Publications by authors named "Raimond Heukers"

31 Publications

Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies.

Nat Commun 2021 07 21;12(1):4436. Epub 2021 Jul 21.

Amsterdam Institute for Molecular and Life Sciences (AIMMS), Division of Medicinal Chemistry, Faculty of Science, VU University, De Boelelaan 1108, Amsterdam, The Netherlands.

Latent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients.
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http://dx.doi.org/10.1038/s41467-021-24608-5DOI Listing
July 2021

Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies.

Nat Commun 2021 07 16;12(1):4357. Epub 2021 Jul 16.

Amsterdam Institute of Molecular and Life Sciences (AIMMS), Division of Medicinal Chemistry, Faculty of Sciences, VU University, Amsterdam, The Netherlands.

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gα protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling.
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http://dx.doi.org/10.1038/s41467-021-24574-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285524PMC
July 2021

Revealing the spatio-phenotypic patterning of cells in healthy and tumor tissues with mLSR-3D and STAPL-3D.

Nat Biotechnol 2021 Jun 3. Epub 2021 Jun 3.

Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning-based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor's spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors.
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http://dx.doi.org/10.1038/s41587-021-00926-3DOI Listing
June 2021

Viral G Protein-Coupled Receptors: Attractive Targets for Herpesvirus-Associated Diseases.

Pharmacol Rev 2021 04;73(2):828-846

In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium (T.W.M.D.G.); Department of Medicine, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom (E.G.E., J.H.S.); Division of Medicinal Chemistry, Faculty of Sciences, Amsterdam Institute for Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (M.S., R.H., M.J.S.); and QVQ Holding B.V., Utrecht, The Netherlands (R.H.)

Herpesviruses are ubiquitous pathogens that establish lifelong, latent infections in their host. Spontaneous reactivation of herpesviruses is often asymptomatic or clinically manageable in healthy individuals, but reactivation events in immunocompromised or immunosuppressed individuals can lead to severe morbidity and mortality. Moreover, herpesvirus infections have been associated with multiple proliferative cardiovascular and post-transplant diseases. Herpesviruses encode viral G protein-coupled receptors (vGPCRs) that alter the host cell by hijacking cellular pathways and play important roles in the viral life cycle and these different disease settings. In this review, we discuss the pharmacological and signaling properties of these vGPCRs, their role in the viral life cycle, and their contribution in different diseases. Because of their prominent role, vGPCRs have emerged as promising drug targets, and the potential of vGPCR-targeting therapeutics is being explored. Overall, these vGPCRs can be considered as attractive targets moving forward in the development of antiviral, cancer, and/or cardiovascular disease treatments. SIGNIFICANCE STATEMENT: In the last decade, herpesvirus-encoded G protein-coupled receptors (GPCRs) have emerged as interesting drug targets with the growing understanding of their critical role in the viral life cycle and in different disease settings. This review presents the pharmacological properties of these viral receptors, their role in the viral life cycle and different diseases, and the emergence of therapeutics targeting viral GPCRs.
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http://dx.doi.org/10.1124/pharmrev.120.000186DOI Listing
April 2021

Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design.

Sci Rep 2021 Feb 2;11(1):2751. Epub 2021 Feb 2.

MassBiologics, University of Massachusetts Medical School, 460 Walk Hill Road, Boston, MA, USA.

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.
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http://dx.doi.org/10.1038/s41598-021-81895-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854682PMC
February 2021

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists.

Proc Natl Acad Sci U S A 2020 11 4;117(46):29144-29154. Epub 2020 Nov 4.

Receptor Signaling Group, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany;

Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.
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http://dx.doi.org/10.1073/pnas.2013319117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682396PMC
November 2020

Monitoring Allosteric Interactions with CXCR4 Using NanoBiT Conjugated Nanobodies.

Cell Chem Biol 2020 10 30;27(10):1250-1261.e5. Epub 2020 Jun 30.

Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK; Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, The Midlands, UK. Electronic address:

Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15-kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognizes the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C terminus to the 11-amino-acid HiBiT tag (VUN400-HiBiT) which complements LgBiT protein, forming a full-length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect allosteric interactions with CXCR4. These data demonstrate that the high specificity offered by extracellular targeted nanobodies can be utilized to probe receptor pharmacology.
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http://dx.doi.org/10.1016/j.chembiol.2020.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573392PMC
October 2020

Natural Killer Cell Hypo-responsiveness in Chronic Lymphocytic Leukemia can be Circumvented In Vitro by Adequate Activating Signaling.

Hemasphere 2019 Dec 30;3(6):e308. Epub 2019 Oct 30.

Amsterdam UMC, University of Amsterdam, Department of Experimental Immunology, Amsterdam Infection and Immunology Institute, Cancer Center Amsterdam, Meibergdreef 9, Amsterdam, the Netherlands.

Chronic lymphocytic leukemia (CLL) is characterized by an acquired immune dysfunction, which may underlie the hampered efficacy of cellular immunotherapy. Most data on dampened immune responses in CLL come from studies investigating CLL and T cell interactions. Natural killer (NK) cells may be an attractive alternative source of effector cells in immunotherapy in CLL, provided that functionality is retained within the CLL micro-environment. Despite their important role in anti-tumor responses, NK cells are not extensively characterized in CLL. Here, we studied the expression of activating and inhibitory receptors on CLL-derived and healthy control (HC) NK cells, and their functional response towards several stimuli. NK cells from CLL patients have an increased maturation stage, with an expansion of NKG2C NK cells in CMV seropositive individuals. The cytotoxicity receptor NKG2D is downregulated, and the killing capacity through this receptor was markedly reduced in CLL-derived NK cells. In contrast, activation via CD16 (FCγRIII) led to adequate activation and functional responses in CLL-derived NK cells. These findings indicate that NK cells in CLL are not intrinsically defect and still perform effector functions upon adequate activating signaling. Clinical relevance of this finding was shown by treatment with novel nanobody-Fc constructs, which induced cytotoxic responses in both CLL- and HC-derived NK cells via CD16. Our results show that NK cells, in contrast to the T cell compartment, retain their function within the CLL micro-environment, provided that they receive an adequate activating signal. These findings warrant future studies on NK cell mediated immunotherapeutic strategies in CLL.
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http://dx.doi.org/10.1097/HS9.0000000000000308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924557PMC
December 2019

VHH-Photosensitizer Conjugates for Targeted Photodynamic Therapy of Met-Overexpressing Tumor Cells.

Antibodies (Basel) 2019 Apr 4;8(2). Epub 2019 Apr 4.

Cell Biology Division, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT.
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http://dx.doi.org/10.3390/antib8020026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640711PMC
April 2019

The human cytomegalovirus-encoded G protein-coupled receptor UL33 exhibits oncomodulatory properties.

J Biol Chem 2019 11 13;294(44):16297-16308. Epub 2019 Sep 13.

Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit, 1081 HZ Amsterdam, The Netherlands

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gα, Gα, and Gα proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth and accelerated tumor growth in different tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.
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http://dx.doi.org/10.1074/jbc.RA119.007796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827316PMC
November 2019

Nanobody-Targeted Photodynamic Therapy Selectively Kills Viral GPCR-Expressing Glioblastoma Cells.

Mol Pharm 2019 07 19;16(7):3145-3156. Epub 2019 Jun 19.

Division of Cell Biology, Department of Biology , Utrecht University , 3584 CH Utrecht , The Netherlands.

Photodynamic therapy (PDT) eradicates tumors by the local activation of a photosensitizer with near-infrared light. One of the aspects hampering the clinical use of PDT is the poor selectivity of the photosensitizer. To improve this, we have recently introduced a new approach for targeted PDT by conjugating photosensitizers to nanobodies. Diverse G protein-coupled receptors (GPCRs) show aberrant overexpression in tumors and are therefore interesting targets in cancer therapy. Here we show that GPCR-targeting nanobodies can be used in targeted PDT. We have developed a nanobody binding the extracellular side of the viral GPCR US28, which is detected in tumors like glioblastoma. The nanobody was site-directionally conjugated to the water-soluble photosensitizer IRDye700DX. This nanobody-photosensitizer conjugate selectively killed US28-expressing glioblastoma cells both in 2D and 3D cultures upon illumination with near-infrared light. This is the first example employing a GPCR as target for nanobody-directed PDT. With the emerging role of GPCRs in cancer, this data provides a new angle for exploiting this large family of receptors for targeted therapies.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728091PMC
July 2019

Nanobodies detecting and modulating GPCRs outside in and inside out.

Curr Opin Cell Biol 2019 04 5;57:115-122. Epub 2019 Mar 5.

Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Sciences, VU University, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands. Electronic address:

G protein-coupled receptors (GPCRs), belonging to the largest class of membrane proteins, play a prominent role in many (patho)physiological processes and are, therefore, important drug targets. Although most often targeted by small molecules, these receptors have become interesting targets for antibodies and antibody fragments, especially camelid-derived heavy chain-only antibodies and fragments thereof (nanobodies). The small size and molecular structure of nanobodies allow GPCR-binding and modulation, from both the intracellular and extracellular sides. These molecular features make nanobodies attractive tools to study, modulate, and exploit GPCRs. Besides modulating GPCR activity as monovalent or multivalent constructs, nanobodies can also be functionalized for imaging and therapy. Moreover, GPCR-binding nanobodies have been instrumental in obtaining crystal structures of GPCRs, facilitating structure-based drug discovery. Here, we describe the current status and future perspectives of nanobodies targeting GPCRs intra and extracellularly.
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http://dx.doi.org/10.1016/j.ceb.2019.01.003DOI Listing
April 2019

Nanobodies: New avenues for imaging, stabilizing and modulating GPCRs.

Mol Cell Endocrinol 2019 03 26;484:15-24. Epub 2019 Jan 26.

Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1108, 1081 HZ, Amsterdam, the Netherlands. Electronic address:

The family of G protein-coupled receptors (GPCRs) is the largest class of membrane proteins and an important drug target due to their role in many (patho)physiological processes. Besides small molecules, GPCRs can be targeted by biologicals including antibodies and antibody fragments. This review describes the use of antibodies and in particular antibody fragments from camelid-derived heavy chain-only antibodies (nanobodies/VHHs/sdAbs) for detecting, stabilizing, modulating and therapeutically targeting GPCRs. Altogether, it becomes increasingly clear that the small size, structure and protruding antigen-binding loops of nanobodies are favorable features for the development of selective and potent GPCRs-binding molecules. This makes them attractive tools to modulate GPCR activity but also as targeting modalities for GPCR-directed therapeutics. In addition, these antibody-fragments are important tools in the stabilization of particular conformations of these receptors. Lastly, nanobodies, in contrast to conventional antibodies, can also easily be expressed intracellularly which render nanobodies important tools for studying GPCR function. Hence, GPCR-targeting nanobodies are ideal modalities to image, stabilize and modulate GPCR function.
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http://dx.doi.org/10.1016/j.mce.2019.01.021DOI Listing
March 2019

Heterogeneity assessment of antibody-derived therapeutics at the intact and middle-up level by low-flow sheathless capillary electrophoresis-mass spectrometry.

Anal Chim Acta 2018 Dec 15;1044:181-190. Epub 2018 Aug 15.

Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands.

Antibody-based pharmaceuticals often encompass a complex structural heterogeneity requiring enhanced analytical methods for reliable characterization of variants and degradation products. We have explored the capabilities of low-flow sheathless capillary electrophoresis-mass spectrometry (CE-MS) for the high-resolution and sensitive profiling of antibody therapeutics. Near-zero electroosmotic flow was achieved by employing a novel neutral capillary coating that also prevents protein adsorption. CE-MS analysis of intact model proteins using an acidic background electrolyte demonstrated satisfactory performance, with overall migration-time RSDs below 2.2% from three different capillaries tested. For system evaluation, three nanobody preparations, including mono- and bivalent forms, and three monoclonal antibodies (mAbs) were analyzed. Intact nanobodies were resolved from their degradation products, which could be assigned to deamidated, cleaved, and truncated forms at the C-terminal tag. Excellent resolution of isomeric deamidated products was obtained. The mAbs were analyzed intact and after digestion by the endoproteinase IdeS (middle-up approach). CE-MS of intact mAbs provided resolution of clipped species (e.g. light chain and light chain-heavy chain fragments) from the native protein. Moreover, glycoforms containing sialic acids were resolved from their non-sialylated counterparts. For IdeS-digested, F (ab) and Fc/2 portions where efficiently resolved for the three mAbs. Whereas the migration time of the Fc/2 fragments was fairly similar, the migration time of the F (ab) part was strongly varied among the mAbs. For all mAbs, separation of Fc/2 charge variants - including sialylated glycoforms and other post-translational modifications, such as loss of C-terminal lysine or asparagine deamidation - was achieved. This allowed a detailed and reliable assessment of the Fc/2 heterogeneity (18-33 proteoforms) of the three analyzed mAbs.
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http://dx.doi.org/10.1016/j.aca.2018.08.024DOI Listing
December 2018

CXCR4-targeting nanobodies differentially inhibit CXCR4 function and HIV entry.

Biochem Pharmacol 2018 12 17;158:402-412. Epub 2018 Oct 17.

Department of Microbiology and Immunology, Laboratory of Virology and Chemotherapy, Rega Institute, KU Leuven, Herestraat 49, 3000 Leuven, Belgium. Electronic address:

The chemokine receptor CXCR4 and its ligand CXCL12 contribute to a variety of human diseases, such as cancer. CXCR4 is also a major co-receptor facilitating HIV entry. Accordingly, CXCR4 is considered as an attractive therapeutic target. Drug side effects and poor pharmacokinetic properties have been major hurdles that have prevented the implementation of CXCR4-directed inhibitors in treatment regimes. We evaluated the activity of a new and promising class of biologics, namely CXCR4-targeting nanobodies, with the purpose of identifying nanobodies that would preferentially inhibit HIV infection, while minimally disturbing other CXCR4-related functions. All CXCR4-interacting nanobodies inhibited CXCL12 binding and receptor-mediated calcium mobilization with comparable relative potencies. Importantly, the anti-HIV-1 activity of the nanobodies did not always correlate with their ability to modulate CXCR4 signaling and function, indicating that the anti-HIV and anti-CXCR4 activity are not entirely overlapping and may be functionally separated. Three nanobodies with divergent activity profiles (VUN400, VUN401 and VUN402) were selected for in depth biological evaluation. While all three nanobodies demonstrated inhibitory activity against a wide range of HIV (X4) strains, VUN402 poorly blocked CXCL12-induced CXCR4 internalization, chemotaxis and changes in cell morphology. Each of these nanobodies recognized distinct, although partially overlapping epitopes on CXCR4, which might underlie their distinct activity profiles. Our results demonstrate the potential of CXCR4-targeting nanobody VUN402 as a novel lead and starting point for the development of a more potent and selective anti-HIV agent.
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http://dx.doi.org/10.1016/j.bcp.2018.10.015DOI Listing
December 2018

Nanobody-Fc constructs targeting chemokine receptor CXCR4 potently inhibit signaling and CXCR4-mediated HIV-entry and induce antibody effector functions.

Biochem Pharmacol 2018 12 17;158:413-424. Epub 2018 Oct 17.

Division of Medicinal Chemistry, Amsterdam Institute for Molecules Medicines and Systems (AIMMS), VU University Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, the Netherlands. Electronic address:

Upregulation of the chemokine receptor CXCR4 contributes to the progression and metastasis of both solid and hematological malignancies, rendering this receptor an attractive therapeutic target. Besides the only FDA-approved CXCR4 antagonist Plerixafor (AMD3100), multiple other classes of CXCR4-targeting molecules are under (pre-)clinical development. Nanobodies (Nb), small single variable domains of heavy-chain only antibodies from Camelids, have appeared to be ideal antibody-fragments for targeting a broad range of epitopes and cavities within GPCRs such as CXCR4. Compared to conventional antibodies, monovalent nanobodies show fast blood clearance and no effector functions. In order to further increase their binding affinities and to restore antibody-mediated effector functions, we have constructed three different bivalent nanobody Fc-fusion molecules (Nb-Fc), targeting distinct epitopes on CXCR4, via fusion of Nbs to a Fc domain of a human IgG1 antibody. Most Nb-Fc constructs show increased binding affinity and enhanced potency in CXCL12 displacement, inhibition of CXCL12-induced signaling and CXCR4-mediated HIV entry, when compared to their monovalent Nb counterparts. Moreover, Nb-Fc induced ADCC- and CDC-mediated cell-death of CXCR4-overexpressing CCRF-CEM leukemia cells and did not affect cells expressing low levels or no CXCR4. These highly potent CXCR4 Nb-Fc constructs with Fc-mediated effector functions are attractive molecules to therapeutically target CXCR4-overexpressing tumors.
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http://dx.doi.org/10.1016/j.bcp.2018.10.014DOI Listing
December 2018

The constitutive activity of the virally encoded chemokine receptor US28 accelerates glioblastoma growth.

Oncogene 2018 07 30;37(30):4110-4121. Epub 2018 Apr 30.

Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1108, Amsterdam, 1081 HZ, The Netherlands.

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.
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http://dx.doi.org/10.1038/s41388-018-0255-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062493PMC
July 2018

CXCR4-Specific Nanobodies as Potential Therapeutics for WHIM syndrome.

J Pharmacol Exp Ther 2017 10 2;363(1):35-44. Epub 2017 Aug 2.

Division of Medicinal Chemistry, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam, Amsterdam, The Netherlands (R.H.d.W., R.H., H.J.B., A.A., D.M., H.F.V, M.J.S.); Inflammation Chemokines and Immunopathology, INSERM, Faculté de Médicine-Université Paris-Sud, Université Paris-Saclay, Clamart, France (P.C., F.B.); and Ablynx N.V., Zwijnaarde, Belgrium (B.S.)

WHIM syndrome is a rare congenital immunodeficiency disease, named after its main clinical manifestations: warts, hypogammaglobulinemia, infections, and myelokathexis, which refers to abnormal accumulation of mature neutrophils in the bone marrow. The disease is primarily caused by C-terminal truncation mutations of the chemokine receptor CXCR4, giving these CXCR4-WHIM mutants a gain of function in response to their ligand CXCL12. Considering the broad functions of CXCR4 in maintaining leukocyte homeostasis, patients are panleukopenic and display altered immune responses, likely as a consequence of impairment in the differentiation and trafficking of leukocytes. Treatment of WHIM patients currently consists of symptom relief, leading to unsatisfactory clinical responses. As an alternative and potentially more effective approach, we tested the potency and efficacy of CXCR4-specific nanobodies on inhibiting CXCR4-WHIM mutants. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies. They combine the advantages of small-molecule drugs and antibody-based therapeutics due to their relative small size, high stability, and high affinity. We compared the potential of monovalent and bivalent CXCR4-specific nanobodies to inhibit CXCL12-induced CXCR4-WHIM-mediated signaling with the small-molecule clinical candidate AMD3100. The CXCR4-targeting nanobodies displace CXCL12 binding and bind CXCR4-wild type and CXCR4-WHIM (R334X/S338X) mutants and with (sub-) nanomolar affinities. The nanobodies' epitope was mapped to extracellular loop 2 of CXCR4, overlapping with the binding site of CXCL12. Monovalent, and in particular bivalent, nanobodies were more potent than AMD3100 in reducing CXCL12-mediated G protein activation. In addition, CXCR4-WHIM-dependent calcium flux and wound healing of human papillomavirus-immortalized cell lines in response to CXCL12 was effectively inhibited by the nanobodies. Based on these in vitro results, we conclude that CXCR4 nanobodies hold significant potential as alternative therapeutics for CXCR4-associated diseases such as WHIM syndrome.
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http://dx.doi.org/10.1124/jpet.117.242735DOI Listing
October 2017

Class III antiarrhythmic drugs amiodarone and dronedarone impair K 2.1 backward trafficking.

J Cell Mol Med 2017 10 19;21(10):2514-2523. Epub 2017 Apr 19.

Division of Heart & Lungs, Department of Medical Physiology, UMCU, Utrecht, The Netherlands.

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. K 2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (I ), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in K 2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited I in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK-KWGF cells, both drugs dose- and time-dependently increased K 2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 μM, 24 hrs) and late-endosomal/lysosomal K 2.1 accumulation. Increased K 2.1 expression level was also observed in the presence of Na 1.5 co-expression. Augmented K 2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on K 11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at -120 mV, 5 μM) enhanced I upon 24-hrs treatment, whereas dronedarone tended to increase I and it did not reach significance (43.8 ± 5.5%, P = 0.26 at -120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced I by inhibiting K 2.1 degradation.
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http://dx.doi.org/10.1111/jcmm.13172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618701PMC
October 2017

EGFR targeted nanobody-photosensitizer conjugates for photodynamic therapy in a pre-clinical model of head and neck cancer.

J Control Release 2016 05 15;229:93-105. Epub 2016 Mar 15.

Molecular Oncology, Cell Biology Division, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Electronic address:

Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer (PS) and has been used to treat head and neck cancers. Yet, common PS lack tumor specificity, which leads to collateral damage to normal tissues. Targeted delivery of PS via antibodies has pre-clinically improved tumor selectivity. However, antibodies have long half-lives and relatively poor tissue penetration, which could limit therapeutic efficacy and lead to long photosensitivity. Here, in this feasibility study, we evaluate at the pre-clinical level a recently introduced format of targeted PDT, which employs nanobodies as targeting agents and a water-soluble PS (IRDye700DX) that is traceable through optical imaging. In vitro, the PS solely binds to cells and induces phototoxicity on cells overexpressing the epidermal growth factor receptor (EGFR), when conjugated to the EGFR targeted nanobodies. To investigate whether this new format of targeted PDT is capable of inducing selective tumor cell death in vivo, PDT was applied on an orthotopic mouse tumor model with illumination at 1h post-injection of the nanobody-PS conjugates, as selected from quantitative fluorescence spectroscopy measurements. In parallel, and as a reference, PDT was applied with an antibody-PS conjugate, with illumination performed 24h post-injection. Importantly, EGFR targeted nanobody-PS conjugates led to extensive tumor necrosis (approx. 90%) and almost no toxicity in healthy tissues, as observed through histology 24h after PDT. Overall, results show that these EGFR targeted nanobody-PS conjugates are selective and able to induce tumor cell death in vivo. Additional studies are now needed to assess the full potential of this approach to improving PDT.
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http://dx.doi.org/10.1016/j.jconrel.2016.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116242PMC
May 2016

Depleting MET-Expressing Tumor Cells by ADCC Provides a Therapeutic Advantage over Inhibiting HGF/MET Signaling.

Cancer Res 2015 Aug 3;75(16):3373-83. Epub 2015 Jul 3.

Department of Oncology, University of Torino Medical School, Candiolo, Turin, Italy. Laboratory of Experimental Therapy, Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Turin, Italy.

Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-0356DOI Listing
August 2015

Nanobody-photosensitizer conjugates for targeted photodynamic therapy.

Nanomedicine 2014 Oct 3;10(7):1441-51. Epub 2014 Jan 3.

Molecular Oncology, Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, the Netherlands. Electronic address:

Photodynamic therapy (PDT) induces cell death through light activation of a photosensitizer (PS). Targeted delivery of PS via monoclonal antibodies has improved tumor selectivity. However, these conjugates have long half-lives, leading to relatively long photosensitivity in patients. In an attempt to target PS specifically to tumors and to accelerate PS clearance, we have developed new conjugates consisting of nanobodies (NB) targeting the epidermal growth factor receptor (EGFR) and a traceable PS (IRDye700DX). These fluorescent conjugates allow the distinction of cell lines with different expression levels of EGFR. Results show that these conjugates specifically induce cell death of EGFR overexpressing cells in low nanomolar concentrations, while PS alone or the NB-PS conjugates in the absence of light induce no toxicity. Delivery of PS using internalizing biparatopic NB-PS conjugates results in even more pronounced phototoxicities. Altogether, EGFR-targeted NB-PS conjugates are specific and potent, enabling the combination of molecular imaging with cancer therapy. From the clinical editor: This study investigates the role of EGFR targeting nanobodies to deliver traceable photosensitizers to cancer molecules for therapeutic exploitation and concomitant imaging. Altogether, EGFR-targeted NB-PS conjugates combine molecular imaging with cancer therapy, the method is specific and potent, paving the way to clinical application of this technology.
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http://dx.doi.org/10.1016/j.nano.2013.12.007DOI Listing
October 2014

Analysis of EGF receptor oligomerization by homo-FRET.

Methods Cell Biol 2013 ;117:305-21

Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, Netherlands.

Growth factor receptors are present in the plasma membrane of resting cells as monomers or (pre)dimers. Ligand binding results in higher-order oligomerization of ligand-receptor complexes. To study the regulation of receptor clustering, several experimental techniques have been developed in the last decades. However, many involve invasive approaches that are likely to disturb the integrity of the membrane, thereby affecting receptor interactions. In this chapter, we describe the use of a noninvasive approach to study receptor dimerization and oligomerization. This method is based upon the Förster energy transfer between identical adjacent fluorescent proteins (homo-FRET) and is determined by analyzing the change in fluorescence anisotropy. Homo-FRET takes place within a distance of 10nm, making this an excellent approach for studying receptor-receptor interactions in intact cells. After excitation of monomeric GFP (mGFP) with polarized light, limiting anisotropy values (r(inf)) of the emitted light are determined, where proteins with known cluster sizes are used as references. Dimerization and oligomerization of the epidermal growth factor receptor (EGFR) in response to ligand binding is determined by using receptors that have been fused with mGFP at their C-terminus. In this chapter, we describe the involved technology and discuss the feasibility of homo-FRET experiments for the determination of cluster sizes of growth factor receptors like EGFR.
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http://dx.doi.org/10.1016/B978-0-12-408143-7.00016-5DOI Listing
June 2014

Targeting hepatocyte growth factor receptor (Met) positive tumor cells using internalizing nanobody-decorated albumin nanoparticles.

Biomaterials 2014 Jan 16;35(1):601-10. Epub 2013 Oct 16.

Cell Biology, Department of Biology, Science Faculty, Utrecht University, 3584 CH Utrecht, The Netherlands.

The hepatocyte growth factor receptor (HGFR, c-Met or Met) is a receptor tyrosine kinase that is involved in embryogenesis, tissue regeneration and wound healing. Abnormal activation of this proto-oncogene product is implicated in the development, progression and metastasis of many cancers. Current therapies directed against Met, such as ligand- or, dimerization-blocking antibodies or kinase inhibitors, reduce tumor growth but hardly eradicate the tumor. In order to improve anti-Met therapy, we have designed a drug delivery system consisting of crosslinked albumin nanoparticles decorated with newly selected anti-Met nanobodies (anti-Met-NANAPs). The anti-Met NANAPs bound specifically to and were specifically taken up by Met-expressing cells and transported to lysosomes for degradation. Treatment of tumor cells with anti-Met NANAPs also resulted in downregulation of the total Met protein. This study shows that anti-Met NANAPs offer a potential system for lysosomal delivery of drugs into Met-positive tumor cells.
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http://dx.doi.org/10.1016/j.biomaterials.2013.10.001DOI Listing
January 2014

Targeting tumors with nanobodies for cancer imaging and therapy.

J Control Release 2013 Dec 11;172(3):607-17. Epub 2013 Sep 11.

Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.

The use of monoclonal antibodies has revolutionized both cancer therapy and cancer imaging. Antibodies have been used to directly inhibit tumor cell proliferation or to target drugs to tumors. Also in molecular imaging, monoclonal antibodies have found their way to the clinic. Nevertheless, distribution within tumors is hampered by their size, leading to insufficient efficacy of cancer treatment and irregular imaging. An attractive alternative for monoclonal antibodies are nanobodies or VHHs. These are the variable domain of heavy-chain antibodies from animals from the Camelidae family that were first discovered in 1993. Stimulated by the ease of nanobody selection, production, and low immunogenicity potential, a number of nanobodies specific to different disease-related targets have been developed. For cancer therapy, nanobodies have been employed as antagonistic drugs, and more recently, as targeting moieties of effector-domaINS and of drug delivery systems. In parallel, nanobodies have also been employed for molecular imaging with modalities such as nuclear and optical imaging. In this review, we discuss recent developments in the application of nanobodies as targeting moieties in cancer therapy and cancer imaging. With such a wide range of successful applications, nanobodies have become much more than simple antagonists.
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http://dx.doi.org/10.1016/j.jconrel.2013.08.298DOI Listing
December 2013

Endocytosis of EGFR requires its kinase activity and N-terminal transmembrane dimerization motif.

J Cell Sci 2013 Nov 13;126(Pt 21):4900-12. Epub 2013 Aug 13.

Cell Biology, Department of Biology, Science Faculty, Utrecht University, 3584 CH Utrecht, The Netherlands.

EGFR signaling is attenuated by endocytosis and degradation of receptor-ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple post-translational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization motif eventually lead to reduced EGFR degradation and sustained signaling. We propose a novel role for the TMD dimerization motif in the negative-feedback control of EGFR. The widely conserved nature of GG4-like dimerization motifs in transmembrane proteins suggests a general role for these motifs in clustering-induced internalization.
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http://dx.doi.org/10.1242/jcs.128611DOI Listing
November 2013

Inhibition of tumor growth by targeted anti-EGFR/IGF-1R nanobullets depends on efficient blocking of cell survival pathways.

Mol Pharm 2013 Oct 23;10(10):3717-27. Epub 2013 Aug 23.

Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University , Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.

The clinical efficacy of epidermal growth factor receptor (EGFR)-targeted inhibitors is limited due to resistance mechanisms of the tumor such as activation of compensatory pathways. Crosstalk between EGFR and insulin-like growth factor 1 (IGF-1R) signaling has been frequently described to be involved in tumor proliferation and resistance. One of the attractive features of nanomedicines is the possibility to codeliver agents that inhibit different molecular targets in one nanocarrier system, thereby strengthening the antitumor effects of the individual agents. Additionally, exposure to healthy tissues and related unwanted side-effects can be reduced. To this end, we have recently developed anti-EGFR nanobody (Nb)-liposomes loaded with the anti-IGF-1R kinase inhibitor AG538, which showed promising antiproliferative effects in vitro. In the present study, we have further evaluated the potential of this dual-active nanomedicine in vitro and for the first time in vivo. As intended, the nanomedicine inhibited EGFR and IGF-1R signaling and subsequent activation of downstream cell proliferation and survival pathways. The degree of inhibition induced by the nanomedicine on a molecular level correlated with cytotoxicity in tumor cell proliferation assays and may even be predictive of the response to nanomedicine treatment in tumor xenograft models. Combination therapy with kinase inhibitor-loaded Nb-liposomes is therefore an appealing strategy for inhibiting the proliferation of tumors that are highly dependent on EGFR and IGF-1R signaling.
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http://dx.doi.org/10.1021/mp400212vDOI Listing
October 2013

Inhibiting the clathrin-mediated endocytosis pathway rescues K(IR)2.1 downregulation by pentamidine.

Pflugers Arch 2013 Feb 29;465(2):247-59. Epub 2012 Nov 29.

Department of Medical Physiology, Division of Heart & Lungs, UMCU, Yalelaan, Utrecht, The Netherlands.

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. We showed that the antiprotozoic pentamidine decreased K(IR)2.x carried I(K1) current and that inhibiting protein degradation in the lysosome increased intracellular K(IR)2.1 levels. In this study, we aim to identify and then inhibit preceding steps in clathrin-mediated endocytosis of K(IR)2.1 to further restore normal levels of functional K(IR)2.1 channels. K(IR)2.1 trafficking in HEK293 cells was studied by live cell imaging, immunofluorescence microscopy, and Western blot following pharmacological intervention with dynasore (Dyn), chlorpromazine (CPZ), bafilomycin A1 (Baf), or chloroquine (CQ). K(IR)2.1 function was determined by patch-clamp electrophysiology. CQ induced lysosomal build-up of full length (3.8 ± 0.8-fold) and N-terminal cleaved K(IR)2.1 protein. Baf induced late endosomal build-up of full length protein only (6.1 ± 1.6-fold). CPZ and Dyn increased plasma membrane-localized channel and protein levels (2.6 ± 0.4- and 4.2 ± 1.1-fold, respectively). Dyn increased I(K1) (at -60 mV) from 31 ± 6 to 55 ± 7 pA/pF (N = 9 and 13 respectively, p < 0.05), while the CPZ effect on current density was not testable due to acute I(K1) block. Baf and CQ did not significantly enhance I(K1) densities. Pentamidine (10 μM, 48 h) reduced K(IR)2.1 levels to 0.6 ± 0.1-fold, which could be rescued by Baf (3.2 ± 0.9), CPZ (1.2 ± 0.3), or Dyn (1.2 ± 0.3). Taken together, the clathrin-mediated endocytosis pathway functions in K(IR)2.1 degradation. Pentamidine-induced downregulation of K(IR)2.1 can be rescued at the level of the plasma membrane, implying that acquired trafficking defects can be rescued.
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http://dx.doi.org/10.1007/s00424-012-1189-5DOI Listing
February 2013

Nanobody-albumin nanoparticles (NANAPs) for the delivery of a multikinase inhibitor 17864 to EGFR overexpressing tumor cells.

J Control Release 2013 Jan 16;165(2):110-8. Epub 2012 Nov 16.

Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.

A novel, EGFR-targeted nanomedicine has been developed in the current study. Glutaraldehyde crosslinked albumin nanoparticles with a size of approximately 100nm were loaded with the multikinase inhibitor 17864-L(x)-a platinum-bound sunitinib analogue-which couples the drug to methionine residues of albumin and is released in a reductive environment. Albumin nanoparticles were surface-coated with bifunctional polyethylene glycol 3500 (PEG) and a nanobody-the single variable domain of an antibody-(Ega1) against the epidermal growth factor receptor (EGFR). EGa1-PEG functionalized nanoparticles showed a 40-fold higher binding to EGFR-positive 14C squamous head and neck cancer cells in comparison to PEGylated nanoparticles. 17864-L(x) loaded EGa1-PEG nanoparticles were internalized by clathrin-mediated endocytosis and ultimately digested in lysosomes. The intracellular routing of EGa1 targeted nanoparticles leads to a successful release of the kinase inhibitor in the cell and inhibition of proliferation whereas the non-targeted formulations had no antiproliferative effects on 14C cells. The drug loaded targeted nanoparticles were as effective as the free drug in vitro. These results demonstrate that multikinase inhibitor loaded nanoparticles are interesting nanomedicines for the treatment of EGFR-positive cancers.
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http://dx.doi.org/10.1016/j.jconrel.2012.11.007DOI Listing
January 2013

Indole-3-carbinol-induced modulation of NF-kappaB signalling is breast cancer cell-specific and does not correlate with cell death.

Breast Cancer Res Treat 2008 Jun 26;109(3):451-62. Epub 2007 Jul 26.

Departments of Biochemistry and Cancer Studies, Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, Leicester, LE1 7RH, UK.

Indole-3-carbinol (I3C), a dietary chemopreventive compound, induces cell death in human breast cancer cells by modulating activities of Src and epidermal growth factor receptor (EGFR). The effect of I3C on NF-kappaB, constitutively activated in breast cancer cells, was investigated. Nuclear extracts of MDA-MB-468, MDA-MB-231 and HBL100 cells contained all of the Rel proteins with similar expression patterns in the latter two. The level of NF-kappaB-regulated reporter gene expression was in the order HBL100 << MDA-MB-468 << MDA-MB-231. Upstream inhibition, using PI3K, EGFR or IKKbeta inhibitors, resulted in cell-specific effects on expression of the NF-kappaB-regulated reporter gene and endogenous genes Bcl-xL, IkappaBalpha and IL-6, as well as on cell viability. The expression patterns of Rel and several NF-kappaB-regulated genes and the response to LY249002 in MDA-MB-468 cells contrasted with those in other cells. I3C induced NF-kappaB-regulated reporter gene expression at 12 h in MDA-MB-468 cells. Conversely, it was reduced at 24 h in HBL100 cells. I3C treatment for 6 h alone or in combination with TNFalpha induced NF-kappaB-regulated reporter gene expression, detected 5 h later, in MDA-MB-468, but not HBL100 cells. I3C induced NF-kappaB p65/p50 DNA binding at 6.5 h, preceded by association of IKKbeta with the Src/EGFR complex and increased phospho-IkappaBalpha in MDA-MB468 cells. TNFalpha increased I3C-induced apoptosis in MDA-MB-468 and MDA-MB-231 cells. It also induced apoptosis, enhanced by I3C, in HBL100 cells. Hence, regulation of constitutive NF-kappaB was cell-specific. I3C influenced the NF-kappaB pathway in a cell-specific manner, which was not related to apoptosis. However, the combination of I3C and TNFalpha increased apoptosis in all cell lines.
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http://dx.doi.org/10.1007/s10549-007-9669-6DOI Listing
June 2008
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