| 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. |
- 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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| 3. |
- Bragina, Olga, et al.
(författare)
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Direct Intra-Patient Comparison of Scaffold Protein-Based Tracers, [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3, for Imaging of HER2-Positive Breast Cancer
- 2023
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 15:12
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary The receptor HER2 is overexpressed in some breast cancers. Tumours with a high HER2 expression can be successfully treated with the antibodies trastuzumab and pertuzumab. The radionuclide imaging of HER2 in disseminated cancer could help to select patients for treatment using these antibodies. Novel radiolabelled small-sized tracers, scaffold proteins, have shown excellent imaging properties in preclinical studies. The scaffold proteins [Tc-99m]Tc-ADAPT6 and DARPin [Tc-99m]Tc-(HE)(3)-G3 have been found to be safe in Phase I clinical trials. They showed promising results in the imaging of HER2. In this study, we compared the distribution of both tracers in the same patients with breast cancer to evaluate whether one of them has any decisive advantage. We found that both tracers provide an excellent visualization of tumours, but the accumulation of [Tc-99m]Tc-ADAPT6 in tumours is higher. The data from this study are essential for researchers developing imaging agents. Previous Phase I clinical evaluations of the radiolabelled scaffold proteins [Tc-99m]Tc-ADAPT6 and DARPin [Tc-99m]Tc-(HE)(3)-G3 in breast cancer patients have demonstrated their safety and indicated their capability to discriminate between HER2-positive and HER2-negative tumours. The objective of this study was to compare the imaging of HER2-positive tumours in the same patients using [Tc-99m]Tc-ADAPT6 and [Tc-99m]Tc-(HE)(3)-G3. Eleven treatment-naive female patients (26-65 years) with HER2-positive primary and metastatic breast cancer were included in the study. Each patient was intravenously injected with [Tc-99m]Tc-ADAPT6, followed by an [Tc-99m]Tc-(HE)(3)-G3 injection 3-4 days later and chest SPECT/CT was performed. All primary tumours were clearly visualized using both tracers. The uptake of [Tc-99m]Tc-ADAPT6 in primary tumours (SUVmax = 4.7 & PLUSMN; 2.1) was significantly higher (p < 0.005) than the uptake of [Tc-99m]Tc-(HE)(3)-G3 (SUVmax = 3.5 & PLUSMN; 1.7). There was no significant difference in primary tumour-to-contralateral site values for [Tc-99m]Tc-ADAPT6 (15.2 & PLUSMN; 7.4) and [Tc-99m]Tc-(HE)(3)-G3 (19.6 & PLUSMN; 12.4). All known lymph node metastases were visualized using both tracers. The uptake of [Tc-99m]Tc-ADAPT6 in all extrahepatic soft tissue lesions was significantly (p < 0.0004) higher than the uptake of [Tc-99m]Tc-(HE)(3)-G3. In conclusion, [Tc-99m]Tc-ADAPT6 and [Tc-99m]Tc-(HE)(3)-G3 are suitable for the visualization of HER2-positive breast cancer. At the selected time points, [Tc-99m]Tc-ADAPT6 has a significantly higher uptake in soft tissue lesions, which might be an advantage for the visualization of small metastases.
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| 4. |
- Bragina, Olga, et al.
(författare)
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Evaluation of Approaches for the Assessment of HER2 Expression in Breast Cancer by Radionuclide Imaging Using the Scaffold Protein [99mTc]Tc-ADAPT6
- 2024
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 16:4
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Tidskriftsartikel (refereegranskat)abstract
- Due to its small size and high affinity binding, the engineered scaffold protein ADAPT6 is a promising targeting probe for radionuclide imaging of human epidermal growth factor receptor type 2 (HER2). In a Phase I clinical trial, [Tc-99m]Tc-ADAPT6 demonstrated safety, tolerability and capacity to visualize HER2 expression in primary breast cancer. In this study, we aimed to select the optimal parameters for distinguishing between breast cancers with high and low expression of HER2 using [Tc-99m]Tc-ADAPT6 in a planned Phase II study. HER2 expression was evaluated in primary tumours and metastatic axillary lymph nodes (mALNs). SPECT/CT imaging of twenty treatment-naive breast cancer patients was performed 2 h after injection of [Tc-99m]Tc-ADAPT6. The imaging data were compared with the data concerning HER2 expression obtained by immunohistochemical evaluation of samples obtained by core biopsy. Maximum Standard Uptake Values (SUVmax) afforded the best performance for both primary tumours and mALNs (areas under the receiver operating characteristic curve (ROC AUC) of 1.0 and 0.97, respectively). Lesion-to-spleen ratios provided somewhat lower performance. However, the ROC AUCs were still over 0.90 for both primary tumours and mALNs. Thus, lesion-to-spleen ratios should be further evaluated to find if these could be applied to imaging using stand-alone SPECT cameras that do not permit SUV calculations.
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| 5. |
- 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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| 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. |
- Oroujeni, Maryam, PhD, 1982-
(författare)
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Radiolabelled affibody molecules for imaging EGFR expression in tumours
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Affibody molecules are promising scaffold-based targeting proteins for radionuclide imaging and cancer therapy. This thesis is based on 5 original research articles (Papers I-V), with the primary focus being placed on the optimization of molecular design of EGFR-binding affibody variants for high contrast imaging of epidermal growth factor receptor (EGFR) expression in tumours. The goal of my studies was to investigate the effect of labelling chemistry on the targeting properties of the anti-EGFR affibody molecule ZEGFR:2377 labelled with technetium-99m (99mTc) for single-photon emission computed tomography (SPECT), gallium-68 (68Ga), zirconium-89 (89Zr) and gallium-66 (66Ga) for positron-emission tomography (PET) to select radiolabelled variants providing the best imaging contrast.In Paper I, we showed the feasibility of stably labelling the anti-EGFR affibody molecule ZEGFR:2377 with 99mTc using a peptide-based cysteine-containing chelator and evaluated the imaging of EGFR expression in tumours using the [99mTc]Tc-ZEGFR:2377 affibody molecule.In Paper II, the effect of the composition of cysteine-containing peptide-based chelators on the biodistribution of 99mTc-labelled anti-EGFR affibody molecules was investigated. We evaluated whether the use of glutamate-based chelators improved the imaging properties of 99mTc-labelled ZEGFR:2377.In Paper III, the use of cyclic (FSC) versus noncyclic chelators (DFO) as bifunctional chelators for radiolabelling the anti-EGFR affibody molecule ZEGFR:2377 with 89Zr was investigated. The in vitro and in vivo properties of the resulting DFO- and FSC-ZEGFR:2377 molecules labelled with 89Zr were studied.In Paper IV, the targeting properties of [68Ga]Ga-DFO-ZEGFR:2377 were evaluated and compared directly with the properties of [89Zr]Zr-DFO-ZEGFR:2377 at 3 h after injection.In Paper V, the targeting properties of [66Ga]Ga-DFO-ZEGFR:2377 were evaluated and compared directly with the properties of [68Ga]Ga-DFO-ZEGFR:2377 and [89Zr]Zr-DFO-ZEGFR:2377 at 3 and 24 h after injection, respectively.
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| 10. |
- Tolmachev, Vladimir, et al.
(författare)
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Direct In Vivo Comparison of Tc-99m-Labeled Scaffold Proteins, DARPin G3 and ADAPT6, for Visualization of HER2 Expression and Monitoring of Early Response for Trastuzumab Therapy
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:23
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Tidskriftsartikel (refereegranskat)abstract
- Non-invasive radionuclide molecular visualization of human epidermal growth factor receptor type 2 (HER2) can provide stratification of patients for HER2-targeting therapy. This method can also enable monitoring of the response to such therapies, thereby making treatment personalized and more efficient. Clinical evaluation in a phase I study demonstrated that injections of two scaffold protein-based imaging probes, [Tc-99m]Tc-(HE)(3)-G3 and [Tc-99m]Tc-ADAPT6, are safe, well-tolerated and cause a low level of radioactivity in healthy tissue. The goal of this preclinical study was to select the best probe for stratification of patients and response monitoring. Biodistribution of both tracers was compared in mice bearing SKOV-3 xenografts with high HER2 expression or MDA-MB-468 xenografts with very low expression. Changes in accumulation of the probes in SKOV-3 tumors 24 h after injection of trastuzumab were evaluated. Both [Tc-99m]Tc-ADAPT6 and [Tc-99m]Tc-(HE)(3)-G3 permitted high contrast imaging of HER2-expressing tumors and a clear discrimination between tumors with high and low HER2 expression. However, [Tc-99m]Tc-ADAPT6 has better preconditions for higher sensitivity and specificity of stratification. On the other hand, [Tc-99m]Tc-(HE)(3)-G3 is capable of detecting the decrease of HER2 expression on response to trastuzumab therapy only 24 h after injection of the loading dose. This indicates that the [Tc-99m]Tc-(HE)(3)-G3 tracer would be better for monitoring early response to such treatment. The results of this study should be considered in planning of further clinical development of HER2 imaging probes.
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