was further shown that an N-terminally placed HEHEHE-tag on ZHER2 provided lower unspecific uptake of radioactivity in liver compared to its H6-tagged counterpart even when radiolabeling was at the C-terminus using alternative chemistries to attach 99mTc, 111In or 125I. In paper III, the H6-tag’s composition and position was varied with regards to charge, hydrophobicity and its C- or N-terminal placement on ZHER2. Among the ten variants investigated, it was found that an N-terminal HEHEHE-tag provided the most favorable overall biodistribution profile and that introduction of hydrophobic and positively charged amino acids provoked liver uptake of radioactivity. In paper IV, the HEHEHE-tag was shown to enable IMAC purification and tri-carbonyl 99mTc-labeling of an IGF-1R-specific Affibody molecule and improved its overall biodistribution when compared to the same construct with a H6-tag. In paper V, the aim was to develop an intracellular receptor-entrapment system to reduce the surface levels of EGFR. An EGFR-specific Affibody molecule was expressed as a fusion to different mutants of an intracellular transport protein in SKOV-3 cells, resulting in a collection of cell lines with 50%, 60%, 80% and 96% reduced surface level of EGFR. Analysis of the proliferation rate of these cell lines showed that a modest reduction (15%) in proliferation occurs between 60% and 80% reduction of the surface level of EGFR. / <p>QC 20130129</p>

Affibody molecules have lately shown great potential as tools for in vivo molecular imaging. These small, 3-helical bundles, with their highly stable protein scaffold, are well suited for the often harsh conditions of radiolabeling. Their small size allows for rapid clearance from the blood circulation which permits the collection of images already within hours after injection. This thesis includes four papers aimed at engineering different variants of a HER2-binding Affibody molecule to enable effective  and  flexible  radiolabeling  and  enhancing  the  molecular  imaging  in  terms  of  imaging contrast and resolution. In paper I an Affibody molecule was engineered to function as a multifunctional platform for site-specific labeling with different nuclides for radionuclide imaging. This was done using only natural amino  acids,  thereby  allowing  for  both  synthetic  and  recombinant  production.  By  grafting  the amino acid sequence -GSECG to the C-terminal of our model-protein, a HER2-binding Affibody molecule, we enabled site specific labeling with both trivalent radiometals and with  99m Tc. Maleim-ide-DOTA was conjugated to the cysteine residue for labeling with  111 In, while the peptide sequence was able to chelate  99m Tc directly. This approach can also be used for site-specific labeling with other probes available for thiol-chemistry, and is applicable also to other protein scaffolds. In paper II we investigated the impact of size and affinity of radiolabeled Affibody molecules on tumor targeting and image contrast. Two HER2-targeting Affibody molecules, a two-helix (~5 kDa) and a three-helix (~7 kDa) counterpart, were synthetically produced, labeled with  111 In via chelation by  DOTA  and  directly  compared  in  terms  of  biodistribution  and  targeting  properties.  Results showed  that  the  smaller  variant  can  provide  higher  contrast  images,  at  the  cost  of  lower  tumor uptake,  in  high-expressing  HER2-tumors.  However,  neither  the  tumor  uptake  nor  the  contrast of the two-helix variant is sufficient to compete with the three-helix molecule in tumors with low expression of HER2. In paper III and IV we were aiming to find methods to improve the labeling of Affibody molecules with  18 F for PET imaging. Current methods are either complex, time-consuming or generate heavily lipophilic conjugates. This results in low yields of radiolabeled tracer, low specific activity left for imaging, undesirable biodistribution or a combination thereof. In paper III we demonstrate a swift and efficient 2-step, 1-pot method for labeling HER2-binding Affibody molecules by the formation of aluminum  18 F-fluoride (Al 18 F) and its chelation by NOTA, all in 30 min. The results show that the  18 F-NOTA-approach is a very promising method of labeling Affibody molecules with  18 F and further investigation of this scheme is highly motivated. In the last paper we pursued the possibility of decreasing the high kidney retention that is common among small radiotracers with residual-izing radiometabolites. In this work  18 F-4-fluorobenzaldehyde (FBA) was conjugated to a synthetic HER2-targeting Affibody molecule via oxime ligation. However, to avoid elevated liver retention, as seen in previous studies with this kind of label, a hydrophilic triglutamyl spacer between the aminooxy moiety and the N-terminal was introduced. A comparison of the two constructs (with and without the triglutamyl spacer) showed a clear reduction of retention in both kidney and liver in NMRI mice at 2 h p.i. when the spacer was included. In the light of these promising results, further studies including tumor-bearing mice, are in preparation. / <p>QC 20130203</p>

HER2/neu is an oncogenic tumor-associated antigen over-expressed in several human tumors including breast and ovarian cancer. The selective expression of HER2/neu and its role in epithelial carcinogenesis makes HER2/neu an ideal target for immunotherapy. Tumor-derived chaperone-rich cell lysate (CRCL), containing numerous heat shock proteins, has successfully been used to generate tumor-specific immunity against a wide range of murine tumors and is a great candidate for an effective vaccine against HER2/neu positive tumors. In the first part of this study, the potency of human ovarian cancer-derived CRCL to activate dendritic cells (DCs) and to generate tumor-specific T cells in vitro has been investigated. Chaperone-rich cell lysate was generated from primary ovarian cancer tissues and SKOV3-A2, a HER2/neu, Wilm's tumor gene 1 (WT1) and HLA-A2 positive human ovarian tumor cell line. T cells from healthy donors and from ovarian cancer patients secreted higher amounts of interferon-&#947; following in vitro re-stimulation with ovarian cancer-derived CRCL compared to HER2/neu or WT1 peptide-pulsed DCs. We were also able to generate cytotoxic T lymphocyte activity against cancer-specific antigens such as HER2/neu and WT1 from all healthy donors, but from only one of the four ovarian cancer patients with bulky disease. In the second part of the study, the potency of tumor-derived CRCL to elicit the humoral immune response against a murine HER2/neu positive tumor (TUBO) has been examined. Vaccination of mice bearing a palpable tumor efficiently delayed the development of the tumor. In the vaccinated mice, CRCL vaccination induced significant anti-HER2/neu antibodies. Using B cell deficient mice and antibody transfer experiments, we have shown that the induction of anti-HER2/neu antibodies is both necessary and sufficient for the anti-tumor effect. Further, we have demonstrated that serum from TUB0 CRCL-vaccinated mice stimulated the internalization of the HER2/neu molecules, resulting in the down-regulation of their surface expression. Moreover, antibody-dependent cellular cytotoxicity has been observed against TUBO cells when presented with sera from vaccinated mice. These results indicate that CRCL may be a potent adjuvant for women suffering from HER2/neu positive ovarian or breast cancer and that this personalized vaccine may be a promising approach for active immunotherapy.

Affibody molecules are promising candidates for targeted radionuclide-based imaging and therapy applications. Optimisation of targeting properties would permit the in vivo visualization of cancer-specific surface receptors with high contrast. In therapy, this may increase the ratio of radioactivity uptake between tumour and normal tissues.  This thesis work is based on 5 original research articles (papers I-V) and focuses on optimisation of targeting properties of anti-HER2 affibody molecules by optimising the labelling chemistry. Paper I and II report the comparative evaluation of the anti-HER2 ZHER2:2395 affibody molecule site specifically labelled with 111In (suitable for SPECT imaging) and 68Ga (suitable for PET imaging) using the thiol reactive derivatives of DOTA and NODAGA as chelators. The incorporation of different macrocyclic chelators and labelling with different radionuclides modified the biodistribution properties of affibody molecules. This indicates that the labelling strategy may have a profound effect on the targeting properties of radiotracers and must be carefully optimized. Paper III reports the study of the mechanism of renal reabsorption of anti-HER2 ZHER2:2395 affibody molecule. An unknown receptor (not HER2) is suspected to be responsible for the high reabsorption of ZHER2:2395 molecules in the kidneys. Paper IV reports the optimization and development of in vivo targeting properties of 188Re-labelled anti-HER2 affibody molecules. By using an array of peptide based chelators, it was found that substitution of one amino acid by another or changing its position can have a dramatic effect on the biodistribution properties of 188Re-labelled affibody molecules. This permitted the selection of –GGGC chelator whichdemonstrated the lowest retention of radioactivity in kidneys compared to other variants and showed excellent tumour targeting properties. Paper V reports the preclinical evaluation of 188Re-ZHER2:V2 as a potential candidate for targeted radionuclide therapy of HER2-expressing tumours. In vivo experiments in mice along with dosimetry assessment in both murine and human models revealed that future human radiotherapy studies using 188Re-ZHER2:V2 may be feasible. It would be reasonable to believe that the results of optimisation of anti-HER2 affibody molecules summarized in this thesis can be of importance for the development of other scaffold protein-based targeting agents.