| 1. |
- Yin, Wen, 1993-, et al.
(författare)
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The Influence of Domain Permutations of an Albumin-Binding Domain-Fused HER2-Targeting Affibody-Based Drug Conjugate on Tumor Cell Proliferation and Therapy Efficacy
- 2021
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 13:11, s. 1974-1974
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Tidskriftsartikel (refereegranskat)abstract
- Human epidermal growth factor receptor 2 (HER2) is a clinically validated target for breast cancer therapy. Previously, a drug-fused HER2-targeting affinity protein construct successfully extended the survival of mice bearing HER2-expressing xenografts. The aim of this study was to evaluate the influence of the number and positioning of the protein domains in the drug conjugate. Seven HER2-targeting affibody-based constructs, including one or two affibody molecules (Z) with or without an albumin-binding domain (ABD), namely Z, Z-ABD, ABD-Z, Z-Z, Z-Z-ABD, Z-ABD-Z, and ABD-Z-Z, were evaluated on their effects on cell growth, in vivo targeting, and biodistribution. The biodistribution study demonstrated that the monomeric constructs had longer blood retention and lower hepatic uptake than the dimeric ones. A dimeric construct, specifically ABD-Z-Z, could stimulate the proliferation of HER2 expressing SKOV-3 cells in vitro and the growth of tumors in vivo, whereas the monomeric construct Z-ABD could not. These two constructs demonstrated a therapeutic effect when coupled to mcDM1; however, the effect was more pronounced for the non-stimulating Z-ABD. The median survival of the mice treated with Z-ABD-mcDM1 was 63 days compared to the 37 days for those treated with ABD-Z-Z-mcDM1 or for the control animals. Domain permutation of an ABD-fused HER2-targeting affibody-based drug conjugate significantly influences tumor cell proliferation and therapy efficacy. The monomeric conjugate Z-ABD is the most promising format for targeted delivery of the cytotoxic drug DM1.
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| 2. |
- 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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| 3. |
- Honarvar, Hadis, et al.
(författare)
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Position for site-specific attachment of a DOTA chelator to synthetic affibody molecules has a different influence on the targeting properties of 68Ga-Compared to 111in-labeled conjugates
- 2014
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Ingår i: Molecular Imaging. - : SAGE Publications. - 1535-3508 .- 1536-0121. ; 13:10
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules, small (7 kDa) scaffold proteins, are a promising class of probes for radionuclide molecular imaging. Radiolabeling of Affibody molecules with the positron-emitting nuclide 68Ga would permit the use of positron emission tomography (PET), providing better resolution, sensitivity, and quantification accuracy than single-photon emission computed tomography (SPECT). The synthetic anti-HER2 ZHER2:S1 Affibody molecule was conjugated with DOTA at the N-terminus, in the middle of helix 3, or at the Cterminus. The biodistribution of 68Ga-and 111In-labeled Affibody molecules was directly compared in NMRI nu/nu mice bearing SKOV3 xenografts. The position of the chelator strongly influenced the biodistribution of the tracers, and the influence was more pronounced for 68Ga-labeled Affibody molecules than for the 111In-labeled counterparts. The best 68Ga-labeled variant was 68Ga-[DOTA-A1]-ZHER2:S1, which provided a tumor uptake of 13 ± 1 %ID/g and a tumor to blood ratio of 39 ± 12 at 2 hours after injection. 111In-[DOTA-A1]-ZHER2:S1 and 111In-[DOTA-K58]-ZHER2:S1 were equally good at this time point, providing a tumor uptake of 15 to 16 %ID/g and a tumor to blood ratio in the range of 60 to 80. In conclusion, the selection of the best position for a chelator in Affibody molecules can be used for optimization of their imaging properties. This may be important for the development of Affibody-based and other protein-based imaging probes.
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| 4. |
- Dahlsson Leitao, Charles, et al.
(författare)
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Molecular Design of HER3-Targeting Affibody Molecules : Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68Ga-Labeled Tracers
- 2019
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 20:5
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Tidskriftsartikel (refereegranskat)abstract
- Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)3-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)3-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)3-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)3-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)3-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.
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| 5. |
- Garousi, Javad, et al.
(författare)
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Radionuclide Therapy Using ABD-fused ADAPT Scaffold Protein: Proof of Principle
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The molecular recognition characteristics of targeted therapeutics is typically attributed to immunoglobulins. However, recent development of engineered scaffold proteins (ESPs) has provided additional opportunities for the improvement of these targeted therapies. ESPs offer inexpensive production in prokaryotic hosts and high molecular stability as well as convenient approaches to modify the biodistribution. In this study, we have demonstrated successful modification of the biodistribution of a particular ESP known as ADAPT (Albumin-binding domain Derived Affinity ProTein). These ADAPTs are generated through screening of combinatorial libraries based on the rigid scaffold of ABD (Albumin Binding Domain) of protein G. As one of these ADAPTs, ADAPT6 binds to human epidermal growth factor 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 re-absorption have prevented its use in radionuclide therapy. To modify the biodistribution of ADAPT6 and allow for a therapeutic use, we present here an ADAPT6 genetically fused to ABD. The non-covalent binding of this fusion protein to the host 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. The injections were not associated with any observable toxicity. 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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| 6. |
- Lindbo, Sarah, et al.
(författare)
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Optimized Molecular Design of ADAPT-Based HER2-Imaging Probes Labeled with 111In and 68Ga
- 2018
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 15:7, s. 2674-2683
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide molecular imaging is a promising tool for visualization of cancer associated molecular abnormalities in vivo and stratification of patients for specific therapies. ADAPT is a new type of small engineered proteins based on the scaffold of an albumin binding domain of protein G. ADAPTs have been utilized to select and develop high affinity binders to different proteinaceous targets. ADAPT6 binds to human epidermal growth factor 2 (HER2) with low nanomolar affinity and can be used for its in vivo visualization. Molecular design of 111In-labeled anti-HER2 ADAPT has been optimized in several earlier studies. In this study, we made a direct comparison of two of the most promising variants, having either a DEAVDANS or a (HE)3DANS sequence at the N-terminus, conjugated with a maleimido derivative of DOTA to a GSSC amino acids sequence at the C-terminus. The variants (designated DOTA-C59-DEAVDANS-ADAPT6-GSSC and DOTA-C61-(HE)3DANS-ADAPT6-GSSC) were stably labeled with 111In for SPECT and 68Ga for PET. Biodistribution of labeled ADAPT variants was evaluated in nude mice bearing human tumor xenografts with different levels of HER2 expression. Both variants enabled clear discrimination between tumors with high and low levels of HER2 expression. 111In-labeled ADAPT6 derivatives provided higher tumor-to-organ ratios compared to 68Ga-labeled counterparts. The best performing variant was DOTA-C61-(HE)3DANS-ADAPT6-GSSC, which provided tumor-to-blood ratios of 208 ± 36 and 109 ± 17 at 3 h for 111In and 68Ga labels, respectively.
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| 7. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(författare)
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Preclinical Evaluation of [Ga-68]Ga-DFO-ZEGFR:2377 : A Promising Affibody-Based Probe for Noninvasive PET Imaging of EGFR Expression in Tumors
- 2018
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Ingår i: Cells. - : MDPI. - 2073-4409. ; 7:9
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Tidskriftsartikel (refereegranskat)abstract
- Radionuclide imaging of epidermal growth factor receptor (EGFR) expression in tumors may stratify patients for EGFR-targeting therapies and predict response or resistance to certain treatments. Affibody molecules, which are nonimmunoglobulin scaffold proteins, have a high potential as probes for molecular imaging. In this study, maleimido derivative of desferrioxamine B (DFO) chelator was site-specifically coupled to the C-terminal cysteine of the anti-EGFR affibody molecule ZEGFR:2377, and the DFO-ZEGFR:2377 conjugate was labeled with the generator-produced positron-emitting radionuclide Ga-68. Stability, specificity of binding to EGFR-expressing cells, and processing of [Ga-68]Ga-DFO-ZEGFR:2377 by cancer cells after binding were evaluated in vitro. In vivo studies were performed in nude mice bearing human EGFR-expressing A431 epidermoid cancer xenografts. The biodistribution of [Ga-68]Ga-DFO-ZEGFR:2377 was directly compared with the biodistribution of [Zr-89]Zr-DFO-ZEGFR:2377. DFO-ZEGFR:2377 was efficiently (isolated yield of 73 +/- 3%) and stably labeled with Ga-68. Binding of [Ga-68]Ga-DFO-ZEGFR:2377 to EGFR-expressing cells in vitro was receptor-specific and proportional to the EGFR expression level. In vivo saturation experiment demonstrated EGFR-specific accumulation of [Ga-68]Ga-DFO-ZEGFR:2377 in A431 xenografts. Compared to [Zr-89]Zr-DFO-ZEGFR:2377, [Ga-68]Ga-DFO-ZEGFR:2377 demonstrated significantly (p < 0.05) higher uptake in tumors and lower uptake in spleen and bones. This resulted in significantly higher tumor-to-organ ratios for [Ga-68]Ga-DFO-ZEGFR:2377. In conclusion, [Ga-68]Ga-DFO-ZEGFR:2377 is a promising probe for imaging of EGFR expression.
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| 8. |
- Rinne, Sara S., et al.
(författare)
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Increase in negative charge of Ga-68/chelator complex reduces unspecific hepatic uptake but does not improve imaging properties of HER3-targeting affibody molecules
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Upregulation of the human epidermal growth factor receptor type 3 (HER3) is a common mechanism to bypass HER-targeted cancer therapy. Affibody-based molecular imaging has the potential for detecting and monitoring HER3 expression during treatment. In this study, we compared the imaging properties of newly generated Ga-68-labeled anti-HER3 affibody molecules (HE)(3)-Z(HER3)-DOTA and (HE)(3)-Z(HER3)-DOTAGA with previously reported [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA. We hypothesized that increasing the negative charge of the gallium-68/chelator complex would reduce hepatic uptake, which could lead to improved contrast of anti-HER3 affibody-based PET-imaging of HER3 expression. (HE)(3)-Z(HER3)-X (X = DOTA, DOTAGA) were produced and labeled with gallium-68. Binding of the new conjugates was specific in HER3 expressing BxPC-3 and DU145 human cancer cells. Biodistribution and in vivo specificity was studied in BxPC-3 xenograft bearing Balb/c nu/nu mice 3 h pi. DOTA- and DOTAGA-containing conjugates had significantly higher concentration in blood than [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA. Presence of the negatively charged Ga-68-DOTAGA complex reduced the unspecific hepatic uptake, but did not improve overall biodistribution of the conjugate. [Ga-68]Ga-(HE)(3)-Z(HER3)-DOTAGA and [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA had similar tumor-to-liver ratios, but [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA had the highest tumor uptake and tumor-to-blood ratio among the tested conjugates. In conclusion, [Ga-68] Ga-(HE)(3)-Z(HER3)-NODAGA remains the favorable variant for PET imaging of HER3 expression.
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| 9. |
- Rinne, Sara S., et al.
(författare)
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Influence of Residualizing Properties of the Radiolabel on Radionuclide Molecular Imaging of HER3 Using Affibody Molecules
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 21:4
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Tidskriftsartikel (refereegranskat)abstract
- Human epidermal growth factor receptor type 3 (HER3) is an emerging therapeutic target in several malignancies. To select potential responders to HER3-targeted therapy, radionuclide molecular imaging of HER3 expression using affibody molecules could be performed. Due to physiological expression of HER3 in normal organs, high imaging contrast remains challenging. Due to slow internalization of affibody molecules by cancer cells, we hypothesized that labeling (HE)(3)-Z(HER3:08698)-DOTAGA affibody molecule with non-residualizing [I-125]-N-succinimidyl-4-iodobenzoate (PIB) label would improve the tumor-to-normal organs ratios compared to previously reported residualizing radiometal labels. The [I-125]I-PIB-(HE)(3)-Z(HER3:08698)-DOTAGA was compared side-by-side with [In-111]In-(HE)(3)-Z(HER3:08698)-DOTAGA. Both conjugates demonstrated specific high-affinity binding to HER3-expressing BxPC-3 and DU145 cancer cells. Biodistribution in mice bearing BxPC-3 xenografts at 4 and 24h pi showed faster clearance of the [I-125]I-PIB label compared to the indium-111 label from most tissues, except blood. This resulted in higher tumor-to-organ ratios in HER3-expressing organs for [I-125]I-PIB-(HE)(3)-Z(HER3:08698)-DOTAGA at 4 h, providing the tumor-to-liver ratio of 2.4 +/- 0.3. The tumor uptake of both conjugates was specific, however, it was lower for the [I-125]I-PIB label. In conclusion, the use of non-residualizing [I-125]I-PIB label for HER3-targeting affibody molecule provided higher tumor-to-liver ratio than the indium-111 label, however, further improvement in tumor uptake and retention is needed.
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| 10. |
- Liu, Hao, et al.
(författare)
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Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A
- 2019
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 55:1, s. 309-319
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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