| 1. |
- Rinne, Sara Sophie
(författare)
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Affibody-Based Molecular Imaging and Targeted Therapy of HER3-Expressing Cancer
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human epidermal growth factor receptor type 3 (HER3) is overexpressed in different types of cancer and is a known contributor to disease progression and resistance to cancer therapy. This thesis is based on five original articles, which aimed to improve the diagnostic and therapeutic potential of affibody-based agents for management of HER3-expressing cancers. Papers I-III focused on the development and optimization of radiolabeled affibody molecules for radionuclide molecular imaging of HER3 expression. In particular, they investigated the influence of different radiometal/chelator complexes and hydrophilicity on the biodistribution and imaging properties of the HER3-targeting affibody molecule ZHER3. Paper IV compared the optimized ZHER3-based radiotracer with antibody and antibody-fragment based radiotracers for PET imaging of HER3 expression. In Paper V, a preclinical therapy study was conducted to investigate the efficacy of different monomeric and dimeric HER3-targeting affibody constructs for treatment of HER3-expressing cancer.It was shown that by optimizing the radiometal/chelator complex and incorporation of a hydrophilic (HE)3-tag the imaging properties of ZHER3-based radiotracers could be improved (Papers I-III). Generally, replacing a positively charged radiometal/chelator complex with a neutral or negatively charged complex improved the image contrast by reducing the normal organ uptake, especially in the liver. Further, it was demonstrated that the optimized affibody-based tracer [68Ga]Ga-(HE)3-ZHER3-NODAGA could provide higher contrast PET images of HER3 expression than the 89Zr-labeled antibody seribantumab and a seribantumab-derived F(ab’)2 fragment (Paper IV). The therapy study showed that the arrangement of the molecular building blocks affected the therapeutic efficacy of ZHER3-based affibody constructs. The monomeric and dimeric ABD-conjugated affibody constructs 3A and 3A3 showed the best therapeutic efficacy among the tested constructs and were able to delay tumor growth and prolong survival with the same efficacy as the therapeutic HER3-targeting antibody seribantumab (Paper V).In conclusion, the results described in this thesis show that HER3-targeting affibody-based agents could be well-suited for molecular imaging of HER3 expression and HER3-targeted therapy in cancer. Careful optimization of the molecular design could improve the imaging properties and therapeutic efficacy of HER3-targeting affibody molecules. Most importantly, it was demonstrated that HER3-targeting affibody molecules could provide superior diagnostic images and similar therapeutic effect than more traditional approaches for management of HER3-expressing cancer.
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| 2. |
- Rosestedt, Maria
(författare)
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Affibody Molecules for HER3-targeted Theranostics of Malignant Tumours
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The HER3 receptor plays a strong role in disease progression and resistance to therapies in several cancer types. Due to its endogenous expression and low overexpression in malignant tumours, it is a particularly challenging target. The primary aim of this thesis project was to develop, evaluate and characterize affibody molecules for theranostic applications in HER3-expressing malignant tumours.Paper I investigated the in vivo targeting properties and therapeutic efficacy of a bivalent affibody construct fused with an albumin binding domain, ZHER3-ABD-ZHER3. This construct could slow down the growth of HER3-expressing tumour xenografts without causing health problems or side effects in mice.Paper II compared the in vitro and in vivo properties of two HER3-targeting affibody molecules (Z08698 and Z08699) to select an imaging probe for HER3 diagnostics. While the two constructs had similar properties, Z08698 demonstrated better blood clearance and better radioactivity retention in tumours.Paper III and IV present the development of a HER3 imaging probe for PET using gallium and cobalt isotopes. We demonstrated that imaging of HER3 expression could be obtained as soon as 3 h pi using gallium-68. Additionally, we demonstrated that affibody molecules labelled with a neutral cobalt-NOTA complex had a lower radioactivity uptake in the liver than molecules radiolabelled with a positive gallium-NOTA complex. Imaging contrast increased over time. As the dose of the injected protein increased, the activity uptake in normal organs decreased, whereas the tumour uptake remained the same, which improved the imaging contrast and allowed discrimination between xenografts with high and low HER3 expression. This modification did not influence tumour activity uptake.Paper V presents the HER3-targeting affibody molecule trimer as a tool to block hepatic uptake in order to increase the imaging contrast in the liver. The trimer demonstrated its ability to bind to endogenous receptors in the liver, which decreased the hepatic uptake of the radiolabelled monomer. This phenomenon enabled the monomer to pass the liver barrier, which increased tumour radioactivity uptake and improved imaging contrast.
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| 3. |
- Abouzayed, Ayman, 1992-
(författare)
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Theranostic Targeting of GRPR and PSMA in Prostate Cancer
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is based on five original articles that investigated the theranostics of prostate cancer by gastrin-releasing peptide receptor (GRPR) and prostate-specific membrane antigen (PSMA) targeting. GRPR and PSMA are two extensively evaluated prostate cancer cell markers due to their overexpression in the majority of prostate cancer samples. Theranostic targeting of GRPR and PSMA is an attractive strategy to improve the management of prostate cancer patients.Papers I and II focused on the dual targeting of GRPR and PSMA. The effect of linker modification on the affinity for GRPR and PSMA and the pharmacokinetic profile was evaluated. In Paper III, the effect of the GRPR antagonist RM26 conjugation to an albumin-binding domain on the pharmacokinetic profile and its potential use in therapy was investigated. Paper IV focused on developing a GRPR antagonist that was suitable for single-photon emission computed tomography (SPECT) using technetium-99m. In Paper V, the GRPR antagonist developed in Paper IV was translated into a phase I clinical trial to assess safety and dosimetry.Modifying the linkers in GRPR and PSMA heterodimers can largely impact the affinity for both targets. This modification influenced the in vivo targeting specificity and biodistribution, with [125I]I-BO530 in Paper I and [111In]In-BQ7812 in Paper II outperforming other analogues. Our findings in Paper III indicated that the conjugation of an albumin-binding domain to RM26 increased the blood concentration of the radiotracer. This increase led to elevated and stable tumour uptake of [111In]In-DOTA-ABD-RM26 after several days of injection. However, [111In]In-DOTA-ABD-RM26 was also increasingly taken up by various healthy organs. The GRPR antagonist [99mTc]Tc-maSSS-PEG2-RM26, studied in Paper IV, showed high specificity and affinity for GRPR. This resulted in elevated GRPR-mediated uptake. Additionally, maSSS-PEG2-RM26 could be radiolabelled via a straightforward radiolabelling protocol. Clinical evaluation of [99mTc]Tc-maSSS-PEG2-RM26 in prostate and breast cancer patients (Paper V) demonstrated the safety and tolerability of the radiotracer, with favourable dosimetry and no side effects.In conclusion, this thesis evaluated different tools for the theranostic targeting of GRPR and PSMA. The findings warrant further investigation to optimise the reported radiotracers.
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| 4. |
- Bragina, Olga, et al.
(författare)
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Phase I study of 99mTc-ADAPT6, a scaffold protein-based probe for visualization of HER2 expression in breast cancer
- 2021
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 62:4, s. 493-499
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide molecular imaging of human epidermal growth factor (HER2) expression may be helpful to stratify breast and gastroesophageal cancer patients for HER2-targeting therapies. ADAPTs (albumin-binding domain derived affinity proteins) are a new type of small (46-59 amino acids) proteins useful as probes for molecular imaging. The aim of this first-in-human study was to evaluate biodistribution, dosimetry, and safety of the HER2-specific 99mTc-ADAPT6.METHODS: Twenty-nine patients with primary breast cancerwere included. In 22 patients with HER2-positive (n = 11) or HER2-negative (n = 11) histopathology an intravenous injection with 385±125 MBq 99mTc-ADAPT6 was performed, randomized to an injected protein mass of either 500 µg (n = 11) or 1000 µg (n = 11). Planar scintigraphy followed by SPECT imaging was performed after 2, 4, 6 and 24 h. An additional cohort (n = 7) was injected with 165±29 MBq (injected protein mass 250 µg) and imaging was performed after 2 h only.RESULTS: Injections of 99mTc-ADAPT6 at all injected mass levels were well tolerated and not associated with adverse effects. 99mTc-ADAPT6 cleared rapidly from blood and most other tissues. The normal organs with the highest accumulation were kidney, liver and lung. Effective doses were 0.009±0.002 and 0.010±0.003 mSv/MBq for injected protein masses of 500 and 1000 µg, respectively. Injection of 500 µg resulted in excellent discrimination between HER2-positive and HER2-negative tumors already 2 h after injection (tumor-to-contralateral breast ratio was 37±19 vs 5±2, p<0.01). The tumor-to-contralateral breast ratios for HER2-positive tumors were significantly (p<0.05) higher for injected mass of 500 µg than for both 250 and 1000 µg.CONCLUSION: Injections of 99mTc-ADAPT6 are safe and associated with low absorbed and effective doses. Protein dose of 500 µg is preferable for discrimination between tumors with high and low expression of HER2. Further studies are justified to evaluate if 99mTc-ADAPT6 can be used as an imaging probe for stratification of patients for HER2-targeting therapy in the areas where PET imaging is not readily available.
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| 5. |
- Abouzayed, Ayman, et al.
(författare)
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The GRPR Antagonist [Tc-99m]Tc-maSSS-PEG(2)-RM26 towards Phase I Clinical Trial : Kit Preparation, Characterization and Toxicity
- 2023
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Ingår i: Diagnostics. - : MDPI AG. - 2075-4418. ; 13:9, s. 1611-
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Tidskriftsartikel (refereegranskat)abstract
- Gastrin-releasing peptide receptors (GRPRs) are overexpressed in the majority of primary prostate tumors and in prostatic lymph node and bone metastases. Several GRPR antagonists were developed for SPECT and PET imaging of prostate cancer. We previously reported a preclinical evaluation of the GRPR antagonist [Tc-99m]Tc-maSSS-PEG2-RM26 (based on [D-Phe(6), Sta(13), Leu(14)-NH2]BBN(6-14)) which bound to GRPR with high affinity and had a favorable biodistribution profile in tumor-bearing animal models. In this study, we aimed to prepare and test kits for prospective use in an early-phase clinical study. The kits were prepared to allow for a one-pot single-step radiolabeling with technetium-99m pertechnetate. The kit vials were tested for sterility and labeling efficacy. The radiolabeled by using the kit GRPR antagonist was evaluated in vitro for binding specificity to GRPR on PC-3 cells (GRPR-positive). In vivo, the toxicity of the kit constituents was evaluated in rats. The labeling efficacy of the kits stored at 4 degrees C was monitored for 18 months. The biological properties of [Tc-99m]Tc-maSSS-PEG2-RM26, which were obtained after this period, were examined both in vitro and in vivo. The one-pot (gluconic acid, ethylenediaminetetraacetic acid, stannous chloride, and maSSS-PEG(2)-RM26) single-step radiolabeling with technetium-99m was successful with high radiochemical yields (>97%) and high molar activities (16-24 MBq/nmol). The radiolabeled peptide maintained its binding properties to GRPR. The kit constituents were sterile and non-toxic when tested in living subjects. In conclusion, the prepared kit is considered safe in animal models and can be further evaluated for use in clinics.
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| 6. |
- Garousi, Javad, et al.
(författare)
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Experimental HER2-Targeted Therapy Using ADAPT6-ABD-mcDM1 in Mice Bearing SKOV3 Ovarian Cancer Xenografts : Efficacy and Selection of Companion Imaging Counterpart
- 2022
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 14:8
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cancer is exploited for targeted therapy using monoclonal antibodies and antibody-drug conjugates. Small engineered scaffold proteins, such as the albumin binding domain (ABD) derived affinity proteins (ADAPTs), are a promising new format of targeting probes for development of drug conjugates with well-defined structure and tunable pharmacokinetics. Radiolabeled ADAPT6 has shown excellent tumor-targeting properties in clinical trials. Recently, we developed a drug conjugate based on the HER2-targeting ADAPT6 fused to an albumin binding domain (ABD) for increased bioavailability and conjugated to DM1 for cytotoxic action, designated as ADAPT6-ABD-mcDM1. In this study, we investigated the therapeutic efficacy of this conjugate in mice bearing HER2-expressing SKOV3 ovarian cancer xenografts. A secondary aim was to evaluate several formats of imaging probes for visualization of HER2 expression in tumors. Administration of ADAPT6-ABD-mcDM1 provided a significant delay of tumor growth and increased the median survival of the mice, in comparison with both a non-targeting homologous construct (ADAPT(Neg)-ABD-mcDM1) and the vehicle-treated groups, without inducing toxicity to liver or kidneys. Moreover, the evaluation of imaging probes showed that small scaffold proteins, such as Tc-99m(CO)(3)-ADAPT6 or the affibody molecule Tc-99m-Z(HER2:41071), are well suited as diagnostic companions for potential stratification of patients for ADAPT6-ABD-mcDM1-based therapy.
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| 7. |
- Garousi, Javad, et al.
(författare)
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Radionuclide therapy using ABD-fused ADAPT scaffold protein : Proof of Principle
- 2021
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Ingår i: Biomaterials. - : Elsevier. - 0142-9612 .- 1878-5905. ; 266
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Tidskriftsartikel (refereegranskat)abstract
- Molecular recognition in targeted therapeutics is typically based on immunoglobulins. Development of engineered scaffold proteins (ESPs) has provided additional opportunities for the development of targeted therapies. ESPs offer inexpensive production in prokaryotic hosts, high stability and convenient approaches to modify their biodistribution. In this study, we demonstrated successful modification of the biodistribution of an ESP known as ADAPT (Albumin-binding domain Derived Affinity ProTein). ADAPTs are selected from a library based on the scaffold of ABD (Albumin Binding Domain) of protein G. A particular ADAPT, the ADAPT6, binds to human epidermal growth factor receptor type 2 (HER2) with high affinity. Preclinical and early clinical studies have demonstrated that radiolabeled ADAPT6 can image HER2-expression in tumors with high contrast. However, its rapid glomerular filtration and high renal reabsorption have prevented its use in radionuclide therapy. To modify the biodistribution, ADAPT6 was genetically fused to an ABD. The non-covalent binding to the host's albumin resulted in a 14-fold reduction of renal uptake and appreciable increase of tumor uptake for the best variant, 177Lu-DOTA-ADAPT6-ABD035. Experimental therapy in mice bearing HER2-expressing xenografts demonstrated more than two-fold increase of median survival even after a single injection of 18 MBq 177Lu-DOTA-ADAPT6-ABD035. Thus, a fusion with ABD and optimization of the molecular design provides ADAPT derivatives with attractive targeting properties for radionuclide therapy.
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| 8. |
- Garousi, Javad, et al.
(författare)
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Targeting HER2 Expressing Tumors with a Potent Drug Conjugate Based on an Albumin Binding Domain-Derived Affinity Protein
- 2021
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 13:11, s. 1847-
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Tidskriftsartikel (refereegranskat)abstract
- Albumin binding domain derived affinity proteins (ADAPTs) are a class of small and folded engineered scaffold proteins that holds great promise for targeting cancer tumors. Here, we have extended the in vivo half-life of an ADAPT, targeting the human epidermal growth factor receptor 2 (HER2) by fusion with an albumin binding domain (ABD), and armed it with the highly cytotoxic payload mertansine (DM1) for an investigation of its properties in vitro and in vivo. The resulting drug conjugate, ADAPT6-ABD-mcDM1, retained binding to its intended targets, namely HER2 and serum albumins. Further, it was able to specifically bind to cells with high HER2 expression, get internalized, and showed potent toxicity, with IC50 values ranging from 5 to 80 nM. Conversely, no toxic effect was found for cells with low HER2 expression. In vivo, ADAPT6-ABD-mcDM1, radiolabeled with Tc-99m, was characterized by low uptake in most normal organs, and the main excretion route was shown to be through the kidneys. The tumor uptake was 5.5% ID/g after 24 h, which was higher than the uptake in all normal organs at this time point except for the kidneys. The uptake in the tumors was blockable by pre-injection of an excess of the monoclonal antibody trastuzumab (having an overlapping epitope on the HER2 receptor). In conclusion, half-life extended drug conjugates based on the ADAPT platform of affinity proteins holds promise for further development towards targeted cancer therapy.
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| 9. |
- Lundmark, Fanny
(författare)
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Design, Synthesis, and Preclinical Evaluation of Peptide-based Radioligands Targeting PSMA and GRPR in Prostate Cancer
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Prostate cancer remains the most frequently diagnosed cancer among men worldwide. Enhanced diagnostic strategies are crucial for better patient management and outcomes, ultimately increasing overall survival. Over the past decade, radionuclide-based molecular imaging has emerged as a significant advancement in prostate cancer management. This technique involves the use of radioligands—molecules labelled with radioactive isotopes—that target specific biomarkers associated with the disease. Two key biomarkers are prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) which are overexpressed in prostate cancer tissues and can be effectively targeted using radioligands. Ideal radioligands exhibit high specificity and affinity for their targets, strong retention in tumour tissues, minimal off-target uptake, rapid clearance from healthy organs, and can be synthesised and labelled efficiently. This thesis comprises four original articles focused on enhancing the diagnostic potential of peptide-based radioligands for imaging prostate cancer by optimising the targeting properties.Papers I and II focus on the development of PSMA/GRPR-targeting heterodimeric radioligands. In particular, Paper I investigate the length and hydrophobicity of the functional linkers in indium-111 labelled radioligands for SPECT imaging. It was shown that compound BQ7812 consisting of a shorter GRPR linker in combination with a more hydrophobic PSMA linker was beneficial for the targeting properties. These findings led to Paper II which covers the preclinical evaluation of BQ7812 labelled with gallium-68 for PET imaging. PET imaging provides higher sensitivity compared to SPECT, increasing the possibility to visualise small metastases. Results confirmed [68Ga]Ga-BQ7812 as a promising radioligand for PET imaging of prostate cancer. Paper III covers a SAR study of PSMA-targeting radioligands focusing on the molecular structure of the functional linker. Modifications of the functional linker have been shown to enhance the targeting properties significantly. Radioligand BQ7859 consisting of a 2-naphthyl-L-alanine-L-tyrosine linker, demonstrated improved tumour retention and increased tumour-to-blood ratio. It was concluded that 2-naphthyl-L-alanine was crucial for high affinity while the subsequent linker position was more acceptable for structural changes and could be used for optimising targeting properties. The last study, Paper IV, explores the development of a GRPR-targeting radioligand labelled with fluorine-18 for PET imaging. Fluorine-18 exhibits favourable radionuclide properties well-suited peptide-based radioligands. This study demonstrates the efficient labelling of GRPR-targeting radioligands using the Tz-TCO IEDDA click chemistry approach. This method occurs at physiological pH and room temperature without the need for further purification of the final product, which is favourable compared to other labelling techniques. [18F]F-MeTz-PEG2-RM26 demonstrated specific binding to the target in vivo with stable retention in GRPR-targeted organs and tumours, leading to increased tumour-to-organ ratios over time.In conclusion, the papers included in this thesis demonstrate that careful optimisation of the functional linkers in PSMA and GRPR-targeting radioligands could lead to improved targeting properties for diagnostic imaging of prostate cancer.
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