Journal: bioRxiv

Article Title: PTPN2-KO CAR-T Cells Demonstrate Enhanced Effector Function, CNS Infiltration, and Toxicity in a Non-Human Primate CAR-T Model

doi: 10.1101/2025.09.17.676426

Figure Lengend Snippet: A . CRISPR/Cas9 PTPN2 gene editing efficiency in healthy human T cells quantified by Synthego ICE deconvolution from Sanger sequencing spectra. Each dot represents a different T cell donor. B-C . PTPN2 WT or KO T cells were stimulated with PHA/Ionomycin and stained for IFN ψ (B) and TNF α (C). Fraction of positive cells of total CD8+ T cells is shown. D . IFN ψ secretion in human PTPN2 WT and KO T cells measured by ELISpot immunoassay following limiting dilution stimulation with CD3 agonistic monoclonal antibody for 18-hours. E . CAR-T cell cytotoxicity against NALM6 tumor cells 4 hours after co-culture with PTPN2-WT or KO CD19.28σ CAR-T cells at an effector-to-target ratio of 1:1. F . CAR-T cell cytotoxicity against CD19 low NALM6 tumor cells 4 hours after co-culture with PTPN2-WT or KO CD19.28σ CAR-T cells at an effector-to-target ratio of 1:1. G-I . Fold change in NFAT (G), NFĸB (H), and IRF (I) activity in PTPN2-WT or KO Jurkat reporter cell lines after activation with CD3 monoclonal antibody or PHA for 16h. J-K . Fold change in NFAT (J) and NFĸB (K) in PTPN2-WT or KO CD19.41BBσ (red) and CD19.CD28σ (blue) CAR Jurkat reporter cell lines after CAR-specific stimulation with NALM6 for 18 hours. Representative results from at least 2 independent experiments with different healthy T cell donors as indicated. Unpaired t-test, * indicates p<0.05, ** p<0.01 and *** p<0.001.

Article Snippet: We first identified a CRISPR/Cas9 guide that would selectively and effectively delete PTPN2 in primary human T-cells in a region with low homology to PTPN1 ( Supplemental Fig.1A-B ), achieving a mean editing efficiency of 73.5+/-2.9% in primary T and CAR-T cells measured by interference analysis using Synthego ICE deconvolution ( , Supplemental Fig.1A) and transcriptional analysis (Supplemental Fig.1B) .

Techniques: CRISPR, Sequencing, Staining, Enzyme-linked Immunospot, Co-Culture Assay, Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: PTPN2-KO CAR-T Cells Demonstrate Enhanced Effector Function, CNS Infiltration, and Toxicity in a Non-Human Primate CAR-T Model

doi: 10.1101/2025.09.17.676426

Figure Lengend Snippet: A . Gene alignment of PTPN1 and PTPN2 in human and rhesus macaque at the PTPN2 gRNA binding site. B . PTPN2 expression in single cell transcriptomic analysis of PTPN2-WT CAR-T cell recipients compared to canonical T cell markers ( TRAC, CD4 and CD8 ). Bubble size represents fraction of cells, and color intensity expression level. C . Manufacturing process of autologous CD20-directed BBσ PTPN2-KO CAR-T cell, and subsequent infusion into lymphodepleted recipients. D . Fraction of CAR+ T-cells measured by EGFRt expression in PTPN2-KO CAR-T manufactured products. E . PTPN2 editing rates in PTPN2-KO CAR-T infusion products quantified by Synthego ICE deconvolution from Sanger sequencing spectra. F . CD4 and CD8 fraction of total CD3+ T cells in manufactured products.

Article Snippet: We first identified a CRISPR/Cas9 guide that would selectively and effectively delete PTPN2 in primary human T-cells in a region with low homology to PTPN1 ( Supplemental Fig.1A-B ), achieving a mean editing efficiency of 73.5+/-2.9% in primary T and CAR-T cells measured by interference analysis using Synthego ICE deconvolution ( , Supplemental Fig.1A) and transcriptional analysis (Supplemental Fig.1B) .

Techniques: Binding Assay, Expressing, Sequencing

Journal: bioRxiv

Article Title: PTPN2-KO CAR-T Cells Demonstrate Enhanced Effector Function, CNS Infiltration, and Toxicity in a Non-Human Primate CAR-T Model

doi: 10.1101/2025.09.17.676426

Figure Lengend Snippet: A . Flow cytometry analysis of CAR+ cells of total CD3+ T cells in peripheral lymphoid organs and central nervous system at time of maximum expansion in recipients receiving DL4 PTPN2 WT vs. KO CAR-T cells. Historical control WT CAR-T cell recipient published included for reference. One-way ANOVA, followed by 2-way student t-tests. B . PTPN2 editing rates in fluorescently cell sorted CAR+ CD3+ T cells at time of product thaw, and maximum expansion in PTPN2-KO DL4 CAR-T cell recipient. Editing rates calculated by Synthego ICE deconvolution from Sanger sequencing spectra. C-D . UMAP embedding of T cells defined by expression of CD3E or TRAC transcripts colored by recipient (C) and tissue type (D). E . Cluster cell type annotations based on CD8A/CD4 expression, CAR Transcript expression, and top 50 highly enriched genes in each cluster. G-H . UMAP embedding showing normalized expression of CD8A (G) and CD4 (H) transcripts. I . Heatmap showing normalized expression of differentially expressed transcripts in PTPN2 WT vs KO blood and brain samples, grouped by unsupervised hierarchical clustering into 4 distinct modules. Gene transcripts are listed in order. J . Violin plots show expression of transcripts in Module 1 upregulated in CD8+ PTPN2-KO brain and blood. K-L . NFAT and NF-ĸB Activity in PTPN2 WT and KO CAR-Jurkat reporter cells upon stimulation with NALM6 in media vs. healthy donor CSF. Representative results from 2 independent experiments. Unpaired t-test, * indicates p<0.05, ** p<0.01 and *** p<0.001.

Article Snippet: We first identified a CRISPR/Cas9 guide that would selectively and effectively delete PTPN2 in primary human T-cells in a region with low homology to PTPN1 ( Supplemental Fig.1A-B ), achieving a mean editing efficiency of 73.5+/-2.9% in primary T and CAR-T cells measured by interference analysis using Synthego ICE deconvolution ( , Supplemental Fig.1A) and transcriptional analysis (Supplemental Fig.1B) .

Techniques: Flow Cytometry, Control, Sequencing, Expressing, Activity Assay