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Rapid and effective generation of nanobody based CARs using PCR and Gibson Assembly

Stijn De Munter (UGent) , Alexander Van Parys (UGent) , Layla Bral, Joline Ingels (UGent) , Glenn Goetgeluk (UGent) , Sarah Bonte (UGent) , Melissa Pille (UGent) , Lore Billiet (UGent) , Karin Weening (UGent) , Annick Verhee (UGent) , et al.
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Abstract
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.
Keywords
Physical and Theoretical Chemistry, Inorganic Chemistry, Organic Chemistry, Spectroscopy, Molecular Biology, Catalysis, General Medicine, Computer Science Applications, nanobody, VHH, chimeric antigen receptor, CAR T cell, CD33, CD20, PCR, Gibson Assembly, nanoCAR, CHIMERIC ANTIGEN RECEPTORS, SINGLE-DOMAIN ANTIBODIES, T-CELLS, B-CELL, ADOPTIVE IMMUNOTHERAPY, SPACER DOMAIN, CD19, THERAPY, INTEGRATION, FRAGMENT

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MLA
De Munter, Stijn, et al. “Rapid and Effective Generation of Nanobody Based CARs Using PCR and Gibson Assembly.” INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 21, no. 3, 2020, doi:10.3390/ijms21030883.
APA
De Munter, S., Van Parys, A., Bral, L., Ingels, J., Goetgeluk, G., Bonte, S., … Vandekerckhove, B. (2020). Rapid and effective generation of nanobody based CARs using PCR and Gibson Assembly. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 21(3). https://doi.org/10.3390/ijms21030883
Chicago author-date
De Munter, Stijn, Alexander Van Parys, Layla Bral, Joline Ingels, Glenn Goetgeluk, Sarah Bonte, Melissa Pille, et al. 2020. “Rapid and Effective Generation of Nanobody Based CARs Using PCR and Gibson Assembly.” INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 21 (3). https://doi.org/10.3390/ijms21030883.
Chicago author-date (all authors)
De Munter, Stijn, Alexander Van Parys, Layla Bral, Joline Ingels, Glenn Goetgeluk, Sarah Bonte, Melissa Pille, Lore Billiet, Karin Weening, Annick Verhee, José Van Der Heyden, Tom Taghon, Georges Leclercq, Tessa Kerre, Jan Tavernier, and Bart Vandekerckhove. 2020. “Rapid and Effective Generation of Nanobody Based CARs Using PCR and Gibson Assembly.” INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 21 (3). doi:10.3390/ijms21030883.
Vancouver
1.
De Munter S, Van Parys A, Bral L, Ingels J, Goetgeluk G, Bonte S, et al. Rapid and effective generation of nanobody based CARs using PCR and Gibson Assembly. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. 2020;21(3).
IEEE
[1]
S. De Munter et al., “Rapid and effective generation of nanobody based CARs using PCR and Gibson Assembly,” INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 21, no. 3, 2020.
@article{8663527,
  abstract     = {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.},
  articleno    = {883},
  author       = {De Munter, Stijn and Van Parys, Alexander and Bral, Layla and Ingels, Joline and Goetgeluk, Glenn and Bonte, Sarah and Pille, Melissa and Billiet, Lore and Weening, Karin and Verhee, Annick and Van Der Heyden, José and Taghon, Tom and Leclercq, Georges and Kerre, Tessa and Tavernier, Jan and Vandekerckhove, Bart},
  issn         = {1422-0067},
  journal      = {INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES},
  keywords     = {Physical and Theoretical Chemistry,Inorganic Chemistry,Organic Chemistry,Spectroscopy,Molecular Biology,Catalysis,General Medicine,Computer Science Applications,nanobody,VHH,chimeric antigen receptor,CAR T cell,CD33,CD20,PCR,Gibson Assembly,nanoCAR,CHIMERIC ANTIGEN RECEPTORS,SINGLE-DOMAIN ANTIBODIES,T-CELLS,B-CELL,ADOPTIVE IMMUNOTHERAPY,SPACER DOMAIN,CD19,THERAPY,INTEGRATION,FRAGMENT},
  language     = {eng},
  number       = {3},
  pages        = {18},
  title        = {Rapid and effective generation of nanobody based CARs using PCR and Gibson Assembly},
  url          = {http://dx.doi.org/10.3390/ijms21030883},
  volume       = {21},
  year         = {2020},
}

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