Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly
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 a...
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MDPI AG
2020-01-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/21/3/883 |
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doaj-83a54f189b9d4f34951f075863969ccb2020-11-25T02:06:05ZengMDPI AGInternational Journal of Molecular Sciences1422-00672020-01-0121388310.3390/ijms21030883ijms21030883Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson AssemblyStijn De Munter0Alexander Van Parys1Layla Bral2Joline Ingels3Glenn Goetgeluk4Sarah Bonte5Melissa Pille6Lore Billiet7Karin Weening8Annick Verhee9Jose Van der Heyden10Tom Taghon11Georges Leclercq12Tessa Kerre13Jan Tavernier14Bart Vandekerckhove15Department of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumCytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB-UGent Center for Medical Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Internal Medicine and Pediatrics, Ghent University, 9000 Ghent, BelgiumDepartment of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumCytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB-UGent Center for Medical Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, BelgiumCytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB-UGent Center for Medical Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumCytokine Receptor Laboratory, Flanders Institute of Biotechnology, VIB-UGent Center for Medical Biotechnology, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Diagnostic Sciences, Ghent University, 9000 Ghent, BelgiumRecent 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 transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs.https://www.mdpi.com/1422-0067/21/3/883nanobodyvhhchimeric antigen receptorcar t cellcd33cd20pcrgibson assemblynanocar |
| collection |
DOAJ |
| language |
English |
| format |
Article |
| sources |
DOAJ |
| author |
Stijn De Munter Alexander Van Parys Layla Bral Joline Ingels Glenn Goetgeluk Sarah Bonte Melissa Pille Lore Billiet Karin Weening Annick Verhee Jose Van der Heyden Tom Taghon Georges Leclercq Tessa Kerre Jan Tavernier Bart Vandekerckhove |
| spellingShingle |
Stijn De Munter Alexander Van Parys Layla Bral Joline Ingels Glenn Goetgeluk Sarah Bonte Melissa Pille Lore Billiet Karin Weening Annick Verhee Jose Van der Heyden Tom Taghon Georges Leclercq Tessa Kerre Jan Tavernier Bart Vandekerckhove Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly International Journal of Molecular Sciences nanobody vhh chimeric antigen receptor car t cell cd33 cd20 pcr gibson assembly nanocar |
| author_facet |
Stijn De Munter Alexander Van Parys Layla Bral Joline Ingels Glenn Goetgeluk Sarah Bonte Melissa Pille Lore Billiet Karin Weening Annick Verhee Jose Van der Heyden Tom Taghon Georges Leclercq Tessa Kerre Jan Tavernier Bart Vandekerckhove |
| author_sort |
Stijn De Munter |
| title |
Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly |
| title_short |
Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly |
| title_full |
Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly |
| title_fullStr |
Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly |
| title_full_unstemmed |
Rapid and Effective Generation of Nanobody Based CARs using PCR and Gibson Assembly |
| title_sort |
rapid and effective generation of nanobody based cars using pcr and gibson assembly |
| publisher |
MDPI AG |
| series |
International Journal of Molecular Sciences |
| issn |
1422-0067 |
| publishDate |
2020-01-01 |
| description |
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 transduction in T cells, we observed high cell membrane nanoCAR expression in all cases. Following stimulation of nanoCAR-expressing T cells with antigen-positive cell lines, robust T cell activation, cytokine production and tumor cell lysis both in vitro and in vivo was observed. The use of nanobody technology in combination with PCR and Gibson Assembly allows for the rapid and effective generation of compact CARs. |
| topic |
nanobody vhh chimeric antigen receptor car t cell cd33 cd20 pcr gibson assembly nanocar |
| url |
https://www.mdpi.com/1422-0067/21/3/883 |
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