Tu115 - Deep Characterization of Autologous Living Drug, the Engineered Treg- like CD4LV-FOXP3, from Patients with IPEX Syndrome Using Multidimensional Methods

Šimon Borna – Department of Pediatrics – Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA; Akshaya Ramachandran – Department of Pediatrics – Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA; Jason Nideffer – Department of Pediatrics – Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA; Keri Marie Tate – Laboratory for Cell and Gene Medicine (LCGM) – Laboratory for Cell and Gene Medicine (LCGM) Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive West, Room 3039, Stanford, CA 94305, USA.; Prachi Wani – Laboratory for Cell and Gene Medicine (LCGM) – Laboratory for Cell and Gene Medicine (LCGM) Stanford University School of Medicine, Lorry I. Lokey Stem Cell Research Building, 265 Campus Drive West, Room 3039, Stanford, CA 94305, USA.; Yu-Hsuan Kuo – Center for Definitive and Curative Medicine (CDCM) Stanford University School of Medicine, Stanford, CA; Rajni Agarwal – Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA; Jessie Alexander – Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA; Shweta Namjoshi – Division of Pediatric Gastroenterology, Stanford University School of Medicine & the Lucile Packard Children’s Hospital, 750 Welch Road- Suite 116, Palo Alto, CA, 94304, USA; Maria Grazia Roncarolo – Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA; Rosa Bacchetta – Department of Pediatrics, Division of Hematology, Oncology, Stem Cell Transplantation and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA

Abstract Text: Human CD4+T cells from both healthy subjects and FOXP3 deficient patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) acquire the phenotype and in vitro and in vivo function of regulatory T cells (Treg) upon lentiviral-mediated expression of exogenous FOXP3. The resulting engineered Treg-like cells, termed CD4LV-FOXP3, are potential “living drugs” for autoimmune diseases with dysfunctional Tregs and are now tested in patients with IPEX in a Phase 1 clinical trial (NCT05241444). However, the heterogeneity of the CD4LV-FOXP3 and the cell-subsets changes of the parental CD4+ T during transduction remains ill-defined. Here, we combined single-cell CITE and TCR sequencing, and other methods to investigate the CD4+T cells from IPEX patients before and after manufacturing of CD4LV-FOXP3. We identified 14 conserved subpopulations with distinct transcriptome profiles from 3 NGFR+ drug substances (NGFR, the co-transduced surface marker) and their NGFR- un-transduced counterparts. The composition of the subsets was comparable among different patients-derived cell products. Compared with the NGFR- subsets, NGFR+ subsets had higher expression of the transgene FOXP3-lv, memory marker gene SELL, CD27 and Treg marker genes, including CCR4 and IL2RA, and lower expression of IL7R and the inflammatory cytokines IL17A, IL17F, IL13, IL22, IL5, IFNG. In addition, NGFR+ clusters have constrained TCR clone expansion and cycling status. Thus, our study reveals that CD4LV-FOXP3 products preserved the heterogeneity of the parental population of cells and further confirmed, at the single-cell level, their Treg-like signatures. These findings constitute the baseline for monitoring the persistence and functional stability of the CD4LV-FOXP3 cells in vivo.