| 1. |
- Faresjö, Rebecca, et al.
(författare)
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Single domain antibody conjugated to Aβ-binding scFv penetrates BBB via TfR to interact with Aβ
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: Camelid antibody fragments are interesting for use as radioligands for Positron Emission Tomography (PET), in central nervous system imaging, due to their fast clearance from blood. This study evaluated single variable domain of heavy chain (VHH) antibodies derived from llama, targeting the mouse and human transferrin receptor (TfR) for mediating increased brain uptake. In experiments, VHHs were combined with either a human Fc or with the single chain fragment of the amyloid beta (Aβ) antibody 3D6 (scFv3D6) to investigate intrabrain targeting.Methods: One novel and one previously disclosed species cross-reactive VHH towards murine TfR (mTfR) and human TfR (hTfR), as well as two VHHs with selective reactivity to mTfR and hTfR, respectively, were compared. The TfR binders were evaluated as recombinant fusion protein (FP) constructs fused with either a human Fc-fragment (FPFc) or with the Aβ-binding fragment scFv3D6 (FPscFv) at either C- or N-terminal positions of scFv3D6. The above FPs were radiolabeled with iodine-125 (125I) and biodistribution was studied ex vivo at 2 h, 6 h and 24 h after injection in wild-type (WT) mice and AD mouse model AppNL-G-F. Brain, blood, plasma and organ concentrations of the 125I-FPs were measured in a γ-counter. Autoradiography, nuclear track emulsion, and immunohistofluorescence imaging were used to study the brain distribution of the FPs. Results: The constructs based on Fc fusions (FPFc) with binding affinity to mTfR displayed significantly higher brain uptake (around 1-3% ID/gbrain) in comparison with FPFc specific to only hTfR (control; 0.2% ID/gbrain). The VHHs reactive to mTfR fused to a scFv (FPscFv) showed an increased brain uptake 2 h after injection compared to control (FPscFv reactive to hTfR only). FPscFv with VHH linked to the N-terminus of scFv3D6 showed more efficient brain delivery than those fused with the C-terminal of scFv3D6. There was a 17-fold higher brain uptake in AppNL-G-F than WT mice for one of the cross-species reactive FPscFv (FPscFv1B) at 24 h post-injection, and 2.5-fold higher at 6 h, in ex vivo studies. FPscFv1B also showed consistently higher relative brain parenchymal localization compared to the other FPs, whether as Fc- or scFv fusion. Conclusion: We showed that the novel cross-reactive VHHs tested herein displayed enhanced brain delivery in mice and that these could be successfully fused with an Aβ-binding scFv-fragment, maintaining high brain and preferential parenchymal delivery with increased retention to Aβ in brain. In summary, a FPscFv construct with affinity towards both Aβ and mTfR showed differentiated and favorable distribution in AD-mice compared to WT already after 6 h (measured ex vivo); a relevant time point for clinical brain PET.
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| 2. |
- Gordon, Emma J., et al.
(författare)
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The endothelial adaptor molecule TSAd is required for VEGF-induced angiogenic sprouting through junctional c-Src activation
- 2016
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 9:437
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Tidskriftsartikel (refereegranskat)abstract
- Activation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) by VEGF binding is critical for vascular morphogenesis. In addition, VEGF disrupts the endothelial barrier by triggering the phosphorylation and turnover of the junctional molecule VE-cadherin, a process mediated by the VEGFR2 downstream effectors T cell-specific adaptor (TSAd) and the tyrosine kinase c-Src. We investigated whether the VEGFR2-TSAd-c-Src pathway was required for angiogenic sprouting. Indeed, Tsad-deficient embryoid bodies failed to sprout in response to VEGF. Tsad-deficient mice displayed impaired angiogenesis specifically during tracheal vessel development, but not during retinal vasculogenesis, and in VEGF-loaded Matrigel plugs, but not in those loaded with FGF. The SH2 and proline-rich domains of TSAd bridged VEGFR2 and c-Src, and this bridging was critical for the localization of activated c-Src to endothelial junctions and elongation of the growing sprout, but not for selection of the tip cell. These results revealed that vascular sprouting and permeability are both controlled through the VEGFR2-TSAd-c-Src signaling pathway in a subset of tissues, which may be useful in developing strategies to control tissue-specific pathological angiogenesis.
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| 3. |
- Li, Xiujuan, et al.
(författare)
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VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2(Y949F/Y949F) leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2(Y949F/Y949F) mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2(Y949F/Y949F) mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms.
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| 4. |
- Roche, Francis P., et al.
(författare)
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Leukocyte differentiation by histidine-rich glycoprotein/stanniocalcin-2 complex regulates murine glioma growth through modulation of anti-tumor immunity
- 2018
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Ingår i: Molecular Cancer Therapeutics. - 1535-7163 .- 1538-8514. ; 17:9, s. 1961-1972
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Tidskriftsartikel (refereegranskat)abstract
- The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and anti-tumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and co-localization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG treated gliomas displayed decreased numbers of Interleukin-35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control anti-tumor immunity.
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