| 1. |
- Danielsson, Marie
(author)
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Development of a new protective group for tryptophan
- 2010
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Licentiate thesis (other academic/artistic)abstract
- A new protective group for the indole nitrogen of the amino acid tryptophan, the Boc-sarcosinoyl-sarcosinolyl (Boc-Sar-Sar) is described. This protective group shows good stability to the reaction conditions used in solid phase peptide synthesis and is incorporated into the peptide as FmocTrp(BocSarSar)OH. When the peptide is cleaved from the resin with trifluoroacetic acid, the Boc group is simultaneously cleaved and N-terminal nitrogen in the Sar-Sar group is exposed. At low pH this amino group is protonated and thereby the peptide will have higher solubility in aqueous solution. High solubility of the peptide is an important factor for the purification of peptides with reversed phase HPLC. During HPLC purification, the protonated Sar-Sar group will remain on the peptide. When the purified peptide is exposed to pH 7,4 the Sar-Sar group will be cleaved and the peptide will be obtained in its fully deprotected form. This new protective group was used for the synthesis of the antimicrobial peptide gramicidin A, an extremely hydrophobic peptide with no ionizable groups and only a few exposed polar functional groups.
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| 2. |
- Höjer, Pontus, et al.
(author)
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Identification of Major Immune Cell Lineages with DBS-Pro
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Other publication (other academic/artistic)abstract
- Proteins play a pivotal role in cellular function and heterogeneity. Understanding cellular diversity at the proteome level necessitates sensitive single-cell assays with high throughput. While current sequencing-based methods offer promise, they often face limitations, including reliance on expensive and inaccessible commercial platforms. Here, we have adopted the DBS-Pro method, utilizing site-specific oligonucleotide-conjugated antibodies, to analyze surface proteins in single cells. The method uses cheap degenerated barcode oligonucleotides and a simple microfluidics setup for cell encapsulation. A sample of PBMCs was examined using a panel targeting six separate immune cell markers. Using this panel we could quantify marker expression on 1,307 cells, identifying major immune cell lineages including CD4+ T-cells, CD8+ T-cells, monocytes, and B-cells. While recognizing the need for protocol improvements, our results present a promising approach for single-cell proteomics.
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| 3. |
- Oroujeni, Maryam, PhD, 1982-, et al.
(author)
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Affibody-Mediated PNA-Based Pretargeted Cotreatment Improves Survival of Trastuzumab-Treated Mice Bearing HER2-Expressing Xenografts
- 2022
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In: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 63:7, s. 1046-1051
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Journal article (peer-reviewed)abstract
- Treatment of patients with human epidermal growth factor receptor 2 (HER2)-expressing tumors using the monoclonal antibody trastuzumab increases survival. The Affibody-based peptide nucleic acid (PNA)-mediated pretargeted radionuclide therapy has demonstrated efficacy against HER2-expressing xenografts in mice. Structural studies suggest that Affibody molecules and trastuzumab bind to different epitopes on HER2. The aim of this study was to test the hypothesis that a combination of PNA-mediated pretargeted radionuclide therapy and trastuzumab treatment of HER2-expressing xenografts can extend survival compared with monotherapies. Methods: Mutual interference of the primary pretargeting probe Z(HER2:342)-SR-HP1 and trastuzumab in binding to HER2-expressing cell lines was investigated in vitro. Experimental therapy evaluated the survival of mice bearing HER2-expressing SKOV-3 xenografts after treatment with vehicle, trastuzumab only, pretargeting using Affibody-PNA chimera Z(HER2:342)-SR-HP1 and complementary probe Lu-177-HP2, and combination of trastuzumab and pretargeting. The ethical permit limited the study to 90 d. The animals'weightsweremonitored during the study. After study termination, samples of liver and kidneys were evaluated by a veterinary pathologist for toxicity signs. Results: The presence of a large molar excess of trastuzumab had no influence on the affinity of Z(HER2:342)-SR-HP1 binding to HER2-expressing cells in vitro. The affinity of trastuzumab was not affected by a large excess of Z(HER2:342)-SR-HP1. Themedian survival of mice treated with trastuzumab (75.5 d) was significantly longer than the survival of mice treated with a vehicle (59.5 d). Median survival of mice treated with pretargeting was not reached by day 90. Six mice of 10 in this group survived, and 2 had complete remission. All mice in the combination treatment group survived, and tumors in 7 mice had disappeared at study termination. There was no significant difference between animal weights in the different treatment groups. No significant pathologic alterations were detected in livers and kidneys of treated animals. Conclusion: Treatment of mice bearing HER2-expressing xenografts with the combination of trastuzumab and Affibody-mediated PNA-based radionuclide pretargeting significantly increased survival compared with monotherapies. Cotreatment was not toxic for normal tissues.
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| 4. |
- Tano, Hanna, et al.
(author)
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Comparative Evaluation of Novel Lu-177-Labeled PNA Probes for Affibody-Mediated PNA-Based Pretargeting
- 2021
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In: Cancers. - : MDPI AG. - 2072-6694. ; 13:3
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Journal article (peer-reviewed)abstract
- Simple Summary Affibody molecules are small, engineered affinity proteins based on a nonimmunoglobulin scaffold. Affibody-based radionuclide imaging probes have demonstrated excellent tumor targeting. However, the renal clearance of affibody molecules is accompanied by high reabsorption and retention of activity in the kidney, which prevents their use for radionuclide therapy. We have previously shown the feasibility of overcoming the high renal uptake using a pretargeting approach for affibody-mediated therapy based on peptide nucleic acid (PNA) hybridization. In this study, we test the hypothesis that shortening the PNA pretargeting probes would further increase the difference between the accumulation of radiometals in tumor xenografts and in kidneys. A series of novel PNA probes has been designed and evaluated in vitro and in vivo. We have found that a variant containing 9 nucleobases enables a two-fold increase of the tumor-to-kidney dose ratio compared with a variant containing 15 nucleobases. This creates preconditions for more efficient therapy of cancer. Affibody-mediated PNA-based pretargeting is a promising approach to radionuclide therapy of HER2-expressing tumors. In this study, we test the hypothesis that shortening the PNA pretargeting probes would increase the tumor-to-kidney dose ratio. The primary probe Z(HER2:342)-SR-HP15 and the complementary secondary probes HP16, HP17, and HP18, containing 9, 12, and 15 nucleobases, respectively, and carrying a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator were designed, synthesized, characterized in vitro, and labeled with Lu-177. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution of these novel probes was evaluated in immunodeficient mice bearing SKOV3 xenografts and compared to the previously studied [Lu-177]Lu-HP2. Characterization confirmed the formation of high-affinity duplexes between HP15 and the secondary probes, with the affinity correlating with the length of the complementary PNA sequences. All the PNA-based probes were bound specifically to HER2-expressing cells in vitro. In vivo studies demonstrated HER2-specific uptake of all Lu-177-labeled probes in xenografts in a pretargeting setting. The ratio of cumulated radioactivity in the tumor to the radioactivity in kidneys was dependent on the secondary probe's size and decreased with an increased number of nucleobases. The shortest PNA probe, [Lu-177]Lu-HP16, showed the highest tumor-to-kidney ratio. [Lu-177]Lu-HP16 is the most promising secondary probe for affibody-mediated tumor pretargeting.
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| 5. |
- Westerlund, Kristina, et al.
(author)
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Radionuclide Therapy of HER2-Expressing Human Xenografts Using Affibody-Based Peptide Nucleic Acid-Mediated Pretargeting : In Vivo Proof of Principle
- 2018
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In: Journal of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 59:7, s. 1092-1098
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Journal article (peer-reviewed)abstract
- Affibody molecules are small proteins engineered using a nonanti-body scaffold. Radiolabeled Affibody molecules are excellent imaging probes, but their application to radionuclide therapy has been prevented by high renal reabsorption. The aim of this study was to test the hypothesis that Affibody-based peptide nucleic acid (PNA)-mediated pretargeted therapy of human epidermal growth factor receptor 2 (HER2)-expressing cancer extends survival without accompanying renal toxicity.Methods: A HER2-targeting Affibody molecule ligated with an AGTCGTGATGTAGTC PNA hybridization probe (Z(HER2:342)-SR-HP1) was used as the primary pretargeting agent. A complementary AGTCGTGATGTAGTC PNA conjugated to the chelator DOTA and labeled with the radionuclide Lu-177 (Lu-177-HP2) was used as the secondary agent. The influence of different factors on pretargeting was investigated. Experimental radionuclide therapy in mice bearing SKOV-3 xenografts was performed in 6 cycles separated by 7 d.Results: Optimal tumor targeting was achieved when 16 MBq/3.5 mu g (0.65 nmol) of Lu-177-HP2 was injected 16 h after injection of 100 mu g (7.7 nmol) of Z(HER2:342)-SR-HP1. The calculated absorbed dose to tumors was 1,075 mGy/MBq, whereas the absorbed dose to kidneys was 206 mGy/MBq and the absorbed dose to blood (surrogate of bone marrow) was 4 mGy/MBq. Survival of mice was significantly longer (P < 0.05) in the treatment group (66 d) than in the control groups treated with the same amount of Z(HER2:342)-SR-HP1 only (37 d), the same amount and activity of Lu-177-HP2 only (32 d), or phosphate-buffered saline (37 d).Conclusion: The studied pretargeting system can deliver an absorbed dose to tumors appreciably exceeding absorbed doses to critical organs, making Affibody-based PNA-mediated pretargeted radionuclide therapy highly attractive.
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