Alex Klarenbeek | Vrije Universiteit Amsterdam (original) (raw)

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Papers by Alex Klarenbeek

Nowadays therapeutic antibodies are gaining ground To validate camelid conventional type antibodi... more Nowadays therapeutic antibodies are gaining ground To validate camelid conventional type antibodies as a source for therapeutic antibodies, their human homology in both conformation (shape) and sequence is analyzed. To thoroughly assess (germline) sequence homology, human immunoglobulin variable regions (V regions) counterparts are identified amongst camelid (alpaca (Lama pacos), llama (Lama glama), and camel (Camelis ferus)) species. As human homology controls, cynomolgous monkey and murine immunoglobulin V regions are used (positive and negative respectively). In addition, two earlier selected Lama glama Fab fragments are crystalized and aligned with their corresponding human family members. Results indicate high sequence/structural homology in camelid heavy chain variable domain regions and a sound structural resemblance to human Fab domains. This is however not seen for other “target” proteins, allowing for efficient immunization, and ultimately making camelids an unexpectedly advantageous therapeutic antibody platform with features not seen in other animal platforms. The momentum gained makes delving deeper into the technical aspect of camelid Fab fragment phage-display seem like an obvious step. Based on earlier observations like finding similar heavy/light chain families paired after selection we sought to employ these to design a quick and easy Fab fragment phage-display method for generating highly potent monoclonal antibodies (mAbs). To do so, a llama is immunized using a cytokine (IL-6) as a soluble target. Selection against IL-6 revealed a panel of Fab fragments that are analyzed for binding the IL-6:IL-6R interface to prevent ligand to receptor binding. The Fab fragment that had these characteristics was made up from a VH and Vλ fragment, these fragments were separately captured in a library and randomly brought together in a working phagemid library. We believe that this happened as this was an original pair originating from the immunized llama. Therefore, the isolated Vλ fragment was recombined with the VH library and surely members (affinity variants) of the VH clone were retrieved. The somatic mutations present within these affinity variants were made into a new VH library containing all possible combinations. When joined with their Vλ fragment to form once again full Fab fragments, phage display was set-up to select for low off-rate molecules resulting in a highly potent Fab fragment that in IgG1 format performed second to none when potency and affinity are considered as compared to the known antibodies in therapy. G-protein coupled receptors (GPCRs) or seven transmembrane receptors (7TMR) form a highly desired druggable target that at the time presented a challenge for antibody platforms. To be prepared, a literature investigation resulted in the needed insight to tackle upcoming problems likely to be encountered. Components like lipoparticles were identified as bona vide vehicles for immunizing llama’s and performing selections with. In addition, therapeutic antibodies raised against chemokines and their receptors (7TMR type) are discussed to get a feel for what has been done in the past and present. Following our survey CXCR4 is picked for being a multifaceted receptor that is druggable in both cancer and HIV-1 context. To set the bar high it was decided to aim for modulating CXCR4 receptor function distinguishing between CXCL12 (ligand) signaling and HIV-1 entry blockade using the smallest camelid derived antibody fragment known as nanobodies. After selection on both CXCR4 nanodiscs and lipoparticles a resulting repertoire of nanobodies is put through rigorous testing to see to what degree and by which concentration (IC50) biologic function of CXCR4 was interfered with. CXCR4 driven calcium flux, migration, cell morphology (impedance), receptor internalization were analyzed. The absence of a significant correlation between the performed assays was used to distinguish a set of nanobodies that were further characterized for exhibiting biased receptor modulation

Biochemical Pharmacology, 2018

Upregulation of the chemokine receptor CXCR4 contributes to the progression and metastasis of bot... more 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 antibodymediated 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.

The Journal of biological chemistry, Jan 27, 2016

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and can... more Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The X-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies, and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side-chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe229 and Phe279 of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to 'hot spot' residue P...

mAbs, 2016

The identification of functional monoclonal antibodies directed against G-protein coupled recepto... more The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

Protein Engineering Design and Selection, 2016

Highly potent human antibodies are required to therapeutically neutralize cytokines such as inter... more Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.

mAbs, 2015

Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; ... more Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1b and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelidderived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform.

Drug Discovery Today: Technologies, 2012

Chemokines and their receptors are highly interesting therapeutic targets for pharmaceutical and ... more Chemokines and their receptors are highly interesting therapeutic targets for pharmaceutical and biotechnology companies. In particular, industrial development pipelines are filled with new chemokine-targeting drugs to treat inflammatory diseases and malignancies. In this review, we specifically highlight antibody-based therapeutics and monoclonal antibodies (mAbs) targeting the chemokine system. Besides discussing pitfalls inherently linked to their discovery, we will elaborate on where progress can be made in the development of novel human therapeutic antibodies directed at the chemokine system.

In twenty first century, urban agriculture has been gaining importance and potential in developin... more In twenty first century, urban agriculture has been gaining importance and potential in developing countries. The objective of the present study was to investigate the developments and problems of urban agriculture on the special emphasis on ward numbers 28,29 and 30 of Chandernagore Municipal Corporation (CMC) of Hooghly district of West Bengal. These three wards have been chosen due to the fact that all the said three wards were composed of areas that were rural before 1995, the years in which these wards were added to CMC to provide for expansion of the town. The significant part of population still dependent on agriculture. Particular crops, especially fruits and vegetables to ensure food security to the urban consumers. Highlights m From the data and information collected from field survey, I advocates that the agriculture is moderately developed in ward no. 28,29 and 30 of Chandernagore Municipal Corporation. m Agriculture can be further developed if the corporation provides good supply of seeds, fertilizers at low cost. m Irrigational development as well as the hygienic market should be formed for the selling purpose.

Nowadays therapeutic antibodies are gaining ground To validate camelid conventional type antibodi... more Nowadays therapeutic antibodies are gaining ground To validate camelid conventional type antibodies as a source for therapeutic antibodies, their human homology in both conformation (shape) and sequence is analyzed. To thoroughly assess (germline) sequence homology, human immunoglobulin variable regions (V regions) counterparts are identified amongst camelid (alpaca (Lama pacos), llama (Lama glama), and camel (Camelis ferus)) species. As human homology controls, cynomolgous monkey and murine immunoglobulin V regions are used (positive and negative respectively). In addition, two earlier selected Lama glama Fab fragments are crystalized and aligned with their corresponding human family members. Results indicate high sequence/structural homology in camelid heavy chain variable domain regions and a sound structural resemblance to human Fab domains. This is however not seen for other “target” proteins, allowing for efficient immunization, and ultimately making camelids an unexpectedly advantageous therapeutic antibody platform with features not seen in other animal platforms. The momentum gained makes delving deeper into the technical aspect of camelid Fab fragment phage-display seem like an obvious step. Based on earlier observations like finding similar heavy/light chain families paired after selection we sought to employ these to design a quick and easy Fab fragment phage-display method for generating highly potent monoclonal antibodies (mAbs). To do so, a llama is immunized using a cytokine (IL-6) as a soluble target. Selection against IL-6 revealed a panel of Fab fragments that are analyzed for binding the IL-6:IL-6R interface to prevent ligand to receptor binding. The Fab fragment that had these characteristics was made up from a VH and Vλ fragment, these fragments were separately captured in a library and randomly brought together in a working phagemid library. We believe that this happened as this was an original pair originating from the immunized llama. Therefore, the isolated Vλ fragment was recombined with the VH library and surely members (affinity variants) of the VH clone were retrieved. The somatic mutations present within these affinity variants were made into a new VH library containing all possible combinations. When joined with their Vλ fragment to form once again full Fab fragments, phage display was set-up to select for low off-rate molecules resulting in a highly potent Fab fragment that in IgG1 format performed second to none when potency and affinity are considered as compared to the known antibodies in therapy. G-protein coupled receptors (GPCRs) or seven transmembrane receptors (7TMR) form a highly desired druggable target that at the time presented a challenge for antibody platforms. To be prepared, a literature investigation resulted in the needed insight to tackle upcoming problems likely to be encountered. Components like lipoparticles were identified as bona vide vehicles for immunizing llama’s and performing selections with. In addition, therapeutic antibodies raised against chemokines and their receptors (7TMR type) are discussed to get a feel for what has been done in the past and present. Following our survey CXCR4 is picked for being a multifaceted receptor that is druggable in both cancer and HIV-1 context. To set the bar high it was decided to aim for modulating CXCR4 receptor function distinguishing between CXCL12 (ligand) signaling and HIV-1 entry blockade using the smallest camelid derived antibody fragment known as nanobodies. After selection on both CXCR4 nanodiscs and lipoparticles a resulting repertoire of nanobodies is put through rigorous testing to see to what degree and by which concentration (IC50) biologic function of CXCR4 was interfered with. CXCR4 driven calcium flux, migration, cell morphology (impedance), receptor internalization were analyzed. The absence of a significant correlation between the performed assays was used to distinguish a set of nanobodies that were further characterized for exhibiting biased receptor modulation

Biochemical Pharmacology, 2018

Upregulation of the chemokine receptor CXCR4 contributes to the progression and metastasis of bot... more 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 antibodymediated 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.

The Journal of biological chemistry, Jan 27, 2016

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and can... more Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The X-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies, and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side-chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe229 and Phe279 of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to 'hot spot' residue P...

mAbs, 2016

The identification of functional monoclonal antibodies directed against G-protein coupled recepto... more The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

Protein Engineering Design and Selection, 2016

Highly potent human antibodies are required to therapeutically neutralize cytokines such as inter... more Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.

mAbs, 2015

Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; ... more Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1b and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelidderived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform.

Drug Discovery Today: Technologies, 2012

Chemokines and their receptors are highly interesting therapeutic targets for pharmaceutical and ... more Chemokines and their receptors are highly interesting therapeutic targets for pharmaceutical and biotechnology companies. In particular, industrial development pipelines are filled with new chemokine-targeting drugs to treat inflammatory diseases and malignancies. In this review, we specifically highlight antibody-based therapeutics and monoclonal antibodies (mAbs) targeting the chemokine system. Besides discussing pitfalls inherently linked to their discovery, we will elaborate on where progress can be made in the development of novel human therapeutic antibodies directed at the chemokine system.

In twenty first century, urban agriculture has been gaining importance and potential in developin... more In twenty first century, urban agriculture has been gaining importance and potential in developing countries. The objective of the present study was to investigate the developments and problems of urban agriculture on the special emphasis on ward numbers 28,29 and 30 of Chandernagore Municipal Corporation (CMC) of Hooghly district of West Bengal. These three wards have been chosen due to the fact that all the said three wards were composed of areas that were rural before 1995, the years in which these wards were added to CMC to provide for expansion of the town. The significant part of population still dependent on agriculture. Particular crops, especially fruits and vegetables to ensure food security to the urban consumers. Highlights m From the data and information collected from field survey, I advocates that the agriculture is moderately developed in ward no. 28,29 and 30 of Chandernagore Municipal Corporation. m Agriculture can be further developed if the corporation provides good supply of seeds, fertilizers at low cost. m Irrigational development as well as the hygienic market should be formed for the selling purpose.