Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

Abstract

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.

In vivo generated human CAR T cells eradicate tumor cells

“…Approaches to optimize the conventional CAR T cell product currently focus on the identification of conditions that favor the enrichment of specific T cell phenotypes such as central memory T cells (T CM ) or effector memory T cells (T EM ) in order to obtain a product exhibiting long-lasting antitumor efficacy. [16][17][18] Since most of the anti-tumoral activities take place in the bone marrow we evaluated the phenotype of in vivo generated CAR T cells in bone marrow based on CD45RA and CD62L expression. In vivo generated CD8- targeted CAR T cells displayed predominantly effector T cell (T eff ) or effector memory T cell (T EM ) phenotype in a donordependent manner (Figure 3(a)).…”

CAR T Cells Redirected to CD44v6 Control Tumor Growth in Lung and Ovary Adenocarcinoma Bearing Mice

“…Moreover, to overcome the CAR T cell product variability related to the different transduction efficiency across individuals, we took advantage of the human low affinity Nerve Growth Factor receptor ( LNGFR)-spacer present in the CD44v6.CAR (13). The LNGFR enables to efficiently enrich CAR T cells with a validated clinical-grade technology (12,13), thus allowing the manufacturing of more standardized CAR T cell products with narrowed variability.…”

“…The CD44v6 CAR includes the single chain variable fragment (scFv) of the humanized mAb BIWA-8 (15), specific for the CD44v6 antigen, a spacer formed by the extracellular domain of the human low-affinity nerve growth factor receptor ( LNGFr), that allows recognition and selection of CAR transduced cells, the transmembrane and intracellular domain of the human costimulatory molecule CD28, and the intracellular signaling domain of the human CD3ζ chain. The same cloning strategy was applied to generate the control vector encoding a CAR specific for the CD19 antigen (CD19 CAR) (13) and the HSV-TK Mut2 suicide gene. In some experiments as control we used the SFCMM3 Mut2 retroviral vector, where the CAR was substituted by the extracellular domain of the LNGFr (12).…”

Recent Advances in Allogeneic CAR-T Cells

“…In this system, injection of rimiducid induced caspase-9-dependent CAR-T cell apoptosis, enhancing cell proliferation, cytokine secretion, and anti-tumor efficacy [94]. Another suicide gene is Herpes Simplex Virus thymidine kinase (HSV-TK), which is inserted into the CD44 isoform variant 6 (CD44v6) CAR plasmid and conditionally co-expressed when needed [95]. Rituximab has been used to treat many cancers, such as non-Hodgkin's lymphoma, rheumatoid arthritis, and chronic lymphocytic leukemia, by targeting CD20 on the B-cells of the related disease [96][97][98][99][100].…”