Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene
Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and s...
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doaj-2fbad32be4d1496aa39b24a192390c942020-11-24T23:15:17ZengFrontiers Media S.A.Frontiers in Immunology1664-32242018-03-01910.3389/fimmu.2018.00507329239Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide GeneMonica Casucci0Laura Falcone1Barbara Camisa2Margherita Norelli3Simona Porcellini4Anna Stornaiuolo5Fabio Ciceri6Fabio Ciceri7Catia Traversari8Claudio Bordignon9Chiara Bonini10Chiara Bonini11Attilio Bondanza12Attilio Bondanza13Attilio Bondanza14Innovative Immunotherapies Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Hospital Scientific Institute, Milano, ItalyInnovative Immunotherapies Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Hospital Scientific Institute, Milano, ItalyInnovative Immunotherapies Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Hospital Scientific Institute, Milano, ItalyInnovative Immunotherapies Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Hospital Scientific Institute, Milano, ItalyMolmed Spa, Milano, ItalyMolmed Spa, Milano, ItalyVita-Salute San Raffaele University, Milano, ItalyHematology and Bone Marrow Transplantation Unit, San Raffaele Hospital Scientific Institute, Milano, ItalyMolmed Spa, Milano, ItalyMolmed Spa, Milano, ItalyVita-Salute San Raffaele University, Milano, ItalyExperimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Hospital Scientific Institute, Milano, ItalyInnovative Immunotherapies Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Hospital Scientific Institute, Milano, ItalyVita-Salute San Raffaele University, Milano, ItalyHematology and Bone Marrow Transplantation Unit, San Raffaele Hospital Scientific Institute, Milano, ItalyChimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.http://journal.frontiersin.org/article/10.3389/fimmu.2018.00507/fullCAR-T cellsCAR spacercell sortinggood manufacturing procedures-manufacturingantitumor efficacysuicide gene |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Monica Casucci Laura Falcone Barbara Camisa Margherita Norelli Simona Porcellini Anna Stornaiuolo Fabio Ciceri Fabio Ciceri Catia Traversari Claudio Bordignon Chiara Bonini Chiara Bonini Attilio Bondanza Attilio Bondanza Attilio Bondanza |
spellingShingle |
Monica Casucci Laura Falcone Barbara Camisa Margherita Norelli Simona Porcellini Anna Stornaiuolo Fabio Ciceri Fabio Ciceri Catia Traversari Claudio Bordignon Chiara Bonini Chiara Bonini Attilio Bondanza Attilio Bondanza Attilio Bondanza Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene Frontiers in Immunology CAR-T cells CAR spacer cell sorting good manufacturing procedures-manufacturing antitumor efficacy suicide gene |
author_facet |
Monica Casucci Laura Falcone Barbara Camisa Margherita Norelli Simona Porcellini Anna Stornaiuolo Fabio Ciceri Fabio Ciceri Catia Traversari Claudio Bordignon Chiara Bonini Chiara Bonini Attilio Bondanza Attilio Bondanza Attilio Bondanza |
author_sort |
Monica Casucci |
title |
Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene |
title_short |
Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene |
title_full |
Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene |
title_fullStr |
Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene |
title_full_unstemmed |
Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene |
title_sort |
extracellular ngfr spacers allow efficient tracking and enrichment of fully functional car-t cells co-expressing a suicide gene |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Immunology |
issn |
1664-3224 |
publishDate |
2018-03-01 |
description |
Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting. |
topic |
CAR-T cells CAR spacer cell sorting good manufacturing procedures-manufacturing antitumor efficacy suicide gene |
url |
http://journal.frontiersin.org/article/10.3389/fimmu.2018.00507/full |
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