| 1. |
- Deyev, Sergey M., et al.
(författare)
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Influence of the Position and Composition of Radiometals and Radioiodine Labels on Imaging of Epcam Expression in Prostate Cancer Model Using the DARPin Ec1
- 2021
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:14
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Ding, Haozhong, et al.
(författare)
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Affibody-Derived Drug Conjugates Targeting HER2 : Effect of Drug Load on Cytotoxicity and Biodistribution
- 2021
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923 .- 1999-4923. ; 13:3
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Ding, Haozhong, et al.
(författare)
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HER2-Specific Pseudomonas Exotoxin A PE25 Based Fusions : Influence of Targeting Domain on Target Binding, Toxicity, and In Vivo Biodistribution
- 2020
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923 .- 1999-4923. ; 12:4
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Tidskriftsartikel (refereegranskat)abstract
- The human epidermal growth factor receptor 2 (HER2) is a clinically validated target for cancer therapy, and targeted therapies are often used in regimens for patients with a high HER2 expression level. Despite the success of current drugs, a number of patients succumb to their disease, which motivates development of novel drugs with other modes of action. We have previously shown that an albumin binding domain-derived affinity protein with specific affinity for HER2, ADAPT(6), can be used to deliver the highly cytotoxic protein domain PE25, a derivative of Pseudomonas exotoxin A, to HER2 overexpressing malignant cells, leading to potent and specific cell killing. In this study we expanded the investigation for an optimal targeting domain and constructed two fusion toxins where a HER2-binding affibody molecule, Z(HER2:2891), or the dual-HER2-binding hybrid Z(HER2:2891)-ADAPT(6) were used for cancer cell targeting. We found that both targeting domains conferred strong binding to HER2; both to the purified extracellular domain and to the HER2 overexpressing cell line SKOV3. This resulted in fusion toxins with high cytotoxic potency toward cell lines with high expression levels of HER2, with EC50 values between 10 and 100 pM. For extension of the plasma half-life, an albumin binding domain was also included. Intravenous injection of the fusion toxins into mice showed a profound influence of the targeting domain on biodistribution. Compared to previous results, with ADAPT(6) as targeting domain, Z(HER2:2891) gave rise to further extension of the plasma half-life and also shifted the clearance route of the fusion toxin from the liver to the kidneys. Collectively, the results show that the targeting domain has a major impact on uptake of PE25-based fusion toxins in different organs. The results also show that PE25-based fusion toxins with high affinity to HER2 do not necessarily increase the cytotoxicity beyond a certain point in affinity. In conclusion, Z(HER2:2891) has the most favorable characteristics as targeting domain for PE25.
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| 4. |
- 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 .- 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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| 5. |
- 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 .- 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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| 6. |
- Leitao, Charles Dahlsson, et al.
(författare)
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Conditionally activated affibody-based prodrug targeting EGFR demonstrates improved tumor selectivity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Safety and efficacy of cancer-targeting treatments can be improved by conditional activation conferred by the distinct milieu of the tumour microenvironment. Proteases are intricately involved in tumorigenesis and commonly dysregulated with elevated expression and activity. Design of prodrug molecules with protease-dependent activation has the potential to increase tumor-selective targeting, while decreasing the exposure to healthy tissues, thus improving safety, allowing for administration of higher doses or use of more aggressive treatment options, leading to higher therapeutic efficacy. We have previously performed in vitro characterizations of an affibody-based prodrug approach for protease-mediated targeting of EGFR. In this study we demonstrate the potential for selective tumor-targeting and shielded uptake in healthy tissues in vivo using tumor-bearing mice for an EGFR-targeting affibody prodrug.
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| 7. |
- Leitao, Charles Dahlsson, 1992-, et al.
(författare)
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Conditionally activated affibody-based prodrug targeting EGFR demonstrates improved tumour selectivity
- 2023
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 357, s. 185-195
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Tidskriftsartikel (refereegranskat)abstract
- Safety and efficacy of cancer-targeting treatments can be improved by conditional activation enabled by the distinct milieu of the tumour microenvironment. Proteases are intricately involved in tumourigenesis and commonly dysregulated with elevated expression and activity. Design of prodrug molecules with protease -dependent activation has the potential to increase tumour-selective targeting while decreasing exposure to healthy tissues, thus improving the safety profile for patients. Higher selectivity could also allow for adminis-tration of higher doses or use of more aggressive treatment options, leading to higher therapeutic efficacy. We have previously developed an affibody-based prodrug with conditional targeting of EGFR conferred by an anti-idiotypic affibody masking domain (ZB05). We could show that binding to endogenous EGFR on cancer cells in vitro was restored following proteolytic removal of ZB05. In this study we evaluate a novel affibody-based pro -drug design, which incorporates a protease substrate sequence recognized by cancer-associated proteases and demonstrate the potential of this approach for selective tumour-targeting and shielded uptake in healthy tissues in vivo using tumour-bearing mice. This may widen the therapeutic index of cytotoxic EGFR-targeted thera-peutics by decreasing side effects, improving selectivity of drug delivery, and enabling the use of more potent cytotoxic drugs.
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| 8. |
- Liu, Yongsheng, et al.
(författare)
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Comparative Preclinical Evaluation of HER2-Targeting ABD-Fused Affibody® Molecules 177Lu-ABY-271 and 177Lu-ABY-027 : Impact of DOTA Position on ABD Domain
- 2021
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923 .- 1999-4923. ; 13:6
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Tidskriftsartikel (refereegranskat)abstract
- Radiolabeled Affibody-based targeting agent 177Lu-ABY-027, a fusion of an anti-HER2 Affibody molecule with albumin binding domain (ABD) site-specifically labeled at the C-terminus, has demonstrated a promising biodistribution profile in mice; binding of the construct to albumin prevents glomerular filtration and significantly reduces renal uptake. In this study, we tested the hypothesis that site-specific positioning of the chelator at helix 1 of ABD, at a maximum distance from the albumin binding site, would further increase the strength of binding to albumin and decrease the renal uptake. The new construct, ABY-271 with DOTA conjugated at the back of ABD, has been labelled with 177Lu. Targeting properties of 177Lu-ABY-271 and 177Lu-ABY-027 were compared directly. 177Lu-ABY-271 specifically accumulated in SKOV-3 xenografts in mice. The tumor uptake of 177Lu-ABY-271 exceeded uptake in any other organ 24 h and later after injection. However, the renal uptake of 177Lu-ABY-271 was two-fold higher than the uptake of 177Lu-ABY-027. Thus, the placement of chelator on helix 1 of ABD does not provide desirable reduction of renal uptake. To conclude, minimal modification of the design of Affibody molecules has a strong effect on biodistribution, which cannot be predicted a priori. This necessitates extensive structure-properties relationship studies to find an optimal design of Affibody-based targeting agents for therapy.
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| 9. |
- Liu, Yongsheng, et al.
(författare)
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Experimental Therapy of HER2-Expressing Xenografts Using the Second-Generation HER2-Targeting Affibody Molecule 188Re-ZHER2:41071
- 2022
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923 .- 1999-4923. ; 14:5
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Tidskriftsartikel (refereegranskat)abstract
- HER2-targeted radionuclide therapy might be helpful for the treatment of breast, gastric, and ovarian cancers which have developed resistance to antibody and antibody-drug conjugate-based therapies despite preserved high HER2-expression. Affibody molecules are small targeting proteins based on a non-immunoglobulin scaffold. The goal of this study was to test in an animal model a hypothesis that the second-generation HER2-targeting Affibody molecule 188Re-ZHER2:41071 might be useful for treatment of HER2-expressing malignant tumors. ZHER2:41071 was efficiently labeled with a beta-emitting radionuclide rhenium-188 (188Re). 188Re-ZHER2:41071 demonstrated preserved specificity and high affinity (KD = 5 ± 3 pM) of binding to HER2-expressing cells. In vivo studies demonstrated rapid washout of 188Re from kidneys. The uptake in HER2-expressing SKOV-3 xenografts was HER2-specific and significantly exceeded the renal uptake 4 h after injection and later. The median survival of mice, which were treated by three injections of 16 MBq 188Re-ZHER2:41071 was 68 days, which was significantly longer (<0.0001 in the log-rank Mantel-Cox test) than survival of mice in the control groups treated with vehicle (29 days) or unlabeled ZHER2:41071 (27.5 days). In conclusion, the experimental radionuclide therapy using 188Re-ZHER2:41071 enabled enhancement of survival of mice with human tumors without toxicity to the kidneys, which is the critical organ.
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| 10. |
- Liu, Yongsheng, et al.
(författare)
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Preclinical Evaluation of a New Format of Ga-68- and In-111-Labeled Affibody Molecule Z(IGF-1R:4551) for the Visualization of IGF-1R Expression in Malignant Tumors Using PET and SPECT
- 2022
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923 .- 1999-4923. ; 14:7
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Tidskriftsartikel (refereegranskat)abstract
- The Insulin-like growth factor-1 receptor (IGF-1R) is a molecular target for several monoclonal antibodies undergoing clinical evaluation as anticancer therapeutics. The non-invasive detection of IGF-1R expression in tumors might enable stratification of patients for specific treatment and improve the outcome of both clinical trials and routine treatment. The affibody molecule Z(IGF-1R:4551) binds specifically to IGF-1R with subnanomolar affinity. The goal of this study was to evaluate the Ga-68 and In-111-labeled affibody construct NODAGA-(HE)(3)-Z(IGF-1R:4551) for the imaging of IGF-1R expression, using PET and SPECT. The labeling was efficient and provided stable coupling of both radionuclides. The two imaging probes, [Ga-68]Ga-NODAGA-(HE)(3)-Z(IGF-1R:4551) and [In-111]In-NODAGA-(HE)(3)-Z(IGF-1R:4551), demonstrated specific binding to IGF-1R-expressing human cancer cell lines in vitro and to IGF-1R-expressing xenografts in mice. Preclinical PET and SPECT/CT imaging demonstrated visualization of IGF-1R-expressing xenografts already one hour after injection. The tumor-to-blood ratios at 3 h after injection were 7.8 +/- 0.2 and 8.0 +/- 0.6 for [Ga-68]Ga-NODAGA-(HE)(3)-Z(IGF-1R:4551) and [In-111]In-NODAGA-(HE)(3)-Z(IGF-1R:4551), respectively. In conclusion, a molecular design of the Z(IGF-1R:4551) affibody molecule, including placement of a (HE)(3)-tag on the N-terminus and site-specific coupling of a NODAGA chelator on the C-terminus, provides a tracer with improved imaging properties for visualization of IGF-1R in malignant tumors, using PET and SPECT.
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