| 471. |
- Winberg, Karl Johan, et al.
(författare)
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Radiobromination of anti-HER2/neu/ErbB-2 monoclonal antibody using the p-isothiocyanatobenzene derivative of the [76Br]undecahydro-bromo-7,8-dicarba-nido-undecaborate(1-) ion
- 2004
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Ingår i: Nuclear Medicine and Biology. - : Elsevier BV. - 0969-8051 .- 1872-9614. ; 31:4, s. 425-33
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Tidskriftsartikel (refereegranskat)abstract
- The monoclonal humanized anti-HER2 antibody trastuzumab was radiolabeled with the positron emitter (76)Br (T(1/2) =16.2 h). Indirect labeling was performed using the p-isothiocyanatobenzene derivative of the [(76)Br]undecahydro-bromo-7,8-dicarba-nido-undecaborate(1-) ((76)Br-NBI) as a precursor molecule. (76)Br-NBI was prepared by bromination of the 7-(p-isothiocyanato-phenyl)dodecahydro-7,8-dicarba-nido-undecaborate(1-) ion (NBI) with a yield of 93-95% using Chloramine-T (CAT) as an oxidant. Coupling of radiobrominated NBI to antibody was performed without intermediate purification, in an "one pot" reaction. An overall labeling yield of 55.7 +/- 4.8% (mean +/- maximum error) was achieved when 300 microg of antibody was labeled. The label was stable in vitro in physiological and denaturing conditions. In a cell binding test, trastuzumab remained immunoreactive after labeling.
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| 472. |
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| 473. |
- Wållberg, Helena, et al.
(författare)
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Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand
- 2011
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 76:1, s. 127-135
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new beta-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (Tc-99m)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.
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| 474. |
- Wållberg, H, et al.
(författare)
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Engineering of C-terminal cysteine-containing peptide-based chelators improves biodistribution of HER2-targeting 99mTc-labeled Affibody molecules
- 2011
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Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 52:3, s. 461-469
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are a recently developed class of targeting proteins based on a nonimmunoglobulin scaffold. The small size (7 kDa) and subnanomolar affinity of Affibody molecules enables high-contrast imaging of tumor-associated molecular targets, particularly human epidermal growth factor receptor type 2 (HER2). 99mTc as a label offers advantages in clinical practice, and earlier studies demonstrated that 99mTc-labeled recombinant Affibody molecules with a C-terminal cysteine could be used for HER2 imaging. However, the renal retention of radioactivity exceeded tumor uptake, which might complicate imaging of metastases in the lumbar region. The aim of this study was to develop an agent with low renal uptake and preserved tumor targeting. Methods: A series of recombinant derivatives of the HER2-binding ZHER2:342 Affibody molecule with a C-terminal chelating sequence, –GXXC (X denoting glycine, serine, lysine, or glutamate), was designed. The constructs were labeled with 99mTc and evaluated in vitro and in vivo. Results: All variants were stably labeled with 99mTc, with preserved capacity to bind specifically to HER2-expressing cells in vitro and in vivo. The composition of the chelating sequence had a clear influence on the cellular processing and biodistribution properties of the Affibody molecules. The best variant, 99mTc-ZHER2:V2, with the C-terminal chelating sequence –GGGC, provided the lowest radioactivity retention in all normal organs and tissues including the kidneys. 99mTc-ZHER2:V2 displayed high uptake of radioactivity in HER2-expressing xenografts, 22.6 ± 4.0 and 7.7 ± 1.5 percentage injected activity per gram of tissue at 4 h after injection in SKOV-3 (high HER2 expression) and DU-145 (low HER2 expression) tumors, respectively. In both models, the tumor uptake exceeded the renal uptake. Conclusion: These results demonstrate that the biodistribution properties of recombinant 99mTc-labeled Affibody molecules can be optimized by modification of the C-terminal cysteine-containing chelating sequence. 99mTc-ZHER2:V2 is a promising candidate for further development as a diagnostic radiopharmaceutical for imaging of HER2-expressing tumors. These results may be useful for the development of imaging agents based on other Affibody molecules and, hopefully, other scaffolds.
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| 475. |
- Wållberg, Helena, et al.
(författare)
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Molecular Design and Optimization of Tc-99m-Labeled Recombinant Affibody Molecules Improves Their Biodistribution and Imaging Properties
- 2011
-
Ingår i: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 52:3, s. 461-469
-
Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are a recently developed class of targeting proteins based on a nonimmunoglobulin scaffold. The small size (7 kDa) and subnanomolar affinity of Affibody molecules enables high-contrast imaging of tumor-associated molecular targets, particularly human epidermal growth factor receptor type 2 (HER2). Tc-99m as a label offers advantages in clinical practice, and earlier studies demonstrated that Tc-99m-labeled recombinant Affibody molecules with a C-terminal cysteine could be used for HER2 imaging. However, the renal retention of radioactivity exceeded tumor uptake, which might complicate imaging of metastases in the lumbar region. The aim of this study was to develop an agent with low renal uptake and preserved tumor targeting. Methods: A series of recombinant derivatives of the HER2-binding Z(HER2:342) Affibody molecule with a C-terminal chelating sequence, -GXXC (X denoting glycine, serine, lysine, or glutamate), was designed. The constructs were labeled with Tc-99m and evaluated in vitro and in vivo. Results: All variants were stably labeled with Tc-99m, with preserved capacity to bind specifically to HER2-expressing cells in vitro and in vivo. The composition of the chelating sequence had a clear influence on the cellular processing and biodistribution properties of the Affibody molecules. The best variant, Tc-99m-Z(HER2:V2), with the C-terminal chelating sequence -GGGC, provided the lowest radioactivity retention in all normal organs and tissues including the kidneys. Tc-99m-Z(HER2:V2) displayed high uptake of radioactivity in HER2-expressing xenografts, 22.6 +/- 4.0 and 7.7 +/- 1.5 percentage injected activity per gram of tissue at 4 h after injection in SKOV-3 (high HER2 expression) and DU-145 (low HER2 expression) tumors, respectively. In both models, the tumor uptake exceeded the renal uptake. Conclusion: These results demonstrate that the biodistribution properties of recombinant Tc-99m-labeled Affibody molecules can be optimized by modification of the C-terminal cysteine-containing chelating sequence. Tc-99m-Z(HER2:V2) is a promising candidate for further development as a diagnostic radiopharmaceutical for imaging of HER2-expressing tumors. These results may be useful for the development of imaging agents based on other Affibody molecules and, hopefully, other scaffolds.
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| 476. |
- Xu, Tianqi, et al.
(författare)
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Drug Conjugates Based on a Monovalent Affibody Targeting Vector Can Efficiently Eradicate HER2 Positive Human Tumors in an Experimental Mouse Model
- 2021
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Ingår i: Cancers. - : MDPI. - 2072-6694. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 477. |
- Xu, Tianqi, et al.
(författare)
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Effect of Inter-Domain Linker Composition on Biodistribution of ABD-Fused Affibody-Drug Conjugates Targeting HER2
- 2022
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923 .- 1999-4923. ; 14:3, s. 522-
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Tidskriftsartikel (refereegranskat)abstract
- Targeted drug conjugates based on Affibody molecules fused to an albumin-binding domain (ABD) for half-life extension have demonstrated potent anti-tumor activity in preclinical therapeutic studies. Furthermore, optimization of their molecular design might increase the cytotoxic effect on tumors and minimize systemic toxicity. This study aimed to investigate the influence of length and composition of a linker between the human epidermal growth factor receptor 2 (HER2)-targeted affibody molecule (Z(HER2:2891)) and the ABD domain on functionality and biodistribution of affibody-drug conjugates containing a microtubulin inhibitor mertansin (mcDM1) (AffiDCs). Two conjugates, having a trimeric (S(3)G)(3) linker or a trimeric (G(3)S)(3) linker were produced, radiolabeled with Tc-99m(CO)(3), and compared side-by-side in vitro and in vivo with the original Z(HER2:2891)-G(4)S-ABD-mcDM1 conjugate having a monomeric G(4)S linker. Both conjugates with longer linkers had a decreased affinity to HER2 and mouse and human serum albumin in vitro, however, no differences in blood retention were observed in NMRI mice up to 24 h post injection. The use of both (S(3)G)(3) and (G(3)S)(3) linkers reduced liver uptake of AffiDCs by approximately 1.2-fold compared with the use of a G(4)S linker. This finding provides important insights into the molecular design for the development of targeted drug conjugates with reduced hepatic uptake.
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| 478. |
- Xu, Tianqi, et al.
(författare)
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Epithelial cell adhesion molecule-targeting designed ankyrin repeat protein-toxin fusion Ec1-LoPE exhibits potent cytotoxic action in prostate cancer cells
- 2022
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Ingår i: Oncology Reports. - : Spandidos Publications. - 1021-335X .- 1791-2431. ; 47:5
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Tidskriftsartikel (refereegranskat)abstract
- Targeted anticancer therapeutics offer the advantage of reducing cytotoxic side effects to normal cells by directing the cytotoxic payload selectively to cancer cells. Designed ankyrin repeat proteins (DARPins) are promising non-immunoglobulin-based scaffold proteins for payload delivery to cancer-associated molecular targets. Epithelial cell adhesion molecule (EpCAM) is overexpressed in 40-60% of prostate cancers (PCs) and is associated with metastasis, increased risk of PC recurrence and resistance to treatment. Here, we investigated the use of DARPin Ec1 for targeted delivery of Pseudomonas exotoxin A variant (LoPE) with low immunogenicity and low non-specific toxicity to EpCAM-expressing prostate cancer cells. Ec1-LoPE fusion protein was radiolabeled with tricarbonyl technetium-99m and its binding specificity, binding kinetics, cellular processing, internalization and cytotoxicity were evaluated in PC-3 and DU145 cell lines. Ec1-LoPE showed EpCAM-specific binding to EpCAM-expressing prostate cancer cells. Rapid internalization mediated potent cytotoxic effect with picomolar IC50 values in both studied cell lines. Taken together, these data support further evaluation of Ec1-LoPE in a therapeutic setting in a prostate cancer model in vivo.
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| 479. |
- Xu, Tianqi, et al.
(författare)
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Feasibility of Co-Targeting HER3 and EpCAM Using Seribantumab and DARPin-Toxin Fusion in a Pancreatic Cancer Xenograft Model
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 24:3
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Tidskriftsartikel (refereegranskat)abstract
- Pancreatic cancer (PC) is one of the most aggressive malignancies. A combination of targeted therapies could increase the therapeutic efficacy in tumors with heterogeneous target expression. Overexpression of the human epidermal growth factor receptor type 3 (HER3) and the epithelial cell adhesion molecule (EpCAM) in up to 40% and 30% of PCs, respectively, is associated with poor prognosis and highlights the relevance of these targets. Designed ankyrin repeat protein (DARPin) Ec1 fused with the low immunogenic bacterial toxin LoPE provides specific and potent cytotoxicity against EpCAM-expressing cancer cells. Here, we investigated whether the co-targeting of HER3 using the monoclonal antibody seribantumab (MM-121) and of EpCAM using Ec1-LoPE would improve the therapeutic efficacy in comparison to the individual agents. Radiolabeled Tc-99m(CO)(3)-Ec1-LoPE showed specific binding with rapid internalization in EpCAM-expressing PC cells. MM-121 did not interfere with the binding of Ec1-LoPE to EpCAM. Evaluation of cytotoxicity indicated synergism between Ec1-LoPE and MM-121 in vitro. An experimental therapy study using Ec1-LoPE and MM-121 in mice bearing EpCAM- and HER3-expressing BxPC3 xenografts demonstrated the feasibility of the therapy. Further development of the co-targeting approach using HER3 and EpCAM could therefore be justified.
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| 480. |
- Xu, Tianqi, et al.
(författare)
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Imaging-Guided Therapy Simultaneously Targeting HER2 and EpCAM with Trastuzumab and EpCAM-Directed Toxin Provides Additive Effect in Ovarian Cancer Model
- 2021
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:16
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Targeted therapeutics provide cytostatic or cytotoxic action selectively to tumor cells while reducing the toxicity to normal cells. Targeting two molecular receptors overexpressed on tumor cells is a way to overcome heterogeneity of expression and improve therapeutic efficacy. Combining drugs with different modes of action might also increase the cytotoxic effect and decrease the chance for the cancer cells to develop resistance to treatment. In this work, we investigated a combination of the clinically used monoclonal antibody trastuzumab with a potent targeting protein-toxin fusion, directed at two different targets present in a large fraction of ovarian cancers. Co-targeted treatment provided a significant reduction in tumor growth and extended the survival of mice compared with the control and monotherapy groups. Our findings support further development of targeted combination therapies for treatment of aggressive and resistant cancer types. Efficient treatment of disseminated ovarian cancer (OC) is challenging due to its heterogeneity and chemoresistance. Overexpression of human epidermal growth factor receptor 2 (HER2) and epithelial cell adhesion molecule (EpCAM) in approx. 30% and 70% of ovarian cancers, respectively, allows for co-targeted treatment. The clinical efficacy of the monoclonal antibody trastuzumab in patients with HER2-positive breast, gastric and gastroesophageal cancers makes it readily available as the HER2-targeting component. As the EpCAM-targeting component, we investigated the designed ankyrin repeat protein (DARPin) Ec1 fused to a truncated variant of Pseudomonas exotoxin A with reduced immunogenicity and low general toxicity (LoPE). Ec1-LoPE was radiolabeled, evaluated in ovarian cancer cells in vitro and its biodistribution and tumor-targeting properties were studied in vivo. The therapeutic efficacy of Ec1-LoPE alone and in combination with trastuzumab was studied in mice bearing EpCAM- and HER2-expressing SKOV3 xenografts. SPECT/CT imaging enabled visualization of EpCAM and HER2 expression in the tumors. Co-treatment using Ec1-LoPE and trastuzumab was more effective at reducing tumor growth and prolonged the median survival of mice compared with mice in the control and monotherapy groups. Repeated administration of Ec1-LoPE was well tolerated without signs of hepatic or kidney toxicity. Co-treatment with trastuzumab and Ec1-LoPE might be a potential therapeutic strategy for HER2- and EpCAM-positive OC.
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