Karin Weening | UGent - Academia.edu (original) (raw)

Papers by Karin Weening

ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificu... more ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof.mr. P.F. van der Heijden,

OncoImmunology

ABSTRACT T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms’ tu... more ABSTRACT T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms’ tumor 1 (WT1), a tumor-associated antigen overexpressed in several malignancies, including acute myeloid leukemia (AML). For both chimeric antigen receptor (CAR)- and TCR-redirected T cells, several clinical studies indicate that T cell subsets with a less-differentiated phenotype (e.g. stem cell memory T cells, TSCM) survive longer and mediate superior anti-tumor effects in vivo as opposed to more terminally differentiated T cells. Cytokines added during in vitro and ex vivo culture of T cells play an important role in driving the phenotype of T cells for adoptive transfer. Using the OP9-DL1 co-culture system, we have shown previously that we are able to generate in vitro, starting from clinically relevant stem cell sources, T cells with a single tumor antigen (TA)-specific TCR. This method circumvents possible TCR chain mispairing and unwanted toxicities that might occur when introducing a TA-specific TCR in peripheral blood lymphocytes. We now show that we are able to optimize our in vitro culture protocol, by adding IL-21 during maturation, resulting in generation of TA-specific T cells with a less-differentiated phenotype and enhanced in vitro anti-tumor effects. We believe the favorable TSCM-like phenotype of these in vitro generated T cells preludes superior in vivo persistence and anti-tumor efficacy. Therefore, these TA-specific T cells could be of use as a valuable new form of patient-tailored T cell immunotherapy for malignancies for which finding a suitable CAR-T target antigen is challenging, such as AML.

Journal of muscle research and cell motility, 2002

cAMP plays a pivotal role in control of cell movement, differentiation and response to stress in ... more cAMP plays a pivotal role in control of cell movement, differentiation and response to stress in all phases of the Dictyostelium life cycle. The multitudinous functions of cAMP require precise spatial and temporal control of its production, degradation and detection. Many novel proteins have recently been identified that critically modulate the cAMP signal. We focus in this review on the properties and functions of the three adenylyl cyclases and the three cAMP-phosphodiesterases that are present in Dictyostelium, and the network of proteins that regulate the activity of these enzymes. We also briefly discuss the two modes of detection of cAMP.

Oncoimmunology, 2021

T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms’ tumor 1 (WT... more T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms’ tumor 1 (WT1), a tumor-associated antigen overexpressed in several malignancies, including acute myeloid leukemia (AML). For both chimeric antigen receptor (CAR)- and TCR-redirected T cells, several clinical studies indicate that T cell subsets with a less-differentiated phenotype (e.g. stem cell memory T cells, TSCM) survive longer and mediate superior anti-tumor effects in vivo as opposed to more terminally differentiated T cells. Cytokines added during in vitro and ex vivo culture of T cells play an important role in driving the phenotype of T cells for adoptive transfer. Using the OP9-DL1 co-culture system, we have shown previously that we are able to generate in vitro, starting from clinically relevant stem cell sources, T cells with a single tumor antigen (TA)-specific TCR. This method circumvents possible TCR chain mispairing and unwanted toxicities that might occur when introducing a TA-spe...

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic va... more Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. In the present study, we describe the full characterization of the human monoclonal antibody (mAb) 2A5 raised against the HCV envelope. Results of GNA binding ELISA showed high affinity of mAb2A5 towards HCV envelope glycoproteins E1E2 of different strains. mAb2A5 efficiently cross-neutralized HCVpp and HCVcc from all genotypes and completely protected humanized mice from ge...

Molecular Biology of the Cell, 1999

International Journal of Molecular Sciences

Human thymic CD8αα+ CD10+ PD-1+ αβ T cells selected through early agonist selection have been pro... more Human thymic CD8αα+ CD10+ PD-1+ αβ T cells selected through early agonist selection have been proposed as the putative thymic precursors of the human CD8αα+ intestinal intraepithelial lymphocytes (IELs). However, the progeny of these thymic precursor cells in human blood or tissues has not yet been characterized. Here, we studied the phenotypical and transcriptional differentiation of the thymic IEL precursor (IELp) lineage upon in vitro exposure to cytokines prominent in the peripheral tissues such as interleukin-15 (IL-15) and the inflammatory cytokines interleukin-12 (IL-12) and interleukin-18 (IL-18). We showed that only the CD1a− fraction of the CD10+ PD-1+ IELp population was able to proliferate with IL-15, suggesting that this subset had acquired functionality. These cells downregulated PD-1 expression and completely lost CD10 expression, whereas other surface markers such as CD95 and CXCR3 remained highly expressed. RNA-seq analysis of the IL-15-cultured cells clearly showed...

EMBO reports

γδ and αβ T cells have unique roles in immunity and both originate in the thymus from T‐lineage c... more γδ and αβ T cells have unique roles in immunity and both originate in the thymus from T‐lineage committed precursors through distinct but unclear mechanisms. Here, we show that Notch1 activation is more stringently required for human γδ development compared to αβ‐lineage differentiation and performed paired mRNA and miRNA profiling across 11 discrete developmental stages of human T cell development in an effort to identify the potential Notch1 downstream mechanism. Our data suggest that the miR‐17–92 cluster is a Notch1 target in immature thymocytes and that miR‐17 can restrict BCL11B expression in these Notch‐dependent T cell precursors. We show that enforced miR‐17 expression promotes human γδ T cell development and, consistently, that BCL11B is absolutely required for αβ but less for γδ T cell development. This study suggests that human γδ T cell development is mediated by a stage‐specific Notch‐driven negative feedback loop through which miR‐17 temporally restricts BCL11B expression and provides functional insights into the developmental role of the disease‐associated genes BCL11B and the miR‐17–92 cluster in a human context.

International Journal of Molecular Sciences

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agenc... more Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduct...

The Journal of Experimental Medicine

In the mouse thymus, invariant γδ T cells are generated at well-defined times during development ... more In the mouse thymus, invariant γδ T cells are generated at well-defined times during development and acquire effector functions before exiting the thymus. However, whether such thymic programming and age-dependent generation of invariant γδ T cells occur in humans is not known. Here we found that, unlike postnatal γδ thymocytes, human fetal γδ thymocytes were functionally programmed (e.g., IFNγ, granzymes) and expressed low levels of terminal deoxynucleotidyl transferase (TdT). This low level of TdT resulted in a low number of N nucleotide insertions in the complementarity-determining region-3 (CDR3) of their TCR repertoire, allowing the usage of short homology repeats within the germline-encoded VDJ segments to generate invariant/public cytomegalovirus-reactive CDR3 sequences (TRGV8-TRJP1-CATWDTTGWFKIF, TRDV2-TRDD3-CACDTGGY, and TRDV1-TRDD3-CALGELGD). Furthermore, both the generation of invariant TCRs and the intrathymic acquisition of effector functions were due to an intrinsic pr...

Blood

Autologous T cells transduced to express chimeric antigen receptors (CAR) directed against CD19, ... more Autologous T cells transduced to express chimeric antigen receptors (CAR) directed against CD19, a B cell antigen, are reported to induce complete remission in patients with leukemia or lymphoma of the B cell lineage. Although potentially very effective, this treatment strategy has major drawbacks. First, CAR therapy is based on autologous T cells and therefore dependent on the nature and quality of T cells present in the peripheral blood of these patients at the time of treatment. Poor quality of the T cells may cause treatment failure in some patients. In addition, therapy based on autologous cells is tailor-made i.e. CAR+ T cells have to be generated de novo for every patient. Finally, autologous cell therapy requires different, more complicated logistics than conventional therapy. We therefore investigate whether a general purpose, allogeneic CAR therapy based on HLA-matched cord blood obtained from cord blood banks can be devised. Here, we investigated whether functional CAR+ T...

Haematologica

Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid disse... more Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepte d for publication. E-publishing of this PDF file has been approved by the authors.

International journal of molecular sciences, Jan 30, 2018

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is... more Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The u...

Antiviral research, 2017

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic va... more Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization l...

Science Immunology, 2017

Immature thymocytes are diverted to the agonist selection pathway before selection of conventiona... more Immature thymocytes are diverted to the agonist selection pathway before selection of conventional T cells.

OncoImmunology, 2017

Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen rec... more Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5 C CD7 C Tlineage precursors, to CD4 C CD8 C double positive cells and finally to mature AR C T cells. The AR C T cells were largely naive CD45RA C CD62L C T cells. These T cells had mostly germline TCRa and TCRb loci and therefore lacked surface-expressed CD3/TCRab complexes. The CD3 ¡ AR-transgenic cells were monospecific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3 ¡ AR C T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.

Scientific Reports, 2016

To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an inf... more To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein-but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/ His 6-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions.

Over the past years, much information has been gathered about the importance of the individual ke... more Over the past years, much information has been gathered about the importance of the individual key components that control cAMP signalling in eukaryotes and prokaryotes. In mammalians, several different classes of ACs produce cAMP and much is known about their mechanism of stimulation and function. In contrast, much less is known about the enzymes that control degradation, the cAMP-PDEs and their regulatory mechanism. Nonetheless, it has become evident that cAMP is degraded by many isoforms of PDEs and that PDEs play a crucial role in controlling dynamic cAMP signalling. Throughout my PhD I have been interested in several aspects of cAMP signalling in the model organism Dictyostelium discoideum with particular emphasis on the roles of the phosphodiesterases in dynamic cAMP signalling. PdsA is the most critical enzyme in the control of aggregation and post-aggregative morphogenesis. It is controlled by three promoters; the vegetative, aggregative and the late promoter, which direct expression during growth, aggregation and late development, respectively. Despite its known crucial role in multicellular development not much is known about the regulation of PdsA or its expression after aggregation. In chapter 2, I present studies on the patterns of the aggregative and late promoter activity throughout development, the signals that regulate promoter activity and the promoter sequences that mediate regulation by these signals. D. discoideum and related species use extracellular cAMP as chemoattractant for aggregation, but most other species use other chemoattractants than cAMP. In chapter 3, I identified orthologues of the PdsA gene throughout the Dictyostelid phylogeny and studied their expression during development. Additionally, I show that one of them encodes a fully functional PDE. Cyclic AMP waves propagate throughout the Dictyostelium slug. This is somewhat enigmatic since ACA, the enzyme considered to be responsible for pulsatile cAMP signalling, is only expressed at the slug anterior. ACG is expressed at the posterior region of the slug, where it was shown to be responsible for induction of prespore differentiation. In chapter 4, I describe experiments showing that similar to ACA, ACG is transiently activated by stimulation of cAMP receptors. This could explain why cAMP waves can propagate in slug posteriors. In Dictyostelium, cGMP is associated with chemotaxis and plays a major role in myosin II regulation during cytoskeletal reorganisation. In the chapter 5, I describe experiments showing that modulation of the expression of the PdeD gene, a cGMPstimulated cGMP-PDE, has a pronounced effect on the expression of early genes, necessitating re-evaluation of the role of cGMP in Dictyostelium chemotaxis.

Over the past years, much information has been gathered about the importance of the individual ke... more Over the past years, much information has been gathered about the importance of the individual key components that control cAMP signalling in eukaryotes and prokaryotes. In mammalians, several different classes of ACs produce cAMP and much is known about their mechanism of stimulation and function. In contrast, much less is known about the enzymes that control degradation, the cAMP-PDEs and their regulatory mechanism. Nonetheless, it has become evident that cAMP is degraded by many isoforms of PDEs and that PDEs play a crucial role in controlling dynamic cAMP signalling. Throughout my PhD I have been interested in several aspects of cAMP signalling in the model organism Dictyostelium discoideum with particular emphasis on the roles of the phosphodiesterases in dynamic cAMP signalling. PdsA is the most critical enzyme in the control of aggregation and post-aggregative morphogenesis. It is controlled by three promoters; the vegetative, aggregative and the late promoter, which direct e...

ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificu... more ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof.mr. P.F. van der Heijden,

OncoImmunology

ABSTRACT T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms’ tu... more ABSTRACT T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms’ tumor 1 (WT1), a tumor-associated antigen overexpressed in several malignancies, including acute myeloid leukemia (AML). For both chimeric antigen receptor (CAR)- and TCR-redirected T cells, several clinical studies indicate that T cell subsets with a less-differentiated phenotype (e.g. stem cell memory T cells, TSCM) survive longer and mediate superior anti-tumor effects in vivo as opposed to more terminally differentiated T cells. Cytokines added during in vitro and ex vivo culture of T cells play an important role in driving the phenotype of T cells for adoptive transfer. Using the OP9-DL1 co-culture system, we have shown previously that we are able to generate in vitro, starting from clinically relevant stem cell sources, T cells with a single tumor antigen (TA)-specific TCR. This method circumvents possible TCR chain mispairing and unwanted toxicities that might occur when introducing a TA-specific TCR in peripheral blood lymphocytes. We now show that we are able to optimize our in vitro culture protocol, by adding IL-21 during maturation, resulting in generation of TA-specific T cells with a less-differentiated phenotype and enhanced in vitro anti-tumor effects. We believe the favorable TSCM-like phenotype of these in vitro generated T cells preludes superior in vivo persistence and anti-tumor efficacy. Therefore, these TA-specific T cells could be of use as a valuable new form of patient-tailored T cell immunotherapy for malignancies for which finding a suitable CAR-T target antigen is challenging, such as AML.

Journal of muscle research and cell motility, 2002

cAMP plays a pivotal role in control of cell movement, differentiation and response to stress in ... more cAMP plays a pivotal role in control of cell movement, differentiation and response to stress in all phases of the Dictyostelium life cycle. The multitudinous functions of cAMP require precise spatial and temporal control of its production, degradation and detection. Many novel proteins have recently been identified that critically modulate the cAMP signal. We focus in this review on the properties and functions of the three adenylyl cyclases and the three cAMP-phosphodiesterases that are present in Dictyostelium, and the network of proteins that regulate the activity of these enzymes. We also briefly discuss the two modes of detection of cAMP.

Oncoimmunology, 2021

T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms’ tumor 1 (WT... more T cell receptor (TCR)-redirected T cells target intracellular antigens such as Wilms’ tumor 1 (WT1), a tumor-associated antigen overexpressed in several malignancies, including acute myeloid leukemia (AML). For both chimeric antigen receptor (CAR)- and TCR-redirected T cells, several clinical studies indicate that T cell subsets with a less-differentiated phenotype (e.g. stem cell memory T cells, TSCM) survive longer and mediate superior anti-tumor effects in vivo as opposed to more terminally differentiated T cells. Cytokines added during in vitro and ex vivo culture of T cells play an important role in driving the phenotype of T cells for adoptive transfer. Using the OP9-DL1 co-culture system, we have shown previously that we are able to generate in vitro, starting from clinically relevant stem cell sources, T cells with a single tumor antigen (TA)-specific TCR. This method circumvents possible TCR chain mispairing and unwanted toxicities that might occur when introducing a TA-spe...

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic va... more Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. In the present study, we describe the full characterization of the human monoclonal antibody (mAb) 2A5 raised against the HCV envelope. Results of GNA binding ELISA showed high affinity of mAb2A5 towards HCV envelope glycoproteins E1E2 of different strains. mAb2A5 efficiently cross-neutralized HCVpp and HCVcc from all genotypes and completely protected humanized mice from ge...

Molecular Biology of the Cell, 1999

International Journal of Molecular Sciences

Human thymic CD8αα+ CD10+ PD-1+ αβ T cells selected through early agonist selection have been pro... more Human thymic CD8αα+ CD10+ PD-1+ αβ T cells selected through early agonist selection have been proposed as the putative thymic precursors of the human CD8αα+ intestinal intraepithelial lymphocytes (IELs). However, the progeny of these thymic precursor cells in human blood or tissues has not yet been characterized. Here, we studied the phenotypical and transcriptional differentiation of the thymic IEL precursor (IELp) lineage upon in vitro exposure to cytokines prominent in the peripheral tissues such as interleukin-15 (IL-15) and the inflammatory cytokines interleukin-12 (IL-12) and interleukin-18 (IL-18). We showed that only the CD1a− fraction of the CD10+ PD-1+ IELp population was able to proliferate with IL-15, suggesting that this subset had acquired functionality. These cells downregulated PD-1 expression and completely lost CD10 expression, whereas other surface markers such as CD95 and CXCR3 remained highly expressed. RNA-seq analysis of the IL-15-cultured cells clearly showed...

EMBO reports

γδ and αβ T cells have unique roles in immunity and both originate in the thymus from T‐lineage c... more γδ and αβ T cells have unique roles in immunity and both originate in the thymus from T‐lineage committed precursors through distinct but unclear mechanisms. Here, we show that Notch1 activation is more stringently required for human γδ development compared to αβ‐lineage differentiation and performed paired mRNA and miRNA profiling across 11 discrete developmental stages of human T cell development in an effort to identify the potential Notch1 downstream mechanism. Our data suggest that the miR‐17–92 cluster is a Notch1 target in immature thymocytes and that miR‐17 can restrict BCL11B expression in these Notch‐dependent T cell precursors. We show that enforced miR‐17 expression promotes human γδ T cell development and, consistently, that BCL11B is absolutely required for αβ but less for γδ T cell development. This study suggests that human γδ T cell development is mediated by a stage‐specific Notch‐driven negative feedback loop through which miR‐17 temporally restricts BCL11B expression and provides functional insights into the developmental role of the disease‐associated genes BCL11B and the miR‐17–92 cluster in a human context.

International Journal of Molecular Sciences

Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agenc... more Recent approval of chimeric antigen receptor (CAR) T cell therapy by the European Medicines Agency (EMA)/Federal and Drug Administration (FDA) and the remarkable results of CAR T clinical trials illustrate the curative potential of this therapy. While CARs against a multitude of different antigens are being developed and tested (pre)clinically, there is still a need for optimization. The use of single-chain variable fragments (scFvs) as targeting moieties hampers the quick generation of functional CARs and could potentially limit the efficacy. Instead, nanobodies may largely circumvent these difficulties. We used an available nanobody library generated after immunization of llamas against Cluster of Differentiation (CD) 20 through DNA vaccination or against the ectodomain of CD33 using soluble protein. The nanobody specific sequences were amplified by PCR and cloned by Gibson Assembly into a retroviral vector containing two different second-generation CAR constructs. After transduct...

The Journal of Experimental Medicine

In the mouse thymus, invariant γδ T cells are generated at well-defined times during development ... more In the mouse thymus, invariant γδ T cells are generated at well-defined times during development and acquire effector functions before exiting the thymus. However, whether such thymic programming and age-dependent generation of invariant γδ T cells occur in humans is not known. Here we found that, unlike postnatal γδ thymocytes, human fetal γδ thymocytes were functionally programmed (e.g., IFNγ, granzymes) and expressed low levels of terminal deoxynucleotidyl transferase (TdT). This low level of TdT resulted in a low number of N nucleotide insertions in the complementarity-determining region-3 (CDR3) of their TCR repertoire, allowing the usage of short homology repeats within the germline-encoded VDJ segments to generate invariant/public cytomegalovirus-reactive CDR3 sequences (TRGV8-TRJP1-CATWDTTGWFKIF, TRDV2-TRDD3-CACDTGGY, and TRDV1-TRDD3-CALGELGD). Furthermore, both the generation of invariant TCRs and the intrathymic acquisition of effector functions were due to an intrinsic pr...

Blood

Autologous T cells transduced to express chimeric antigen receptors (CAR) directed against CD19, ... more Autologous T cells transduced to express chimeric antigen receptors (CAR) directed against CD19, a B cell antigen, are reported to induce complete remission in patients with leukemia or lymphoma of the B cell lineage. Although potentially very effective, this treatment strategy has major drawbacks. First, CAR therapy is based on autologous T cells and therefore dependent on the nature and quality of T cells present in the peripheral blood of these patients at the time of treatment. Poor quality of the T cells may cause treatment failure in some patients. In addition, therapy based on autologous cells is tailor-made i.e. CAR+ T cells have to be generated de novo for every patient. Finally, autologous cell therapy requires different, more complicated logistics than conventional therapy. We therefore investigate whether a general purpose, allogeneic CAR therapy based on HLA-matched cord blood obtained from cord blood banks can be devised. Here, we investigated whether functional CAR+ T...

Haematologica

Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid disse... more Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepte d for publication. E-publishing of this PDF file has been approved by the authors.

International journal of molecular sciences, Jan 30, 2018

Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is... more Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The u...

Antiviral research, 2017

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic va... more Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the efficacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization l...

Science Immunology, 2017

Immature thymocytes are diverted to the agonist selection pathway before selection of conventiona... more Immature thymocytes are diverted to the agonist selection pathway before selection of conventional T cells.

OncoImmunology, 2017

Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen rec... more Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5 C CD7 C Tlineage precursors, to CD4 C CD8 C double positive cells and finally to mature AR C T cells. The AR C T cells were largely naive CD45RA C CD62L C T cells. These T cells had mostly germline TCRa and TCRb loci and therefore lacked surface-expressed CD3/TCRab complexes. The CD3 ¡ AR-transgenic cells were monospecific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3 ¡ AR C T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.

Scientific Reports, 2016

To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an inf... more To facilitate studies on Vpr function in replicating HIV-1, we aimed to tag the protein in an infectious virus. First we showed that N-, but not C-terminal HA/FLAG tagging of Vpr protein preserves Vpr cytopathicity. Cloning the tags into proviral DNA however ablated viral production and replication. By construction of additional viral variants we could show this defect was not protein-but RNA-dependent and sequence specific, and characterized by oversplicing of the genomic RNA. Simulation of genomic RNA folding suggested that introduction of the tag sequence induced an alternative folding structure in a region enriched in splice sites and splicing regulatory sequences. In silico predictions identified the HA/ His 6-Vpr tagging in HIV-1 to affect mRNA folding less than HA/FLAG-Vpr tagging. In vitro infectivity and mRNA splice pattern improved but did not reach wild-type values. Thus, sequence-specific insertions may interfere with mRNA splicing, possibly due to altered RNA folding. Our results point to the complexity of viral RNA genome sequence interactions. This should be taken into consideration when designing viral manipulation strategies, for both research as for biological interventions.

Over the past years, much information has been gathered about the importance of the individual ke... more Over the past years, much information has been gathered about the importance of the individual key components that control cAMP signalling in eukaryotes and prokaryotes. In mammalians, several different classes of ACs produce cAMP and much is known about their mechanism of stimulation and function. In contrast, much less is known about the enzymes that control degradation, the cAMP-PDEs and their regulatory mechanism. Nonetheless, it has become evident that cAMP is degraded by many isoforms of PDEs and that PDEs play a crucial role in controlling dynamic cAMP signalling. Throughout my PhD I have been interested in several aspects of cAMP signalling in the model organism Dictyostelium discoideum with particular emphasis on the roles of the phosphodiesterases in dynamic cAMP signalling. PdsA is the most critical enzyme in the control of aggregation and post-aggregative morphogenesis. It is controlled by three promoters; the vegetative, aggregative and the late promoter, which direct expression during growth, aggregation and late development, respectively. Despite its known crucial role in multicellular development not much is known about the regulation of PdsA or its expression after aggregation. In chapter 2, I present studies on the patterns of the aggregative and late promoter activity throughout development, the signals that regulate promoter activity and the promoter sequences that mediate regulation by these signals. D. discoideum and related species use extracellular cAMP as chemoattractant for aggregation, but most other species use other chemoattractants than cAMP. In chapter 3, I identified orthologues of the PdsA gene throughout the Dictyostelid phylogeny and studied their expression during development. Additionally, I show that one of them encodes a fully functional PDE. Cyclic AMP waves propagate throughout the Dictyostelium slug. This is somewhat enigmatic since ACA, the enzyme considered to be responsible for pulsatile cAMP signalling, is only expressed at the slug anterior. ACG is expressed at the posterior region of the slug, where it was shown to be responsible for induction of prespore differentiation. In chapter 4, I describe experiments showing that similar to ACA, ACG is transiently activated by stimulation of cAMP receptors. This could explain why cAMP waves can propagate in slug posteriors. In Dictyostelium, cGMP is associated with chemotaxis and plays a major role in myosin II regulation during cytoskeletal reorganisation. In the chapter 5, I describe experiments showing that modulation of the expression of the PdeD gene, a cGMPstimulated cGMP-PDE, has a pronounced effect on the expression of early genes, necessitating re-evaluation of the role of cGMP in Dictyostelium chemotaxis.

Over the past years, much information has been gathered about the importance of the individual ke... more Over the past years, much information has been gathered about the importance of the individual key components that control cAMP signalling in eukaryotes and prokaryotes. In mammalians, several different classes of ACs produce cAMP and much is known about their mechanism of stimulation and function. In contrast, much less is known about the enzymes that control degradation, the cAMP-PDEs and their regulatory mechanism. Nonetheless, it has become evident that cAMP is degraded by many isoforms of PDEs and that PDEs play a crucial role in controlling dynamic cAMP signalling. Throughout my PhD I have been interested in several aspects of cAMP signalling in the model organism Dictyostelium discoideum with particular emphasis on the roles of the phosphodiesterases in dynamic cAMP signalling. PdsA is the most critical enzyme in the control of aggregation and post-aggregative morphogenesis. It is controlled by three promoters; the vegetative, aggregative and the late promoter, which direct e...