| 1. |
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| 2. |
- Altai, Mohamed, et al.
(författare)
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188Re-ZHER2:V2, a promising affibody-based targeting agent against HER2-expressing tumors : preclinical assessment
- 2014
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Ingår i: Journal of nuclear medicine : official publication, Society of Nuclear Medicine. - : Society of Nuclear Medicine. - 0161-5505 .- 1535-5667 .- 2159-662X. ; 55:11, s. 8-1842
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Tidskriftsartikel (refereegranskat)abstract
- UNLABELLED: Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of (99m)Tc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide-based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate (188)Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)-expressing tumors.METHODS: ZHER2:V2 was labeled with (188)Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment.RESULTS: Binding of (188)Re-ZHER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 percentage injected activity per gram [%ID/g] at 1 h after injection). The tumor uptake was 14 ± 2, 12 ± 2, 5 ± 2, and 1.8 ± 0.5 %IA/g at 1, 4, 24, and 48 h after injection, respectively. The in vivo targeting of HER2-expressing xenografts was specific. Already at 4 h after injection, tumor uptake exceeded kidney uptake (2.1 ± 0.2 %IA/g). Scintillation-camera imaging showed that tumor xenografts were the only sites with prominent accumulation of radioactivity at 4 h after injection. Based on the biokinetics, a dosimetry evaluation for humans suggests that (188)Re-ZHER2:V2 would provide an absorbed dose to tumor of 79 Gy without exceeding absorbed doses of 23 Gy to kidneys and 2 Gy to bone marrow. This indicates that future human radiotherapy studies may be feasible.CONCLUSION: (188)Re-ZHER2:V2 can deliver high absorbed doses to tumors without exceeding kidney and bone marrow toxicity limits.
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| 3. |
- Altai, Mohamed, et al.
(författare)
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Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
- 2018
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 7:10
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD 035) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified.
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| 4. |
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| 5. |
- Altai, Mohamed, et al.
(författare)
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Selection of an optimal cysteine-containing peptide-based chelator for labeling of affibody molecules with (188)Re.
- 2014
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Ingår i: European Journal of Medicinal Chemistry. - : Elsevier BV. - 0223-5234 .- 1768-3254. ; 87, s. 519-28
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of (188)Re-ZHER2:V2 (3.1 ± 0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental (188)Re-ZHER2:2395 (172 ± 32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of (188)Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.
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| 6. |
- Altai, Mohamed, et al.
(författare)
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Selection of an optimal cysteine-containing peptide-based chelator for labeling of Affibody molecules with 188-Re
- 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:Suppl. 2, s. S219-S220
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of 188Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all 188Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The 188Re-ZHER2:V2 affibody molecule with a GGGC chelator provided the lowest uptake in all organs and tissues. The renal retention of 188Re-ZHER2:V2 (3.1±0.5 %ID/g at 4 h after injection) was 55-fold lower than retention of the parental 188Re-ZHER2:2395 (172±32 %ID/g). We show that engineering of cysteine-containing peptide-based chelators can be used for significant improvement of biodistribution of 188Re-labeled scaffold proteins, particularly reduction of their uptake in excretory organs.
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| 7. |
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| 8. |
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| 9. |
- Andersson, Ken G., et al.
(författare)
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Autotransporter-mediated display of a naïve Affibody library on the outer membrane of E. coli
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is the most frequently used method, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli have several properties that are valuable for library applications and then in particular the high transformation efficiency. Although the first studies on display of recombinant peptides and proteins on E. coli were reported over 25 years ago, the method is still not fully established for directed evolution of affinity proteins. More recently, the use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and in particular for directed evolution of different enzymes. Here, we report on display of a large naïve Affibody library on the outer membrane of E. coli using the autotransporter AIDA-I. The expression cassette was first engineered by removing non-essential sequences, followed by introduction of an Affibody library, comprising more than 109 variants, into the new display vector. Selections by FACS against five different target molecules resulted in a panel of binders with down to nanomolar affinities.
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| 10. |
- Andersson, Ken G., et al.
(författare)
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Autotransporter-Mediated Display of a Naive Affibody Library on the Outer Membrane of Escherichia coli
- 2019
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Ingår i: Biotechnology Journal. - : WILEY-V C H VERLAG GMBH. - 1860-6768 .- 1860-7314. ; 14:4
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Tidskriftsartikel (refereegranskat)abstract
- Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is most frequently used, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli has several valuable properties for library applications and in particular the high transformation efficiency. The use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and particularly for directed evolution of different enzymes. Here, the authors report on display of a large naive affibody library on the outer membrane of E. coli using the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I). The expression cassette is first engineered by removing non-essential sequences, followed by introduction of an affibody library, comprising more than 10(9) variants, into the new display vector. The quality of the library and general performance of the method is assessed by FACS against five different targets, which resulted in a panel of binders with down to nanomolar affinities, suggesting that the method has potential as a complement to phage display for generation of affibody molecules.
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| 11. |
- Andersson, Ken G., 1987-
(författare)
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Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Directed evolution is today an established strategy for generation of new affinity proteins. This thesis describes the development of a cell-display method using Escherichia coli for directed evolution of Affibody molecules. Further, the thesis describes rational design of Affibody-based tracers, intended for future patient stratification using medical imaging. Fusing recombinant proteins to various autotransporters is a promising approach for efficient surface display on the surface of E. coli, as well as for construction of high-complexity libraries. In paper I, we successfully engineered an expression vector for display of Affibody molecules using the autotransporter AIDA-I. In paper II, a large Affibody library of 2.3x109 variants was constructed and screening using FACS resulted in new specific binders in the nanomolar range. In paper III, we demonstrated Sortase-mediated secretion and conjugation of binders directly from the E. coli surface. The three following studies describe rational design of Affibody-based tracers against two cancer-associated targets for molecular imaging. First, anti-HER3 Affibody molecules were labelled with 111In, and SPECT imaging showed that the conjugates specifically targeted HER3-expressing xenografts. Furthermore, labeling with 68Ga for PET imaging showed that tumor uptake correlated with HER3 expression, suggesting that the tracers have potential for patient stratification. The last study describes the development and investigation of anti-EGFR Affibody-based imaging agents. Labeled with 89Zr, the Affibody tracer demonstrated higher tumor uptake at 3 h post injection than the anti-EGFR antibody cetuximab at 48 h post injection. In conclusion, this thesis describes new tools and knowledge that will hopefully contribute to the development of affinity proteins for biotechnology, therapy and medical imaging in the future.
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| 12. |
- Andersson, Ken G, et al.
(författare)
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Comparative evaluation of 111In-labeled NOTA‑conjugated affibody molecules for visualization of HER3 expression in malignant tumors
- 2015
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Ingår i: Oncology Reports. - : Spandidos Publications. - 1021-335X .- 1791-2431. ; 34:2, s. 1042-8
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Tidskriftsartikel (refereegranskat)abstract
- Expression of human epidermal growth factor receptor type 3 (HER3) in malignant tumors has been associated with resistance to a variety of anticancer therapies. Several anti-HER3 monoclonal antibodies are currently under pre-clinical and clinical development aiming to overcome HER3-mediated resistance. Radionuclide molecular imaging of HER3 expression may improve treatment by allowing the selection of suitable patients for HER3-targeted therapy. Affibody molecules are a class of small (7kDa) high-affinity targeting proteins with appreciable potential as molecular imaging probes. In a recent study, we selected affibody molecules with affinity to HER3 at a low picomolar range. The aim of the present study was to develop an anti-HER3 affibody molecule suitable for labeling with radiometals. The HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA HER3-specific affibody molecules were labeled with indium‑111 (111In) and assessed invitro and invivo for imaging properties using single photon emission computed tomography (SPECT). Labeling of HEHEHE-Z08698-NOTA and HEHEHE-Z08699-NOTA with 111In provided stable conjugates. Invitro cell tests demonstrated specific binding of the two conjugates to HER3-expressing BT‑474 breast carcinoma cells. In mice bearing BT‑474 xenografts, the tumor uptake of the two conjugates was receptor‑specific. Direct invivo comparison of 111In-HEHEHE-Z08698-NOTA and 111In-HEHEHE-Z08699‑NOTA demonstrated that the two conjugates provided equal radioactivity uptake in tumors, although the tumor-to-blood ratio was improved for 111In-HEHEHE-Z08698-NOTA [12±3 vs. 8±1, 4h post injection (p.i.)] due to more efficient blood clearance. 111In-HEHEHE-Z08698-NOTA is a promising candidate for imaging of HER3-expression in malignant tumors using SPECT. Results of the present study indicate that this conjugate could be used for patient stratification for anti-HER3 therapy.
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| 13. |
- Andersson, Ken G., et al.
(författare)
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Coupled release and site-specific conjugation of Affibody molecules from the surface of E. coli using Sortase A
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Combinatorial protein engineering using libraries displayed on various microorganisms is a powerful method forgeneration of new affinity proteins. Successful efforts often result in broad panels of isolated binders, which are thentypically subcloned, produced, purified and characterized in various assays. Many such assays also require conjugation tofor example reporters or other functional molecules and the downstream production and modification thus tends to be verylaborious and limits the number of candidates that can be screened. Staphylococcal sortase A is a natural transpeptidasethat catalyzes the ligation between a LPXTG motif and N-terminal glycines and is today used in a variety of applicationsfor site-specific conjugation of different molecules to recombinant proteins. We have previously developed a surfacedisplay method for combinatorial protein engineering of Affibody molecules on the outer membrane of E. coli usingautodisplay. Here, we introduced a sortase-A recognition motif into the displayed recombinant proteins and evaluatedsortase-mediated release and specific conjugation of various reporters to Affibody molecules. The approach has potentialto significantly increase the flexibility and throughput of downstream characterization of affinity proteins after directedevolution using cell display and FACS.
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| 14. |
- Andersson, Ken G., et al.
(författare)
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Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with Tc-99m using a peptide-based cysteine-containing chelator
- 2016
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Ingår i: International Journal of Oncology. - : SPANDIDOS. - 1019-6439 .- 1791-2423. ; 49:6, s. 2285-2293
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Tidskriftsartikel (refereegranskat)abstract
- The epidermal growth factor receptor (EGFR) is overexpressed in a number of malignant tumors and is a molecular target for several specific anticancer antibodies and tyrosine kinase inhibitors. The overexpression of EGFR is a predictive biomarker for response to several therapy regimens. Radionuclide molecular imaging might enable detection of EGFR overexpression by a non-invasive procedure and could be used repeatedly. Affibody molecules are engineered scaffold proteins, which could be selected to have a high affinity and selectivity to predetermined targets. The anti-EGFR ZEGFR:2377 affibody molecule is a potential imaging probe for EGFR detection. The use of the generator-produced radionuclide Tc-99m should facilitate clinical translation of an imaging probe due to its low price, availability and favorable dosimetry of the radionuclide. In the present study, we evaluated feasibility of ZEGFR:2377 labeling with Tc-99m using a peptide-based cysteine-containing chelator expressed at the C-terminus of ZEGFR:2377. The label was stable in vitro under cysteine challenge. In addition, Tc-99m-ZEGFR:2377 was capable of specific binding to EGFR-expressing cells with high affinity (274 pM). Studies in BALB/C nu/nu mice bearing A431 xenografts demonstrated that Tc-99m-ZEGFR:2377 accumulates in tumors in an EGFR-specific manner. The tumor uptake values were 3.6 1 and 2.5 0.4% ID/g at 3 and 24 h after injection, respectively. The corresponding tumor-to-blood ratios were 1.8 0.4 and 8 3. The xenografts were clearly visualized at both time-points. This study demonstrated the potential of Tc-99m-labeled ZEGFR:2377 for imaging of EGFR in vivo.
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| 15. |
- Bass, Tarek, et al.
(författare)
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In vivo evaluation of a novel format of a bivalent HER3-targeting and albumin- binding therapeutic affibody construct
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression of human epidermal growth factor receptor 3 (HER3) is involved in resistance to several therapies for malignant tumours. Currently, several anti-HER3 monoclonal antibodies are under clinical development. We introduce an alternative approach to HER3-targeted therapy based on engineered scaffold proteins, i.e. affibody molecules. We designed a small construct (22.5 kDa, denoted 3A3), consisting of two high-affinity anti-HER3 affibody molecules flanking an albumin-binding domain ABD, which was introduced for prolonged residence in circulation. In vitro, 3A3 efficiently inhibited growth of HER3-expressing BxPC-3 cells. Biodistribution in mice was measured using 3A3 that was site-specifically labelled with In-111 via a DOTA chelator. The residence time of In-111-DOTA-3A3 in blood was extended when compared with the monomeric affibody molecule. In-111-DOTA-3A3 accumulated specifically in HER3-expressing BxPC-3 xenografts in mice. However, In-111-DOTA-3A3 cleared more rapidly from blood than a size-matched control construct In-111-DOTA-TAT, most likely due to sequestering of 3A3 by mErbB3, the murine counterpart of HER3. Repeated dosing and increase of injected protein dose decreased uptake of In-111-DOTA-3A3 in mErbB3-expressing tissues. Encouragingly, growth of BxPC-3 xenografts in mice was delayed in an experimental (pilot-scale) therapy study using 3A3. We conclude that the 3A3 affibody format seems promising for treatment of HER3-overexpressing tumours.
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| 16. |
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| 17. |
- Boutajangout, Allal, et al.
(författare)
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Affibody-Mediated Sequestration of Amyloid beta Demonstrates Preventive Efficacy in a Transgenic Alzheimer's Disease Mouse Model
- 2019
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Ingår i: Frontiers in Aging Neuroscience. - : Frontiers Media S.A.. - 1663-4365. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Different strategies for treatment and prevention of Alzheimer's disease (AD) are currently under investigation, including passive immunization with anti-amyloid beta (anti-A beta) monoclonal antibodies (mAbs). Here, we investigate the therapeutic potential of a novel type of A beta-targeting agent based on an affibody molecule with fundamentally different properties to mAbs. We generated a therapeutic candidate, denoted Z(SYM73)-albumin-binding domain (ABD; 16.8 kDa), by genetic linkage of the dimeric Z(SYM73) affibody for sequestering of monomeric A beta-peptides and an ABD for extension of its in vivo half-life. Amyloid precursor protein (APP)/PS1 transgenic AD mice were administered with Z(SYM73)-ABD, followed by behavioral examination and immunohistochemistry. Results demonstrated rescued cognitive functions and significantly lower amyloid burden in the treated animals compared to controls. No toxicological symptoms or immunology-related side-effects were observed. To our knowledge, this is the first reported in vivo investigation of a systemically delivered scaffold protein against monomeric A beta, demonstrating a therapeutic potential for prevention of AD.
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| 18. |
- Chaudhary, Himanshu, et al.
(författare)
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Dissecting the structural organization of multiprotein amyloid aggregates using a bottom-up approach
- 2020
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 11:10, s. 1447-1457
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Tidskriftsartikel (refereegranskat)abstract
- Deposition of fibrillar amyloid β (Aβ) in senile plaques is a pathological signature of Alzheimer's disease. However, senile plaques also contain many other components, including a range of different proteins. Although the composition of the plaques can be analyzed in post mortem tissue, knowledge of the molecular details of these multiprotein inclusions and their assembly processes is limited, which impedes the progress in deciphering the biochemical mechanisms associated with Aβ pathology. We here describe a bottom-up approach to monitor how proteins from human cerebrospinal fluid associate with Aβ amyloid fibrils to form plaque particles. The method combines flow cytometry and mass spectrometry proteomics and allowed us to identify and quantify 128 components of the captured multiprotein aggregates. The results provide insights in the functional characteristics of the sequestered proteins and reveal distinct interactome responses for the two investigated Aβ variants, Aβ(1-40) and Aβ(1-42). Furthermore, the quantitative data is used to build models of the structural organization of the multiprotein aggregates, which suggests that Aβ is not the primary binding target for all the proteins; secondary interactions account for the majority of the assembled components. The study elucidates how different proteins are recruited into senile plaques and establishes a new model system for exploring the pathological mechanisms of Alzheimer's disease from a molecular perspective.
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| 19. |
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| 20. |
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| 21. |
- Cheung, Pierre, et al.
(författare)
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Preclinical evaluation of Affibody molecule for PET imaging of human pancreatic islets derived from stem cells
- 2023
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Ingår i: EJNMMI Research. - : Springer Nature. - 2191-219X. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Beta-cell replacement methods such as transplantation of isolated donor islets have been proposed as a curative treatment of type 1 diabetes, but widespread application is challenging due to shortages of donor tissue and the need for continuous immunosuppressive treatments. Stem-cell-derived islets have been suggested as an alternative source of beta cells, but face transplantation protocols optimization difficulties, mainly due to a lack of available methods and markers to directly monitor grafts survival, as well as their localization and function. Molecular imaging techniques and particularly positron emission tomography has been suggested as a tool for monitoring the fate of islets after clinical transplantation. The integral membrane protein DGCR2 has been demonstrated to be a potential pancreatic islet biomarker, with specific expression on insulin-positive human embryonic stem-cell-derived pancreatic progenitor cells. The candidate Affibody molecule ZDGCR2:AM106 was radiolabeled with fluorine-18 using a novel click chemistry-based approach. The resulting positron emission tomography tracer [18F]ZDGCR2:AM106 was evaluated for binding to recombinant human DGCR2 and cryosections of stem-cell-derived islets, as well as in vivo using an immune-deficient mouse model transplanted with stem-cell-derived islets. Biodistribution of the [18F]ZDGCR2:AM106 was also assessed in healthy rats and pigs. Results: [18F]ZDGCR2:AM106 was successfully synthesized with high radiochemical purity and yield via a pretargeting approach. [18F]ZDGCR2:AM106 retained binding to recombinant human DCGR2 as well as to cryosectioned stem-cell-derived islets, but in vivo binding to native pancreatic tissue in both rat and pig was low. However, in vivo uptake of [18F]ZDGCR2:AM106 in stem-cell-derived islets transplanted in the immunodeficient mice was observed, albeit only within the early imaging frames after injection of the radiotracer. Conclusion: Targeting of DGCR2 is a promising approach for in vivo detection of stem-cell-derived islets grafts by molecular imaging. The synthesis of [18F]ZDGCR2:AM106 was successfully performed via a pretargeting method to label a site-specific covalently bonded fluorine-18 to the Affibody molecule. However, the rapid washout of [18F]ZDGCR2:AM106 from the stem-cell-derived islets graft indicates that dissociation kinetics can be improved. Further studies using alternative binders of similar classes with improved binding potential are warranted.
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| 22. |
- Dahlsson Leitao, Charles
(författare)
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Affibody-mediated targeting of HER-family receptors for cancer imaging and therapy
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are remarkable molecules with diverse and specialized functions playing essential roles in most biological processes. One such function is protecting us from diseases by the action of antibodies in our immune system that can recognize and mediate the destruction of invading pathogens by binding to foreign epitopes found on non-self proteins. The concept of utilizing specific protein-protein interactions to achieve a therapeutic effect has for several decades been a cornerstone for the development of cancer-directed treatments. While antibodies have formed a basis for the development of such drugs, other protein alternatives may be engineered to complement current antibody-based treatments, and may even prove to possess superior features. This thesis focuses on the engineering of affibody molecules, a small alternative scaffold protein, for design and development of novel cancer-targeting therapeutic and diagnostic drugs. There are many different strategies that have been investigated for inhibiting cancer progression and tumour growth with perhaps one of the most straightforward involving disruption of dysregulated growth-promoting signalling pathways. Members of the human epidermal growth factor receptor (HER) family is prominently expressed in various cancer types and have been shown to be intricately involved in tumorigenesis. One of the members (HER3) often becomes upregulated in cancer and have been shown to mediate acquired resistance to targeted therapies by the mechanism of ligand-induced activation. We have designed five novel affibody-based HER3-targeting molecules able to prevent ligand-binding and consequently activation of HER3. We investigated the targeting properties and biodistribution profiles of these molecules in vivo and subsequently evaluated the anti-tumour efficacy for the most promising variants in direct comparison to a HER3-targeting antibody with a similar inhibitory mechanism. We observed a large influence of design on both the biodistribution properties and the in vivo efficacy of different affibody molecules. Moreover, we demonstrated that two of the affibody-formats were equally effective as the antibody in inhibiting tumour growth and prolonging survival of mice bearing HER3-positive xenografts. The effectiveness of cancer treatments depends on efficient diagnostic approaches that can reliably stratify patients based on these targetable biomarkers, which is possible using radionuclide molecular imaging. We have performed a direct comparison of the diagnostic potential for visualizing HER3-expressing tumours of affibody- and antibody-based imaging probes. We concluded that affibody molecules provide superior imaging quality with higher diagnostic potential and enable early visualization of HER3-expression in tumours. Another member of the HER family that is of interest for cancer therapy is HER1 (or EGFR) but due to substantial expression in healthy tissues, targeted therapies may lead to severe side-effects. One possible solution to this is taking advantage of the distinct milieu of the tumour microenvironment to design EGFR-targeting drugs that become conditionally activated at the tumour site, but not in normal tissues, with the aim of drastically reducing systemic toxicity. We have generated an affibody molecule with anti-idiotypic binding specificity for a previously generated EGFR-binding affibody molecule, which we used to construct an affibody-based prodrug. We were able to show that, in a proof-of-concept format, this anti-idiotypic masking domain was able to block the binding to EGFR until removed by protease-mediated cleavage. We subsequently developed and characterized a more refined version of this prodrug, which we call a pro-affibody, and could show that activation by cancer-associated proteases confers binding to EGFR-expressing cancer cells and enables conditional cytotoxic payload delivery in vitro. The pro-affibody was further evaluated in vivo using tumour-bearing mice to investigate the feasibility for masked uptake in healthy tissues while retaining binding-activity in tumours. We observed a substantial reduction in EGFR-specific liver uptake compared to a control construct without a masking domain, and a strong indication of protease-mediated EGFR-binding in tumours. In conclusion, the experimental work presented in this thesis provides a rationale for designing effective affibody-based cancer therapeutics and diagnostics with different targeting strategies and demonstrates the potential of such drugs from preclinical in vivo data.
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| 23. |
- 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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| 24. |
- Deyev, S., et al.
(författare)
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Comparative Evaluation of Two DARPin Variants : Effect of Affinity, Size, and Label on Tumor Targeting Properties
- 2019
-
Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 16:3, s. 995-1008
-
Tidskriftsartikel (refereegranskat)abstract
- Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins that can be selected for binding to desirable molecular targets. High affinity and small size of DARPins render them promising probes for radionuclide molecular imaging. However, detailed knowledge on many factors influencing their imaging properties is still lacking. We have evaluated two human epidermal growth factor 2 (HER2)-specific DARPins with different size and binding properties. DARPins 9-29-H 6 and G3-H 6 were radiolabeled with iodine-125 and tricarbonyl technetium-99m and evaluated in vitro. A side-by-side comparison of biodistribution and tumor targeting was performed. HER2-specific tumor accumulation of G3-H 6 was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H 6 in comparison to 9-29-H 6 . Technetium-99m labeled G3-H 6 demonstrated a better biodistribution profile than 9-29-H 6 , with several-fold lower uptake in liver. Radioiodinated G3-H 6 showed the best tumor-to-organ ratios. The combined effect of affinity, molecular weight, scaffold composition, and nonresidualizing properties of iodine label provided radioiodinated G3-H 6 with high clinical potential for imaging of HER2.
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| 25. |
- Faresjö, Rebecca, et al.
(författare)
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Transferrin Receptor Binding BBB-Shuttle Facilitates Brain Delivery of Anti-Aβ-Affibodies
- 2022
-
Ingår i: Pharmaceutical research. - : Springer Nature. - 0724-8741 .- 1573-904X. ; 39:7, s. 1509-1521
-
Tidskriftsartikel (refereegranskat)abstract
- Affibodies targeting amyloid-beta (Aβ) could potentially be used as therapeutic and diagnostic agents in Alzheimer’s disease (AD). Affibodies display suitable characteristics for imaging applications such as high stability and a short biological half-life. The aim of this study was to explore brain delivery and retention of Aβ protofibril-targeted affibodies in wild-type (WT) and AD transgenic mice and to evaluate their potential as imaging agents. Two affibodies, Z5 and Z1, were fused with the blood–brain barrier (BBB) shuttle single-chain variable fragment scFv8D3. In vitro binding of 125I-labeled affibodies with and without scFv8D3 was evaluated by ELISA and autoradiography. Brain uptake and retention of the affibodies at 2 h and 24 h post injection was studied ex vivo in WT and transgenic (tg-Swe and tg-ArcSwe) mice. At 2 h post injection, [125I]I-Z5 and [125I]I-Z1 displayed brain concentrations of 0.37 ± 0.09% and 0.46 ± 0.08% ID/g brain, respectively. [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 showed increased brain concentrations of 0.53 ± 0.16% and 1.20 ± 0.35%ID/g brain. At 24 h post injection, brain retention of [125I]I-Z1 and [125I]I-Z5 was low, while [125I]I-scFv8D3-Z1 and [125I]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [125I]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 compared with the affibodies without scFv8D3, but could not confirm specific affibody accumulation around Aβ deposits. Affibody-scFv8D3 fusions displayed increased brain and parenchymal delivery compared with the non-fused affibodies. However, fast brain washout and a suboptimal balance between Aβ and mTfR1 affinity resulted in low intrabrain retention around Aβ deposits.
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| 26. |
- Fleetwood, Filippa, et al.
(författare)
-
An engineered autotransporter-based surface expression vector enables efficient display of Affibody molecules on OmpT-negative E. coli as well as protease-mediated secretion in OmpT-positive strains
- 2014
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 13, s. 179-
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Cell display technologies (e.g. bacterial display) are attractive in directed evolution as they provide the option to use flow-cytometric cell sorting for selection from combinatorial libraries. The aim of this study was to engineer and investigate an expression vector system with dual functionalities: i) recombinant display of Affibody libraries on Escherichia coli for directed evolution and ii) small scale secreted production of candidate affinity proteins, allowing initial downstream characterizations prior to subcloning. Autotransporters form a class of surface proteins in Gram-negative bacteria that have potential for efficient translocation and tethering of recombinant passenger proteins to the outer membrane. We engineered a bacterial display vector based on the E. coli AIDA-I autotransporter for anchoring to the bacterial surface. Potential advantages of employing autotransporters combined with E. coli as host include: high surface expression level, high transformation frequency, alternative promoter systems available, efficient translocation to the outer membrane and tolerance for large multi-domain passenger proteins. Results: The new vector was designed to comprise an expression cassette encoding for an Affibody molecule, three albumin binding domains for monitoring of surface expression levels, an Outer membrane Protease T (OmpT) recognition site for potential protease-mediated secretion of displayed affinity proteins and a histidine-tag for purification. A panel of vectors with different promoters were generated and evaluated, and suitable cultivation conditions were investigated. The results demonstrated a high surface expression level of the different evaluated Affibody molecules, high correlation between target binding and surface expression level, high signal-to-background ratio, efficient secretion and purification of binders in OmpT-positive hosts as well as tight regulation of surface expression for the titratable promoters. Importantly, a mock selection using FACS from a 1: 100,000 background yielded around 20,000-fold enrichment in a single round and high viability of the isolated bacteria after sorting. Conclusions: The new expression vectors are promising for combinatorial engineering of Affibody molecules and the strategy for small-scale production of soluble recombinant proteins has the potential to increase throughput of the entire discovery process.
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| 27. |
- Fleetwood, Filippa, 1985-
(författare)
-
Bacterial display systems for engineering of affinity proteins
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Directed evolution is a powerful method for engineering of specific affinity proteins such as antibodies and alternative scaffold proteins. For selections from combinatorial protein libraries, robust and high-throughput selection platforms are needed. An attractive technology for this purpose is cell surface display, offering many advantages, such as the quantitative isolation of high-affinity library members using flow-cytometric cell sorting. This thesis describes the development, evaluation and use of bacterial display technologies for the engineering of affinity proteins.Affinity proteins used in therapeutic and diagnostic applications commonly aim to specifically bind to disease-related drug targets. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a critical process in various types of cancer and vascular eye disorders. Vascular Growth Factor Receptor 2 (VEGFR2) is one of the main regulators of angiogenesis. The first two studies presented in this thesis describe the engineering of a biparatopic Affibody molecule targeting VEGFR2, intended for therapeutic and in vivo imaging applications. Monomeric VEGFR2-specific Affibody molecules were generated by combining phage and staphylococcal display technologies, and the engineering of two Affibody molecules, targeting distinct epitopes on VEGFR2 into a biparatopic construct, resulted in a dramatic increase in affinity. The biparatopic construct was able to block the ligand VEGF-A from binding to VEGFR2-expressing cells, resulting in an efficient inhibition of VEGFR2 phosphorylation and angiogenesis-like tube formation in vitro.In the third study, the staphylococcal display system was evaluated for the selection from a single-domain antibody library. This was the first demonstration of successful selection from an antibody-based library on Gram-positive bacteria. A direct comparison to the selection from the same library displayed on phage resulted in different sets of binders, and higher affinities among the clones selected by staphylococcal display. These results highlight the importance of choosing a display system that is suitable for the intended application.The last study describes the development and evaluation of an autotransporter-based display system intended for display of Affibody libraries on E. coli. A dual-purpose expression vector was designed, allowing efficient display of Affibody molecules, as well as small-scale protein production and purification of selected candidates without the need for sub-cloning. The use of E. coli would allow the display of large Affibody libraries due to a high transformation frequency. In combination with the facilitated means for protein production, this system has potential to improve the throughput of the engineering process of Affibody molecules.In summary, this thesis describes the development, evaluation and use of bacterial display systems for engineering of affinity proteins. The results demonstrate great potential of these display systems and the generated affinity proteins for future biotechnological and therapeutic use.
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| 28. |
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| 29. |
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| 30. |
- Fleetwood, Filippa, et al.
(författare)
-
Novel affinity binders for neutralization of vascular endothelial growth factor (VEGF) signaling
- 2016
-
Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Birkhauser Verlag. - 1420-682X .- 1420-9071. ; 73:8, s. 1671-1683
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Tidskriftsartikel (refereegranskat)abstract
- Angiogenesis denotes the formation of new blood vessels from pre-existing vasculature. Progression of diseases such as cancer and several ophthalmological disorders may be promoted by excess angiogenesis. Novel therapeutics to inhibit angiogenesis and diagnostic tools for monitoring angiogenesis during therapy, hold great potential for improving treatment of such diseases. We have previously generated so-called biparatopic Affibody constructs with high affinity for the vascular endothelial growth factor receptor-2 (VEGFR2), which recognize two non-overlapping epitopes in the ligand-binding site on the receptor. Affibody molecules have previously been demonstrated suitable for imaging purposes. Their small size also makes them attractive for applications where an alternative route of administration is beneficial, such as topical delivery using eye drops. In this study, we show that decreasing linker length between the two Affibody domains resulted in even slower dissociation from the receptor. The new variants of the biparatopic Affibody bound to VEGFR2-expressing cells, blocked VEGFA binding, and inhibited VEGFA-induced signaling of VEGFR2 over expressing cells. Moreover, the biparatopic Affibody inhibited sprout formation of endothelial cells in an in vitro angiogenesis assay with similar potency as the bivalent monoclonal antibody ramucirumab. This study demonstrates that the biparatopic Affibody constructs show promise for future therapeutic as well as in vivo imaging applications.
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| 31. |
- Fleetwood, Filippa, et al.
(författare)
-
Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity
- 2014
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 4, s. 7518-
-
Tidskriftsartikel (refereegranskat)abstract
- Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases.
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| 32. |
- Fleetwood, Filippa, et al.
(författare)
-
Surface display of a single-domain antibody library on Gram-positive bacteria
- 2013
-
Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Nature. - 1420-682X .- 1420-9071. ; 70:6, s. 1081-1093
-
Tidskriftsartikel (refereegranskat)abstract
- Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.
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| 33. |
- Garousi, Javad, et al.
(författare)
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Comparative evaluation of affibody- and antibody fragments-based CAIX imaging probes in mice bearing renal cell carcinoma xenografts
- 2019
-
Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE3-ZCAIX:2, was labeled with In-111 and characterized in vitro. Tumor-targeting properties of [In-111]In-DOTA-HE3-ZCAIX:2 were compared head-to-head with properties of the parental variant, [(99)mTc]Tc(CO)(3)-HE3-ZCAIX:2, and the most promising antibody fragment-based tracer, [In-111]In-DTPA-G250(Fab')(2), in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the (99)mTc-labeled parental variant, [In-111]In-DOTA-HE3-ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [In-111]In-DOTA-HE3-ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [In-111]In-G250(Fab']2. In conclusion, [In-111]In-DOTA-HE3-ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.
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| 34. |
- Garousi, Javad, et al.
(författare)
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Comparative Evaluation of Affibody Molecules for Radionuclide Imaging of in Vivo Expression of Carbonic Anhydrase IX
- 2016
-
Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 13:11, s. 3676-3687
-
Tidskriftsartikel (refereegranskat)abstract
- Overexpression of the enzyme carbonic anhydrase IX (CAIX) is documented for chronically hypoxic malignant tumors as well as for normoxic renal cell carcinoma. Radionuclide molecular imaging of CAIX would be useful for detection of hypoxic areas in malignant tumors, for patients' stratification for CAIX-targeted therapies, and for discrimination of primary malignant and benign renal tumors. Earlier, we have reported feasibility of in vivo radionuclide based imaging of CAIX expressing tumors using Affibody molecules, small affinity proteins based on a non-immunoglobulin scaffold. In this study, we compared imaging properties of several anti-CAIX Affibody molecules having identical scaffold parts and competing for the same epitope on CAIX, but having different binding paratopes. Four variants were labeled using residualizing Tc-99m and nonresidualizing I-125 labels. All radiolabeled variants demonstrated high affinity detection of CAIX-expressing cell line SK-RC-52 in vitro and specific accumulation in SK-RC-52 xenografts in vivo. I-125-labeled conjugates demonstrated much lower radioactivity uptake in kidneys but higher radioactivity concentration in blood compared with Tc-99m-labeled counterparts. Although all variants cleared rapidly from blood and nonspecific compartments, there was noticeable difference in their biodistribution. The best variant for imaging of expression of CAIX in disseminated cancer was Tc-99m-(HE)(3)-ZCAIX:2 providing tumor uptake of 16.3 +/- 0.9% ID/g and tumor-to-blood ratio of 44 +/- 7 at 4 h after injection. For primary renal cell carcinoma, the most promising imaging candidate was I-125-ZCAIX:4 providing tumor-kidney ratio of 2.1 0.5. In conclusion, several clones of scaffold proteins should be evaluated to select the best variant for development of an imaging probe with optimal sensitivity for the intended application.
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| 35. |
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| 36. |
- Garousi, Javad, et al.
(författare)
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PET imaging of epidermal growth factor receptor expression in tumours using Zr-89-labelled ZEGFR:2377 affibody molecules
- 2016
-
Ingår i: International Journal of Oncology. - : Spandidos. - 1019-6439 .- 1791-2423. ; 48:4, s. 1325-1332
-
Tidskriftsartikel (refereegranskat)abstract
- Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor, which is overexpressed in many types of cancer. The use of EGFR-targeting monoclonal antibodies and tyrosine-kinase inhibitors improves significantly survival of patients with colorectal, non-small cell lung cancer and head and neck squamous cell carcinoma. Detection of EGFR overexpression provides important prognostic and predictive information influencing management of the patients. The use of radionuclide molecular imaging would enable non-invasive repeatable determination of EGFR expression in disseminated cancer. Moreover, positron emission tomography (PET) would provide superior sensitivity and quantitation accuracy in EGFR expression imaging. Affibody molecules are a new type of imaging probes, providing high contrast in molecular imaging. In the present study, an EGFR-binding affibody molecule (ZEGFR:2377) was site-specifically conjugated with a deferoxamine (DFO) chelator and labelled under mild conditions (room temperature and neutral pH) with a radionuclide Zr-89. The Zr-89-DFO-ZEGFR:2377 tracer demonstrated specific high affinity (160 +/- 60 pM) binding to EGFR-expressing A431 epidermoid carcinoma cell line. In mice bearing A431 xenografts, Zr-89-DFO-ZEGFR: 2377 demonstrated specific uptake in tumours and EGFR-expressing tissues. The tracer provided tumour uptake of 2.6 +/- 0.5% ID/g and tumour-to-blood ratio of 3.7 +/- 0.6 at 24 h after injection. Zr-89-DFO-ZEGFR: 2377 provides higher tumour-to-organ ratios than anti-EGFR antibody Zr-89-DFO-cetuximab at 48 h after injection. EGFR-expressing tumours were clearly visualized by microPET using Zr-89-DFO-ZEGFR: 2377 at both 3 and 24 h after injection. In conclusion, Zr-89-DFO-ZEGFR: 2377 is a potential probe for PET imaging of EGFR-expression in vivo.
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| 37. |
- Garousi, Javad, et al.
(författare)
-
The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule
- 2017
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
-
Tidskriftsartikel (refereegranskat)abstract
- Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The In-111-labelled DOTA-conjugated Z(EGFR:2377) Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-Z(EGFR:2377). DOTA-Z(EGFR:2377) was labelled with Co-57 (T-1/2 = 271.8 d), Co-55 (T-1/2 = 17.5 h), and, for comparison, with the positron-emitting radionuclide Ga-68 (T-1/2 = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope Co-57 was used in animal studies. Both Co-57-DOTA-Z(EGFR:2377) and Ga-68-DOTA-Z(EGFR:2377) demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-Z(EGFR:2377) were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, Co-57-DOTA-Z(EGFR:2377) demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for (68)GaDOTA-Z(EGFR:2377). The results of this study suggest that the positron-emitting cobalt isotope 55Co would be an optimal label for DOTA-Z(EGFR:2377) and further development should concentrate on this radionuclide as a label.
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| 38. |
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| 39. |
- Güler, Rezan
(författare)
-
Affibody Molecules Targeting VEGFR2 - Two Turns Off and Four Turns On
- 2020
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The notion of employing proteins as drugs traces back several decades. As recombinant DNA technology emerged, it became a powerful tool for the tailoring of protein traits via genetic approaches - so-called protein engineering. The application of such tools to develop proteins that bind specific molecular targets has seen remarkable clinical success, and today seven out of the ten top-selling drugs in the world belong to this class of proteins. A well-investigated protein scaffold for generating novel target-binding moieties is the small staphylococcal protein A-derived affibody molecule. This thesis revolves around the engineering of affibody-based binding proteins that aim to influence the signaling network in the biological process of blood vessel formation, so-called angiogenesis. The first study in this thesis describes the engineering of a heterodimeric affibody molecule that targets the principal regulating receptor of angiogenesis, vascular endothelial growth factor receptor-2 (VEGFR2). Two separate affibody molecules that bind adjacent VEGFR2 epitopes were previously fused into one biparatopic construct, leading to a remarkable increase in apparent affinity. Further, the biparatopic protein here demonstrated inhibition of vascular endothelial growth factor A (VEGF-A) binding to VEGFR2, and consequently inhibition of VEGFR2 phosphorylation, proliferation and in vitro sprouting of endothelial cells. In the second study, the aim was to evaluate the biparatopic protein as a molecular imaging probe for in vivo visualization of VEGFR2 expression in a glioblastoma multiforme (GBM) brain tumor-model. The results displayed significantly higher probe uptake in tumor compared to normal brain tissue, with a two-hour post injection tumor-to-brain ratio of 78. In the third study, the goal was to mimic the ability of the natural ligand to agonize VEGFR2 via receptor dimerization, and also simulate presentation as extracellular matrix (ECM) bound factors. To this end, the dimeric antagonist was reformatted into a tetrameric construct, hypothesized to bridge two receptor units, and fused to recombinant spider silk. Interestingly, whereas the tetramer displayed agonistic effects both in soluble and immobilized form, the activity of the dimer shifted from antagonistic to agonistic when immobilized. In the fourth study a combined in silico and directed evolution approach was used to increase the thermal stability and hydrophilicity of the biparatopic protein. The final construct demonstrated an increase in melting temperature of about 15°C, complete refolding after heat-induced denaturation and decreased uptake in normal tissues when evaluated in mouse biodistribution studies. In conclusion, this thesis covers the development, characterization and engineering of VEGFR2-binding affibody molecules, aimed for use in research, therapy and diagnosis.
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| 40. |
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| 41. |
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| 42. |
- Güler, Rezan, et al.
(författare)
-
Increasing thermal stability and improving biodistribution of VEGFR2-binding affibody molecules by a combination of in silico and directed evolution approaches
- 2020
-
Ingår i: Scientific Reports. - : Nature Research. - 2045-2322. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- The family of vascular endothelial growth factor (VEGF) ligands and their interactions with VEGF receptors (VEGFRs) play important roles in both pathological and physiological angiogenesis. Hence, agonistic and antagonistic ligands targeting this signaling pathway have potential for both studies on fundamental biology and for development of therapies and diagnostics. Here, we engineer VEGFR2-binding affibody molecules for increased thermostability, refolding and improved biodistribution. We designed libraries based on the original monomeric binders with the intention of reducing hydrophobicity, while retaining high affinity for VEGFR2. Libraries were displayed on bacteria and binders were isolated by fluorescence-activated cell sorting (FACS). In parallel, we used an automated sequence- and structure-based in silico algorithm to identify potentially stabilizing mutations. Monomeric variants isolated from the screening and the in silico approach, respectively, were characterized by circular dichroism spectroscopy and biosensor assays. The most promising mutations were combined into new monomeric constructs which were finally fused into a dimeric construct, resulting in a 15 degrees C increase in melting temperature, complete refolding capability after heat-induced denaturation, retained low picomolar affinity and improved biodistribution profile in an in vivo mouse model. These VEGFR2-binding affibody molecules show promise as candidates for further in vivo studies to assess their suitability as molecular imaging and therapeutic agents.
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| 43. |
- Güler, Rezan, et al.
(författare)
-
VEGFR2-Specific Ligands Based on Affibody Molecules Demonstrate Agonistic Effects when Tetrameric in the Soluble Form or Immobilized via Spider Silk
- 2019
-
Ingår i: ACS Biomaterials Science & Engineering. - : American Chemical Society (ACS). - 2373-9878. ; 5:12, s. 6474-6484
-
Tidskriftsartikel (refereegranskat)abstract
- Strategies to promote vascularization are being developed in order to improve long-term survival of artificial tissue constructs. Vascular endothelial growth factor A (VEGFA) has an important role in both pathological and physiological angiogenesis, mediated by binding to VEGF receptors (VEGFRs). In nature, signaling can be modulated by presentation of growth factors in either soluble form or bound to the extracellular matrix. Herein, a previously reported VEGFR2-binding antagonistic affibody heterodimer (di-Z(VEGFR2)) was formatted into a tetrameric construct (tetra-Z(VEGFR2)) with the intention of generating artificial agonistic ligands for VEGFR2 signaling. In vitro cell assays demonstrated that tetra-Z(VEGFR2) induced VEGFR2 phosphorylation and increased cell proliferation, in contrast to di-Z(VEGFR2). In order to simulate matrix-bound factors, both constructs were fused at the genetic level to a partial spider silk protein, 4RepCT. Assembly of the silk fusion proteins onto a solid surface was verified by quartz crystal microbalance with dissipation analysis. Moreover, surface plasmon resonance studies demonstrated retained VEGFR2 binding ability of di-Z(VEGFR2) silk and tetra-Z(VEGFR2)-silk after silk-mediated immobilization. Cell culture studies demonstrated that VEGFR2-overexpressing cells adhered to di-Z(VEGFR2) -silk and tetra-Z(VEGFR2) -silk and had activated VEGFR2 signaling. Altogether, we demonstrate the potential of especially tetra-Z(VEGFR2)-silk to promote angiogenesis in tissue-engineering applications. The results from the study also show that molecules can obtain completely new functions when presented on materials, and verifying the biological effects after functionalizing materials is thus always recommended.
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| 44. |
- Göstring, Lovisa, et al.
(författare)
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Cellular Effects of HER3-Specific Affibody Molecules
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:6, s. e40023-
-
Tidskriftsartikel (refereegranskat)abstract
- Recent studies have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy.
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| 45. |
- Hjelm, Barbara, et al.
(författare)
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Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase
- 2010
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Ingår i: NEW BIOTECHNOL. - : Elsevier BV. - 1871-6784. ; 27:2, s. 129-137
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Tidskriftsartikel (refereegranskat)abstract
- There is a need to characterize the epitopes of affinity reagents to develop high quality affinity reagents for research, diagnostics and therapy. Here, we describe the analysis of epitopes of antibodies generated toward human tryptophanyl-tRNA synthetase (WARS) using both combinatorial bacterial display and suspension bead array. The bacterial display revealed that the polyclonal antibody binds to three separate epitopes and peptide scanning using 15-mers revealed binding to a 13 amino acid consensus sequence (ELINRIERATGQR). A mouse monoclonal antibody was generated and the mapping approach revealed binding toward a slightly shifted position of the same epitope. Structural analysis showed that the antibodies bind to a-helical regions on the surface of the target protein. An alanine-scanning experiment showed binding to four specific residues. The implications for the systematic analysis of antibody epitopes on the basis of these results are discussed.
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| 46. |
- Hjelm, Barbara, 1983-, et al.
(författare)
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Immunizations of inbred rabbits using the same antigen yield antibodies with similar, but not identical, epitopes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A problem for the generation of polyclonal antibodies is the potential difficulties to obtain a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of in-bred rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen gene on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several in-bred rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.
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| 47. |
- Hjelm, Barbara, et al.
(författare)
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Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:12, s. e45817-
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Tidskriftsartikel (refereegranskat)abstract
- A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.
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| 48. |
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| 49. |
- Hjelm, Linnea C., 1993-, et al.
(författare)
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Affibody Molecules Intended for Receptor-Mediated Transcytosis via the Transferrin Receptor
- 2023
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Ingår i: Pharmaceuticals. - : MDPI. - 1424-8247. ; 16:7
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Tidskriftsartikel (refereegranskat)abstract
- The development of biologics for diseases affecting the central nervous system has been less successful compared to other disease areas, in part due to the challenge of delivering drugs to the brain. The most well-investigated and successful strategy for increasing brain uptake of biological drugs is using receptor-mediated transcytosis over the blood-brain barrier and, in particular, targeting the transferrin receptor-1 (TfR). Here, affibody molecules are selected for TfR using phage display technology. The two most interesting candidates demonstrated binding to human TfR, cross-reactivity to the murine orthologue, non-competitive binding with human transferrin, and binding to TfR-expressing brain endothelial cell lines. Single amino acid mutagenesis of the affibody molecules revealed the binding contribution of individual residues and was used to develop second-generation variants with improved properties. The second-generation variants were further analyzed and showed an ability for transcytosis in an in vitro transwell assay. The new TfR-specific affibody molecules have the potential for the development of small brain shuttles for increasing the uptake of various compounds to the central nervous system and thus warrant further investigations.
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| 50. |
- Hjelm, Linnea C., et al.
(författare)
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Construction and Validation of a New Naive Sequestrin Library for Directed Evolution of Binders against Aggregation-Prone Peptides
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are small affinity proteins that have excellent properties for many different applications, ranging from biotechnology to diagnostics and therapy. The relatively flat binding surface is typically resulting in high affinity and specificity when developing binding reagents for globular target proteins. For smaller unstructured peptides, the paratope of affibody molecules makes it more challenging to achieve a sufficiently large binding surface for high-affinity interactions. Here, we describe the development of a new type of protein scaffold based on a dimeric form of affibodies with a secondary structure content and mode of binding that is distinct from conventional affibody molecules. The interaction is characterized by encapsulation of the target peptide in a tunnel-like cavity upon binding. The new scaffold was used for construction of a high-complexity phage-displayed library and selections from the library against the amyloid beta peptide resulted in identification of high-affinity binders that effectively inhibited amyloid aggregation.
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