Overactive epidermal growth factor receptor (EGFR) signaling is often involved in driving different types of carcinomas. It is a well-studied target for targeted therapies, with both monoclonal antibodies and kinase inhibitors available for clinical use. Even though these drugs show a clinical benefit, most patients develop resistance over time. The development of new therapeutic modalities is therefore highly motivated. Herein, we describe a new type of drug candidate targeting EGFR, a so-called affibody-based drug conjugate. It consists of an EGFR-targeting affibody molecule, ZEGFR, expressed as a fusion to an albumin-binding domain for half-life extension, and coupled with the potent cytotoxic drug DM1 via a maleimidocaproyl linker. The resulting drug conjugate ZEGFR-ABD-mcDM1, showed strong binding to recombinant EGFR and EGFR-expressing cells. It was found to be highly potent in killing EGFR-expressing A431 cells with an IC50of 3.4 nM. In vivo, it showed moderate uptake in A431-derived xenografts with high EGFR expression. Collectively, the results from this study, demonstrate a potent and EGFR-specific drug candidate that holds promise for further development.

We report development and characterization of small non-immunoglobulin affibody affinity proteins directed to the highly glycosylated human carcinoembryonic antigen-related adhesion molecule 5 (CEACAM5, CEA), and their use in immunohistochemical (IHC) analyses of human pancreatic cancer samples and for in vivo tumor imaging. A total of nineteen unique anti-CEA affibodies were identified from large phage display libraries constructed using combinatorial protein engineering of a small 58 amino acid three-helix bundle protein domain. Molecular modeling suggested that all enriched clones share a binding surface with several clustered tryptophan residues interacting with a hydrophobic patch in the N1 domain of CEA centered around a phenylalanine residue. One variant, designated as C9, exhibited the highest affinity in biosensor analyses and was reformatted into a 15 kDa homodimer expressed in Escherichia coli. The biotinylated form, C9-C9-Bio, was evaluated for its IHC performance on matched frozen and formalin-fixed, paraffin-embedded (FFPE) sections of human pancreatic cancer samples (n = 7). Compared to clinical-grade monoclonal antibodies II-7 and CEA31, as well as a polyclonal reagent, C9-C9-Bio demonstrated highly sensitive CEA detection with minimal background staining. Statistical analyses including intraclass correlation and Bland-Altman assessments revealed excellent agreement between C9-C9-Bio and the two monoclonal antibodies in FFPE tissue samples. Further, a ^99mTc[Tc]-labeled C9-C9 construct showed CEA-dependent binding to human cancer cell lines in vitro, and selectively bound to CEA-expressing BxPC3 xenografts in mice when investigated as a tracer for in vivo imaging, allowing for a visualization of tumors after four hours. In summary, these findings highlight the potential use of the easily produced CEA-binding C9 affibody for various clinical applications, including IHC and medical imaging, and as a targeting moiety for directing various therapeutic modalities to CEA-expressing tumors.

Treatment with antibody drug conjugates targeting receptors over-expressed on cancer cells is well established for clinical use in several types of cancer, however, resistance often occurs motivating the development of novel drugs. We have recently investigated a drug conjugate consisting of an affibody molecule targeting the human epidermal growth factor receptor 2 (HER2), fused to an albumin-binding domain (ABD) for half-life extension, loaded with the cytotoxic maytansine derivative DM1. In this study, we investigated the impact of the cytotoxic payload on binding properties, cytotoxicity and biodistribution by comparing DM1 with the auristatins MMAE and MMAF, as part of the drug conjugate. All constructs had specific and high affinity binding to HER2, human and mouse albumins with values in the low- to sub-nM range. ZHER2-ABD-mcMMAF demonstrated the most potent cytotoxic effect on several HER2-over-expressing cell lines. In an experimental therapy study, the MMAFbased conjugate provided complete tumor regression in 50% of BALB/c nu/nu mice bearing HER2-overexpressing SKOV3 tumors at a 2.9 mg/kg dose, while the same dose of ZHER2-ABD-mcDM1 provided only a moderate anti-tumor effect. A comparison with the non-targeting ZTaq-ABD-mcMMAF control demonstrated HER2-targeting specificity. In conclusion, a combination of potent cytotoxicity in vitro, with minimal uptake in normal organs in vivo, and efficient delivery to tumors provided a superior anti-tumor effect of ZHER2-ABDmcMMAF, while maintaining a favorable toxicity profile with no observed adverse effects.

Targeted therapy is an emerging treatment for a variety of cancers. Small- sized scaffold proteins are an alternative to conventional antibody-based targeting molecules. Two small scaffold proteins—the 58-amino-acid protein class, the affibody molecules, and the 46-amino-acid protein class, the Albumin binding domain Derived Affinity Proteins (ADAPTs)—have previously been engineered to bind to a large variety of tumor-associated molecular targets with a high affinity. 

The human epidermal growth factor receptor 2 (HER2) is a membrane-bound receptor for growth signal transmission. Expression of a high level of HER2 can cause cells to proliferate and may ultimately lead to cancer. It has earlier been shown that HER2 is involved in several different types of cancers, e.g., breast, ovarian, bladder, and gastric cancers. 

HER2-targeted affibody and ADAPT molecules have previously been developed, such as Z_HER2:2891 and ADAPT6 with strong affinity to HER2 with equilibrium dissociation constants of 76 pM and 2.5 nM, respectively. Their small size and high specificity have rendered these two scaffold proteins promising candidates for imaging of HER2-positive breast cancer tumors in clinical trials. 

Delivery of cytotoxic agents to cancer cells, using a cell-targeting domain, may potentially precisely kill the cancer cells while having very low cytotoxic effects on normal cells. Many cancer-targeted antibody drug conjugates (ADCs) and toxic proteins (immunotoxins) have advanced the field of cancer treatment. Small-sized scaffold proteins hold promise as alternative targeting domains to build novel drug conjugates or fusion toxins for cancer treatment. 

In this thesis, I first investigated an affibody-based drug conjugate (AffiDC) composed of an anti-HER2 affibody and an anti-mitotic maytansine-derived drug (DM1) for treatment of HER2-overexpressing cells. I studied a variety of targeting domain formats for efficacy optimization. All Z_HER2:2891-based AffiDCs showed specific anti-tumor activity on HER2-overexpressing cancer cells in vitro as well as in mouse tumor xenografts. The hepatic uptake of the AffiDCs could be reduced by shielding the hydrophobic DM1 using a poly-glutamic-acid spacer, which might help to reduce potential liver toxicity allowing for administration of higher doses. In addition, tuning the valency of the affibody-targeting domain (Z_HER2) from a divalent domain to a monovalent domain showed increased potency and reduced liver uptake. We also investigated the influence of the number of drug payloads on the pharmacokinetic profile of the AffiDCs. An AffiDC bearing three DM1s showed higher delivery of DM1 to the cancer cells in vivo, but fast blood clearance and an elevated liver retention was also observed. 

With regards to fusion toxin design, we constructed a variety of recombinant toxins. The targeting domains were Z_HER2:2891 and/or ADAPT6, which were genetically fused with truncated versions of the highly cytotoxic Pseudomonas Exotoxin A (PE). All fusion toxins we studied showed potent HER2-specific anti-tumor activity. The results suggested that both Z_HER2:2891 and ADAPT6 could direct the PE-based cytotoxins specifically to HER2- overexpressing cancer cells. 

In this work, we have demonstrated the potential of using Z_HER2:2891 and ADAPT6 as targeting domains to carry the small molecule drug DM1, or cytotoxic PE-derived peptides to cancer cells. It can be concluded that careful molecular design of the targeting domain may considerably improve the potency and minimize the off-target uptake. 

Riktad terapi är en framväxande behandlingsmetod för en mängd olika cancerformer. Små affinitetsproteiner är ett alternativ till konventionella antikroppsbaserade målsökarmolekyler. Två små affinitetsproteiner – de 58-aminosyror stora affibodymolekylerna och de 46 aminosyror stora Albumin binding domain derived affinity proteins (ADAPTs) – har tidigare genererats för att binda till en stor mängd olika tumörassocierade molekylära målmolekyler med hög affinitet.

Human epidermal growth factor receptor 2 (HER2) är en membranbunden receptor för överföring av tillväxtsignaler. Uttryck av en hög nivå av HER2 kan få celler att dela sig och kan i slutändan leda till cancer. Det har tidigare visats att HER2 är involverat i flera olika typer av cancer, t.ex. bröst-, äggstocks-, urinblåse- och magcancer.

HER2-riktade affibodymolekyler och ADAPT-molekyler har tidigare utvecklats, t.ex. ZHER2:2891 och ADAPT6, med stark affinitet till HER2 med jämviktsdissociationskonstanter på 76 pM respektive 2,5 nM. Deras ringa storlek och höga specificitet har gjort dessa två affinitetsproteiner till lovande kandidater för avbildning av HER2-positiva bröstcancertumörer i kliniska prövningar.

Leverans av cytotoxiska läkemedel till cancerceller, med användning av en målsökande domän, kan potentiellt döda cancercellerna samtidigt som de har en låg cytotoxisk effekt på normala celler. Många cancerriktade antikroppsläkemedelskonjugat (ADCer) och toxiska proteiner (immunotoxiner) har expanderat cancerbehandlingsmöjligheterna. Små affinitetsproteiner är lovande som alternativa målsökarmolekyler för att bygga nya läkemedelskonjugat eller immunotoxiner för cancerbehandling.

I denna avhandling undersökte jag först ett affibodybaserat läkemedelskonjugat (AffiDC) bestående av en anti-HER2 affibodymolekyl och ett antimitotiskt maytansinoid-derivat (DM1) för behandling av HER2-överuttryckande celler. Jag studerade en mängd olika format för den målsökande domänen för optimering av effektiviteten. Alla ZHER2:2891-baserade AffiDCer visade specifik antitumöraktivitet mot HER2-överuttryckande cancerceller in vitro, såväl som i transplanterade tumörer i möss. Leverupptaget av AffiDCerna kunde minskas genom att skydda den hydrofoba DM1-delen med en poly-glutaminsyra-linker, vilket kunde bidra till att minska potentiell levertoxicitet vilket möjliggör administrering av högre doser. Dessutom visade justering av valensen för den målsökande affibodydomänen (ZHER2) från en divalent domän till en monovalent domän ökad potens och minskat leverupptag. Vi undersökte också påverkan av antalet läkemedelsmolekyler på AffiDCernas farmakokinetiska profil. En AffiDC som bär tre DM1 visade högre leverans av DM1 till cancercellerna in vivo, men en kort halveringstid i blod och ett förhöjt leverupptag observerades samtidigt.

När det gäller design av fusionstoxiner, konstruerade vi en mängd olika rekombinanta toxiner. Måldomänerna var ZHER2:2891 och/eller ADAPT6, som var genetiskt fuserade med trunkerade versioner av den starkt cytotoxiska peptiden Pseudomonas Exotoxin A (PE). Alla fusionstoxiner vi studerade visade potent HER2-specifik antitumöraktivitet. Resultaten antydde att både ZHER2:2891 och ADAPT6 kunde leverera de PE-baserade cellgifterna specifikt till HER2-överuttryckande cancerceller.

I detta arbete har vi visat potentialen i att använda ZHER2:2891 och ADAPT6 som målsökande domäner för att bära det småmolekylära läkemedlet DM1, eller cytotoxiska PE-deriverade peptider till cancerceller. Man kan dra slutsatsen att noggrann molekylär design av målsökardomänen kan förbättra potensen avsevärt och minimera upptaget i normal vävnad.

Affibody molecules hold great promise as carriers of cytotoxic drugs for cancer therapy due to their typically high affinity, easy production, and inherent control of the drug molecules' loading and spatial arrangement. Here, the impact of increasing the drug load from one to three on the properties of an affibody drug conjugate targeting the human epidermal growth factor receptor 2 (HER2) was investigated. The affibody carrier was recombinantly expressed as a fusion to an albumin-binding domain (ABD) for plasma half-life extension. One or three cysteine amino acids were placed at the C-terminus to which cytotoxic mcDM1 molecules were conjugated. The resulting drug conjugates, Z(HER2)-ABD-mcDM1 and Z(HER2)-ABD-mcDM1(3), were characterized in vitro, and their biodistribution in mice carrying HER2-overexpressing SKOV3 xenografts was determined. Increasing the drug load from one to three led to a decrease in affinity for HER2, but a significantly more potent cytotoxic effect on SKOV3 cells with high HER2 expression. The difference in cytotoxic effect on other cell lines with high HER2 expression was not significant. In vivo, an increase in drug load led to a 1.45-fold higher amount of cytotoxic mcDM1 delivered to the tumors. The increase in drug load also led to more rapid hepatic clearance, warranting further optimization of the molecular design.

The human epidermal growth factor receptor 2 (HER2) is frequently overexpressed in a variety of cancers and therapies targeting HER2 are routinely used in the clinic. Recently, small engineered scaffold proteins, such as affibody molecules, have shown promise as carriers of cytotoxic drugs, and these drug conjugates may become complements or alternatives to the current HER2-targeting therapies. Here, we investigated if a monovalent HER2-binding affibody molecule, Z(HER2:2891), fused with a plasma half-life extending albumin binding domain (ABD), may be used as carrier of the cytotoxic maytansine derivate mcDM1. We found that the resulting drug conjugate, Z(HER2:2891)-ABD-E-3-mcDM1, had strong affinity for its cognate molecular targets: HER2 and serum albumin. Z(HER2:2891)-ABD-E-3-mcDM1 displayed potent cytotoxic activity towards cells with high HER2 expression, with IC50 values ranging from 0.6 to 33 nM. In vivo, an unspecific increase in uptake in the liver, imparted by the hydrophobic mcDM1, was counteracted by incorporation of hydrophilic and negatively charged glutamate residues near the site of mcDM1 conjugation. A dose-escalation experiment showed that increasing doses up to 15.1 mg/kg gave a proportional increase in uptake in xenografted HER2-overexpressing SKOV3 tumors, after which the tumors became saturated. Experimental therapy with four once-weekly injection of 10.3 or 15.1 mg/kg led to efficient regression of tumors in all animals and complete regression in some. Weight loss was detected for some animals in the group receiving the highest dose, suggesting that it was close to the maximum tolerated dose. In conclusion, the monovalent HER2-targeting affibody drug conjugate presented herein have potent anti-tumor activity in vivo.

Simple Summary Targeted therapeutics provide cytostatic or cytotoxic action selectively to tumor cells while reducing the toxicity to normal cells. Targeting two molecular receptors overexpressed on tumor cells is a way to overcome heterogeneity of expression and improve therapeutic efficacy. Combining drugs with different modes of action might also increase the cytotoxic effect and decrease the chance for the cancer cells to develop resistance to treatment. In this work, we investigated a combination of the clinically used monoclonal antibody trastuzumab with a potent targeting protein-toxin fusion, directed at two different targets present in a large fraction of ovarian cancers. Co-targeted treatment provided a significant reduction in tumor growth and extended the survival of mice compared with the control and monotherapy groups. Our findings support further development of targeted combination therapies for treatment of aggressive and resistant cancer types. Efficient treatment of disseminated ovarian cancer (OC) is challenging due to its heterogeneity and chemoresistance. Overexpression of human epidermal growth factor receptor 2 (HER2) and epithelial cell adhesion molecule (EpCAM) in approx. 30% and 70% of ovarian cancers, respectively, allows for co-targeted treatment. The clinical efficacy of the monoclonal antibody trastuzumab in patients with HER2-positive breast, gastric and gastroesophageal cancers makes it readily available as the HER2-targeting component. As the EpCAM-targeting component, we investigated the designed ankyrin repeat protein (DARPin) Ec1 fused to a truncated variant of Pseudomonas exotoxin A with reduced immunogenicity and low general toxicity (LoPE). Ec1-LoPE was radiolabeled, evaluated in ovarian cancer cells in vitro and its biodistribution and tumor-targeting properties were studied in vivo. The therapeutic efficacy of Ec1-LoPE alone and in combination with trastuzumab was studied in mice bearing EpCAM- and HER2-expressing SKOV3 xenografts. SPECT/CT imaging enabled visualization of EpCAM and HER2 expression in the tumors. Co-treatment using Ec1-LoPE and trastuzumab was more effective at reducing tumor growth and prolonged the median survival of mice compared with mice in the control and monotherapy groups. Repeated administration of Ec1-LoPE was well tolerated without signs of hepatic or kidney toxicity. Co-treatment with trastuzumab and Ec1-LoPE might be a potential therapeutic strategy for HER2- and EpCAM-positive OC.

Simple Summary Metastasis-targeting therapy might improve outcomes in oligometastatic prostate cancer. Epithelial cell adhesion molecule (EpCAM) is overexpressed in 40-60% of prostate cancer cases and might be used as a target for specific delivery of toxins and drugs. Radionuclide molecular imaging could enable non-invasive detection of EpCAM and stratification of patients for targeted therapy. Designed ankyrin repeat proteins (DARPins) are scaffold proteins, which can be selected for specific binding to different targets. The DARPin Ec1 binds strongly to EpCAM. To determine an optimal design of Ec1-based probes, we labeled Ec1 at two different positions with four different nuclides (Ga-68, In-111, Co-57 and I-125) and investigated the impact on Ec1 biodistribution. We found that the C-terminus is the best position for labeling and that In-111 and I-125 provide the best imaging contrast. This study might be helpful for scientists developing imaging probes based on scaffold proteins. The epithelial cell adhesion molecule (EpCAM) is intensively overexpressed in 40-60% of prostate cancer (PCa) cases and can be used as a target for the delivery of drugs and toxins. The designed ankyrin repeat protein (DARPin) Ec1 has a high affinity to EpCAM (68 pM) and a small size (18 kDa). Radiolabeled Ec1 might be used as a companion diagnostic for the selection of PCa patients for therapy. The study aimed to investigate the influence of radiolabel position (N- or C-terminal) and composition on the targeting and imaging properties of Ec1. Two variants, having an N- or C-terminal cysteine, were produced, site-specifically conjugated to a DOTA chelator and labeled with cobalt-57, gallium-68 or indium-111. Site-specific radioiodination was performed using ((4-hydroxyphenyl)-ethyl)maleimide (HPEM). Biodistribution of eight radiolabeled Ec1-probes was measured in nude mice bearing PCa DU145 xenografts. In all cases, positioning of a label at the C-terminus provided the best tumor-to-organ ratios. The non-residualizing [I-125]I-HPEM label provided the highest tumor-to-muscle and tumor-to-bone ratios and is more suitable for EpCAM imaging in early-stage PCa. Among the radiometals, indium-111 provided the highest tumor-to-blood, tumor-to-lung and tumor-to-liver ratios and could be used at late-stage PCa. In conclusion, label position and composition are important for the DARPin Ec1.