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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 ZHER2:2891 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 ZHER2:2891-based AffiDCs showed specific an, 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, QC 2022-09-02

QC 20221031

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., QC 20210203

QC 20220119

QC 20220119

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-org

QC 20201215

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain-derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA-derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half-life extension, can be used for specific killing of HER2-expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (K-D 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (K-D) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50-values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA-derivatives are promising agents for targeted cancer therapy.

QC 20191126

QC 20191202

QC 20180517

QC 20180517

Auristatin and maytansine-derived drugs are cytotoxic tubulin polymerization inhibitors commonly used as payloads in drug conjugates intended for targeted cancer therapy. We have previously shown that an affibody molecule ZHER2, binding to the human epidermal growth factor receptor 2 (HER2), can be site-specifically conjugated to DM1, a maytansine- derived payload, creating the potent and specific drug conjugate, ZHER2-ABD-mcDM1, where the ABD is an albumin binding domain used for in vivo half-life extension. Here, we investigated the properties of the HER2-binding affibody molecule conjugated with the two auristatin-derived payloads, monomethyl auristatin E and F (MMAE and MMAF), in comparison with the construct with DM1. We found that the drug conjugate ZHER2-ABD- mcMMAF was more potent than ZHER2-ABD-mcDM1, with IC50 values to high-HER2 expressing cell lines ranging from 0.18 to 12 nM. By contrast the IC50 values of ZHER2-ABD- mcMMAE was considerably weaker and this construct would probably benefit from a different linker connecting the drug to the affibody fusion protein. Quantification of uptake in HER2-expressing tumors and normal organs of 99m-technetium labeled drug conjugates showed that they were predominantly cleared by the kidneys, with relatively high tumor uptake, peaking at 11.1 ± 4.1 %ID/g for ZHER2-ABD-mcMMAE at 24 h post-injection, 8.5 ± 1.5 %ID/g for ZHER2-ABD-mcMMAF at 48 h post-injection, and 7.1 ± 1.8 %ID/g for ZHER2- ABD-mcDM1 at 48 h post-injection. Most normal organs, except for the kidneys, had a relatively low uptake. In conclusion, ZHER2-ABD-mcMMAF was the best performing drug conjugate with the highest potency, and lowest uptake in liver; slightly outperforming ZHER2- ABD-mcDM1., QC 20220225