| 11. |
- Mitran, Bogdan, et al.
(författare)
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Evaluation of Tc-99m-Z(IGF1R:4551)-GGGC affibody molecule, a new probe for imaging of insulin-like growth factor type 1 receptor expression
- 2015
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Ingår i: Amino Acids. - : Springer Science and Business Media LLC. - 0939-4451 .- 1438-2199. ; 47:2, s. 303-315
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression of insulin-like growth factor-1 receptor (IGF-1R) in several cancers is associated with resistance to therapy. Radionuclide molecular imaging of IGF-1R expression in tumors may help in selecting the patients that will potentially respond to IGF-1R-targeted therapy. Affibody molecules are small (7 kDa) non-immunoglobulin-based scaffold proteins that are well-suited probes for radionuclide imaging. The aim of this study was the evaluation of an anti-IGF-1R affibody molecule labeled with technetium-99m using cysteine-containing peptide-based chelator GGGC at C-terminus. Z(IGF1R:4551)-GGGC was efficiently and stably labeled with technetium-99m (radiochemical yield 97 +/- A 3 %). Tc-99m-Z(IGF1R:4551)-GGGC demonstrated specific binding to IGF-1R-expressing DU-145 (prostate cancer) and MCF-7 (breast cancer) cell lines and slow internalization in vitro. The tumor-targeting properties were studied in BALB/c nu/nu mice bearing DU-145 and MCF-7 xenografts. [Tc-99m(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) was used for comparison. The biodistribution study demonstrated high tumor-to-blood ratios (6.2 +/- A 0.9 and 6.9 +/- A 1.0, for DU-145 and MCF-7, respectively, at 4 h after injection). Renal radioactivity concentration was 16-fold lower for Tc-99m-Z(IGF1R:4551)-GGGC than for [Tc-99m(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) at 4 h after injection. However, the liver uptake of Tc-99m-Z(IGF1R:4551)-GGGC was 1.2- to 2-fold higher in comparison with [Tc-99m(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551). A possible reason for the elevated hepatic uptake of Tc-99m-Z(IGF1R:4551)-GGGC is a high lipophilicity of amino acids in the binding site of Z(IGF1R:4551), which is not compensated in Tc-99m-Z(IGF1R:4551)-GGGC. In conclusion, Tc-99m-Z(IGF1R:4551)-GGGC can visualize the IGF-1R expression in human tumor xenografts and provides low retention of radioactivity in kidneys. Further development of this imaging agent should include molecular design aimed at reducing the hepatic uptake.
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| 12. |
- Mortensen, Anja, et al.
(författare)
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Selection, characterization and in vivo evaluation of novel CD44v6-targeting antibodies for targeted molecular radiotherapy
- 2023
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Molecular radiotherapy combines the advantages of systemic administration of highly specific antibodies or peptides and the localized potency of ionizing radiation. A potential target for molecular radiotherapy is the cell surface antigen CD44v6, which is overexpressed in numerous cancers, with limited expression in normal tissues. The aim of the present study was to generate and characterize a panel of human anti-CD44v6 antibodies and identify a suitable candidate for future use in molecular radiotherapy of CD44v6-expressing cancers. Binders were first isolated from large synthetic phage display libraries containing human scFv and Fab antibody fragments. The antibodies were extensively analyzed through in vitro investigations of binding kinetics, affinity, off-target binding, and cell binding. Lead candidates were further subjected to in vivo biodistribution studies in mice bearing anaplastic thyroid cancer xenografts that express high levels of CD44v6. Additionally, antigen-dependent tumor uptake of the lead candidate was verified in additional xenograft models with varying levels of target expression. Interestingly, although only small differences were observed among the top antibody candidates in vitro, significant differences in tumor uptake and retention were uncovered in in vivo experiments. A high-affinity anti-CD44v6 lead drug candidate was identified, mAb UU-40, which exhibited favorable target binding properties and in vivo distribution. In conclusion, a panel of human anti-CD44v6 antibodies was successfully generated and characterized in this study. Through comprehensive evaluation, mAb UU-40 was identified as a promising lead candidate for future molecular radiotherapy of CD44v6-expressing cancers due to its high affinity, excellent target binding properties, and desirable in vivo distribution characteristics.
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| 13. |
- Orlova, Anna, et al.
(författare)
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[99mTc(CO)3]+-(HE)3-Z IGF1R45514551 : a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours.
- 2013
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Ingår i: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 40:3, s. 439-449
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: Radionuclide imaging of insulin-like growth factor type 1 receptor (IGF-1R) expression in tumours might be used for selection of patients who would benefit from IGF-1R-targeted therapy. We have previously shown the feasibility of IGF-1R imaging using the Affibody molecule (111)In-DOTA-His(6)-Z(IGF1R:4551). The use of (99m)Tc instead of (111)In should improve sensitivity and resolution of imaging, and reduce the dose burden to patients. We hypothesized that inclusion of a HEHEHE tag instead of a His(6) tag in Z(IGF1R:4551) would permit its convenient purification using IMAC, enable labelling with [(99m)Tc(CO)(3)](+), and improve its biodistribution. METHODS: Z(IGF1R:4551) was expressed with a HEHEHE tag in the N terminus. The resulting (HE)(3)-Z(IGF1R:4551) construct was labelled with [(99m)Tc(CO)(3)](+). Targeting of IGF-1R-expressing cells using [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) was evaluated in vitro and in vivo. RESULTS: (HE)(3)-Z(IGF1R:4551) was stably labelled with (99m)Tc with preserved specific binding to IGF-1R-expressing DU-145 prostate cancer cells in vitro. In mice, [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) accumulated in IGF-1R-expressing organs (pancreas, stomach, lung and salivary gland). [(99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) demonstrated 3.6-fold lower accumulation in the liver and spleen than (111)In-DOTA-Z(IGF1R:4551). In NMRI nu/nu mice with DU-145 prostate cancer xenografts, the tumour uptake was 1.32 ± 0.11 %ID/g and the tumour-to-blood ratio was 4.4 ± 0.3 at 8 h after injection. The xenografts were visualized using a gamma camera 6 h after injection. CONCLUSION: (99m)Tc(CO)(3)](+)-(HE)(3)-Z(IGF1R:4551) is a promising candidate for visualization of IGF-1R expression in malignant tumours.
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| 14. |
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| 15. |
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| 16. |
- Preger, C., et al.
(författare)
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Generation and validation of recombinant antibodies to study human aminoacyl-tRNA synthetases
- 2020
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 295:41, s. 13981-13993
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Tidskriftsartikel (refereegranskat)abstract
- Aminoacyl-tRNA synthetases (aaRSs) have long been viewed as mere housekeeping proteins and have therefore often been overlooked in drug discovery. However, recent findings have revealed that many aaRSs have noncanonical functions, and several of the aaRSs have been linked to autoimmune diseases, cancer, and neurological disorders. Deciphering these roles has been challenging because of a lack of tools to enable their study. To help solve this problem, we have generated recombinant high-affinity antibodies for a collection of thirteen cytoplasmic and one mitochondrial aaRSs. Selected domains of these proteins were produced recombinantly in Escherichia coli and used as antigens in phage display selections using a synthetic human single-chain fragment variable library. All targets yielded large sets of antibody candidates that were validated through a panel of binding assays against the purified antigen. Furthermore, the top-performing binders were tested in immunoprecipitation followed by MS for their ability to capture the endogenous protein from mammalian cell lysates. For antibodies targeting individual members of the multi-tRNA synthetase complex, we were able to detect all members of the complex, co-immunoprecipitating with the target, in several cell types. The functionality of a subset of binders for each target was also confirmed using immunofluorescence. The sequences of these proteins have been deposited in publicly available databases and repositories. We anticipate that this open source resource, in the form of high-quality recombinant proteins and antibodies, will accelerate and empower future research of the role of aaRSs in health and disease.
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| 17. |
- Sahi, Maryam, et al.
(författare)
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Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy
- 2024
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; 2024:203
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) ˂14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.
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| 18. |
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| 19. |
- Sarén, Tina, et al.
(författare)
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Complementarity-determining region clustering may cause CAR-T cell dysfunction
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1, s. 4732-
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Tidskriftsartikel (refereegranskat)abstract
- Chimeric antigen receptor (CAR)-T cell therapy is rapidly advancing as cancer treatment, however, designing an optimal CAR remains challenging. A single-chain variable fragment (scFv) is generally used as CAR targeting moiety, wherein the complementarity-determining regions (CDRs) define its specificity. We report here that the CDR loops can cause CAR clustering, leading to antigen-independent tonic signalling and subsequent CAR-T cell dysfunction. We show via CARs incorporating scFvs with identical framework and varying CDR sequences that CARs may cluster on the T cell surface, which leads to antigen-independent CAR-T cell activation, characterized by increased cell size and interferon (IFN)-γ secretion. This results in CAR-T cell exhaustion, activation-induced cell death and reduced responsiveness to target-antigen-expressing tumour cells. CDR mutagenesis confirms that the CAR-clustering is mediated by CDR-loops. In summary, antigen-independent tonic signalling can be induced by CDR-mediated CAR clustering, which could not be predicted from the scFv sequences, but could be tested for by evaluating the activity of unstimulated CAR-T cells.
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| 20. |
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