Recently, chimeric antigen receptor (CAR) T cell technology has revolutionized cancer immunothera... more Recently, chimeric antigen receptor (CAR) T cell technology has revolutionized cancer immunotherapy. This strategy uses synthetic CARs to redirect T cells to specific antigens expressed on the surface of tumor cells. Despite impressive progress in the treatment of hematological malignancies with CAR T cells, scientific challenges still remain for use of CAR T cell therapy to treat solid tumors. This is mainly due to the hostile tumor microenvironment and CAR-related toxicities. As the glycans decorating the T cell surface are implicated in T cell activation, differentiation, proliferation, and in the interaction of human T cells with tumor cells, we studied the role of human T cell glycosylation in more depth by manipulating their glycome. In this context, there isin vitroevidence that β-galactoside binding lectins (Galectins) can have a strong impact on the functionality of tumor-infiltrating T cells. The high-affinity poly-LacNAc N-linked galectin ligands are mainly synthesized on...
We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and −... more We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and −2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and −2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homoge...
As small and stable high-affinity antigen binders, VHHs boast attractive characteristics both for... more As small and stable high-affinity antigen binders, VHHs boast attractive characteristics both for therapeutic use in various disease indications, and as versatile reagents in research and diagnostics. To further increase the versatility of VHHs, we explored the VHH scaffold in a structure-guided approach to select regions where the introduction of an N-glycosylation N-X-T sequon and its associated glycan should not interfere with protein folding or epitope recognition. We expressed variants of such glycoengineered VHHs in the Pichia pastoris GlycoSwitchM5 strain, allowing us to pinpoint preferred sites at which Man5GlcNAc2-glycans can be introduced at high site occupancy without affecting antigen binding. A VHH carrying predominantly a Man5GlcNAc2 N-glycan at one of these preferred sites showed highly efficient, glycan-dependent uptake by Mf4/4 macrophages in vitro and by alveolar lung macrophages in vivo, illustrating one potential application of glyco-engineered VHHs: a glycan-bas...
Zip file S1. Galaxy workflows and BioPerl algorithm that were used to deconvolute the mutant posi... more Zip file S1. Galaxy workflows and BioPerl algorithm that were used to deconvolute the mutant positions
Table S9. Ortholog of mycobacterial genes between M. tb H37Rv, M. bovis BCG Pasteur, M. bovis BCG... more Table S9. Ortholog of mycobacterial genes between M. tb H37Rv, M. bovis BCG Pasteur, M. bovis BCG Danish WT and sapM KO. (XLSX 880â kb)
A heavy chain–only antibody drug with broad SARS coronavirus neutralizing activity protects mice ... more A heavy chain–only antibody drug with broad SARS coronavirus neutralizing activity protects mice and hamsters.
While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis ele... more While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis elegans ), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of Mycobacterium bovis BCG Danish targets 83% of all nonessential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g., drug resistance research and vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the Mycobacterium tuberculosis complex. The stretch to a full collection of mutants in all nonessential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective metho...
Mutant resources are essential to improve our understanding of the biology of slow-growing mycoba... more Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach; despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian Pooling-Coordinate Sequencing allows the creation of large archivedMycobacteriumtransposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which is undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described, which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporatingFRTsites for FlpE/F...
ABSTRACTMycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) is the only vaccine available... more ABSTRACTMycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). This study reports on an integrated genome analysis workflow for BCG, resulting in the completely assembled genome sequence of BCG Danish 1331 (07/270), one of the WHO reference strains for BCG vaccines. We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of this vaccine strain.
The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine shows variable efficacy in protecti... more The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine shows variable efficacy in protection against adult tuberculosis (TB). Earlier, we have described a BCG mutant vaccine with a transposon insertion in the gene coding for the secreted acid phosphatase SapM, which led to enhanced long-term survival of vaccinated mice challenged with TB infection. To facilitate development of this mutation as part of a future improved live attenuated TB vaccine, we have now characterized the genome and transcriptome of this sapM::Tn mutant versus parental BCG Pasteur. Furthermore, we show that the sapM::Tn mutant had an equal low pathogenicity as WT BCG upon intravenous administration to immunocompromised SCID mice, passing this important safety test. Subsequently, we investigated the clearance of this improved vaccine strain following vaccination and found a more effective innate immune control over the sapM::Tn vaccine bacteria as compared to WT BCG. This leads to a fast contraction of IFN...
A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) e... more A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells that is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared with the wild-type tomato. Given the importance of this fast response in sitiens resistance, we investigated cell wall and cuticle properties of the mutant at the chemical, histological, and ultrastructural levels. We demonstrate that ABA deficiency in the mutant leads to increased cuticle permeability, which is positively correlated with disease resistance. Furthermore, perturbation of ABA levels affects pectin composition. sitiens plants have a relatively higher degree of pectin methylesterification and release different oligosaccharides upon inoculation wi...
Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. ... more Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast. We identified a glycosidase from C. cellulans that 'uncaps' N-glycans modified by yeast-type mannose-Pi-6-mannose to generate mammalian-type N-glycans with a mannose-6-phosphate substitution. Determination of the crystal structure of this glycosidase provided insight into its substrate specificity. We used this uncapping enzyme together with α-mannosidase to produce in yeast a form of the Pompe disease enzyme α-glucosidase rich in mannose-6-phosphate. Compared with the currently used therapeutic version, this form of α-glucosidase was more efficiently taken up by fibroblasts from Pompe disease patients, and it more effectively reduced cardiac muscular glycogen storage in a mouse model of the disease.
Recently, chimeric antigen receptor (CAR) T cell technology has revolutionized cancer immunothera... more Recently, chimeric antigen receptor (CAR) T cell technology has revolutionized cancer immunotherapy. This strategy uses synthetic CARs to redirect T cells to specific antigens expressed on the surface of tumor cells. Despite impressive progress in the treatment of hematological malignancies with CAR T cells, scientific challenges still remain for use of CAR T cell therapy to treat solid tumors. This is mainly due to the hostile tumor microenvironment and CAR-related toxicities. As the glycans decorating the T cell surface are implicated in T cell activation, differentiation, proliferation, and in the interaction of human T cells with tumor cells, we studied the role of human T cell glycosylation in more depth by manipulating their glycome. In this context, there isin vitroevidence that β-galactoside binding lectins (Galectins) can have a strong impact on the functionality of tumor-infiltrating T cells. The high-affinity poly-LacNAc N-linked galectin ligands are mainly synthesized on...
We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and −... more We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and −2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and −2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homoge...
As small and stable high-affinity antigen binders, VHHs boast attractive characteristics both for... more As small and stable high-affinity antigen binders, VHHs boast attractive characteristics both for therapeutic use in various disease indications, and as versatile reagents in research and diagnostics. To further increase the versatility of VHHs, we explored the VHH scaffold in a structure-guided approach to select regions where the introduction of an N-glycosylation N-X-T sequon and its associated glycan should not interfere with protein folding or epitope recognition. We expressed variants of such glycoengineered VHHs in the Pichia pastoris GlycoSwitchM5 strain, allowing us to pinpoint preferred sites at which Man5GlcNAc2-glycans can be introduced at high site occupancy without affecting antigen binding. A VHH carrying predominantly a Man5GlcNAc2 N-glycan at one of these preferred sites showed highly efficient, glycan-dependent uptake by Mf4/4 macrophages in vitro and by alveolar lung macrophages in vivo, illustrating one potential application of glyco-engineered VHHs: a glycan-bas...
Zip file S1. Galaxy workflows and BioPerl algorithm that were used to deconvolute the mutant posi... more Zip file S1. Galaxy workflows and BioPerl algorithm that were used to deconvolute the mutant positions
Table S9. Ortholog of mycobacterial genes between M. tb H37Rv, M. bovis BCG Pasteur, M. bovis BCG... more Table S9. Ortholog of mycobacterial genes between M. tb H37Rv, M. bovis BCG Pasteur, M. bovis BCG Danish WT and sapM KO. (XLSX 880â kb)
A heavy chain–only antibody drug with broad SARS coronavirus neutralizing activity protects mice ... more A heavy chain–only antibody drug with broad SARS coronavirus neutralizing activity protects mice and hamsters.
While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis ele... more While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis elegans ), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of Mycobacterium bovis BCG Danish targets 83% of all nonessential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g., drug resistance research and vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the Mycobacterium tuberculosis complex. The stretch to a full collection of mutants in all nonessential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective metho...
Mutant resources are essential to improve our understanding of the biology of slow-growing mycoba... more Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach; despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian Pooling-Coordinate Sequencing allows the creation of large archivedMycobacteriumtransposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which is undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described, which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporatingFRTsites for FlpE/F...
ABSTRACTMycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) is the only vaccine available... more ABSTRACTMycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). This study reports on an integrated genome analysis workflow for BCG, resulting in the completely assembled genome sequence of BCG Danish 1331 (07/270), one of the WHO reference strains for BCG vaccines. We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of this vaccine strain.
The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine shows variable efficacy in protecti... more The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine shows variable efficacy in protection against adult tuberculosis (TB). Earlier, we have described a BCG mutant vaccine with a transposon insertion in the gene coding for the secreted acid phosphatase SapM, which led to enhanced long-term survival of vaccinated mice challenged with TB infection. To facilitate development of this mutation as part of a future improved live attenuated TB vaccine, we have now characterized the genome and transcriptome of this sapM::Tn mutant versus parental BCG Pasteur. Furthermore, we show that the sapM::Tn mutant had an equal low pathogenicity as WT BCG upon intravenous administration to immunocompromised SCID mice, passing this important safety test. Subsequently, we investigated the clearance of this improved vaccine strain following vaccination and found a more effective innate immune control over the sapM::Tn vaccine bacteria as compared to WT BCG. This leads to a fast contraction of IFN...
A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) e... more A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells that is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared with the wild-type tomato. Given the importance of this fast response in sitiens resistance, we investigated cell wall and cuticle properties of the mutant at the chemical, histological, and ultrastructural levels. We demonstrate that ABA deficiency in the mutant leads to increased cuticle permeability, which is positively correlated with disease resistance. Furthermore, perturbation of ABA levels affects pectin composition. sitiens plants have a relatively higher degree of pectin methylesterification and release different oligosaccharides upon inoculation wi...
Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. ... more Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast. We identified a glycosidase from C. cellulans that 'uncaps' N-glycans modified by yeast-type mannose-Pi-6-mannose to generate mammalian-type N-glycans with a mannose-6-phosphate substitution. Determination of the crystal structure of this glycosidase provided insight into its substrate specificity. We used this uncapping enzyme together with α-mannosidase to produce in yeast a form of the Pompe disease enzyme α-glucosidase rich in mannose-6-phosphate. Compared with the currently used therapeutic version, this form of α-glucosidase was more efficiently taken up by fibroblasts from Pompe disease patients, and it more effectively reduced cardiac muscular glycogen storage in a mouse model of the disease.
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Papers by Annelies Hecke