Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Histogram showing in-silico analysis of CAR T cell-treated patients ( n = 4219) revealed a high relapse rate, with 42.11% ( n = 216 of n = 513 overall relapse patients) experiencing CD19-negative recurrence after monospecific CAR Therapy ( n = 2916). b Schematic overview of the CAR design strategy showing mono, bi, and trispecific constructs targeting CD19, CD20, and CD22. c Experimental workflow illustrating CAR screening: 1452 CARs were transduced into primary T cells and analyzed for signal-1 (activation), signal-2 (exhaustion), and signal-3 (cell death). Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . d Categorization of CARs into low (L), medium (M), and high (H) levels based on fluorescence intensity cutoffs determined by CD19 CARs as reference. e bar graph showing the distribution of 1452 screened CARs across L-, M-, and H-CARMSeD categories using the CARMSeD scoring system. f AI model development pipeline for CAR dysfunction risk prediction, based on 1,452 CAR constructs with an 80:20 split for training and testing. g–j Performance metrics of AI model predicting CARMSeD scores using 1452 CAR constructs. g ML learning curve of model accuracy over 50 epochs, achieving a training accuracy of 0.98 and validation accuracy of 0.95. h Scatter plot comparing measured versus predicted CARMSeD scores for training ( R 2 = 0.87) and validation ( R 2 = 0.83) sets. i Predicted versus measured CARMSeD scores on the validation set, categorized into low (blue), medium (orange), and high (green) CARMSeD. j Box plots show the median (center line), the 25th–75th percentiles (box), and whiskers extending to the minimum and maximum non-outlier values; individual points denote outliers. Numbers above each box indicate sequence counts. k Molecular dynamics simulation of CAR constructs with varying linker lengths, assessing scFv-scFv interaction. Structural conformations at 0 ns, 50 ns and 200 ns for different CAR scFv arrangements highlighting CDR regions (surface transparency 30%), Root Mean Square Deviation (RMSD) plots over 200 ns for both constructs, respectively, indicating structural stability and conformational changes. l Bar graph showing in vitro receptor binding affinity validation for top humanized scFvs of CD19, CD20, and CD22 CARs ( n = 6 biologically independent samples). Data represent mean ± SEM. ** p < 0.01; **** p < 0.001; ns: not significant. A non-parametric t-test was used for statistical analysis between groups. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: In Silico, Construct, Activation Assay, Fluorescence, Biomarker Discovery, Sequencing, In Vitro, Binding Assay

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic illustration of the K562 cell line model expressing individual or triple combinations of CD19 (purple), CD20 (red), and CD22 (yellow) antigens. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bar chart depicting the percentage expression of each antigen in K562 cell lines, both individually and in combination. c–f Line graph of cytotoxicity assays showing antigen-specific killing of K562 target cells. All tested constructs surpassed the performance of second-generation monospecific CD19 (m19) CAR T cells ( n = 3 biologically independent samples). g Heatmap showing comparison of proliferation rates for bispecific; b20/19 or b22/19, and trispecific; t20/19/22 CAR T cells, represented as fold expansion up to Day 17 with respect to the baseline at the time of cell seeding. h Schematic of the Raji WT cell line platform expressing CD19 (purple), CD20 (red), and CD22 (yellow) antigens, edited using CRISPR-Cas9 to generate knockout variants. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . i , j Line graph of cytotoxicity assays demonstrating the superior efficacy of b20/19 CAR T cells in eliminating antigen-negative Raji variants, compared to m19 CARs ( n = 5 biologically independent samples). k Schematic representation of the tumor rechallenge (TR) model using the Raji WT cell line (Raji WT ). Gray circles represent initial engraftment and monitoring phases, pink circle shows the first incubation with Raji WT , while purple circles indicate the timing of the RajiCD19 −/− rechallenge. l Heatmap representation of TR model showing IFN-γ secretion (pg/mL), percentage of tumor lysis (1:10; T: E), and the number of CAR T cells detected on days 7, 9, 11, 15, and 17 post-rechallenge ( n = 5 biologically independent samples). Data represent mean ± SEM. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: Expressing, Construct, Comparison, CRISPR, Knock-Out, Incubation, Lysis

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic timeline of in vivo lymphoma model for evaluation of monospecific and bispecific CAR T cells. Mice were xenografted with RajiWT cells (expressing CD19, CD20, and CD22) (day 0), followed by administration of m19 or b20/19 CAR T cells on day 5 and subsequent RajiCD19 −/− TR on day 12, 19 and 26. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . b Bioluminescent imaging and ( c ) stacked area plot showing tumor burden quantification show effective tumor control by b20/19 CAR T cells versus m19 CARs ( n = 5). d CAR T cell survival over time ( n = 5 mice). e Kaplan-Meier survival curves showing survival outcomes over 70 days ( n = 5 mice). f Analysis of residual tumor CD19 or CD20 tumor cells over time ( n = 5 mice). g , h Bar plot showing Granzyme B and IFN-γ secretion from human CD8 + CAR T cells isolated b20/19 post-treatment to confirm functional cytotoxicity of b20/19 against CD19⁻ targets ( n = 5). The CAR T cells isolated from mice that received conventional monospecific (m)CD19 CAR T cells served as the control for comparison. i , j TR induced upregulation of exhaustion markers PD-1 and LAG-3 ( n = 5 mice). k Immunophenotyping of CAR T cells post-TR shows loss of central memory (T cm ) populations and increased PD-1 expression, consistent with functional exhaustion and limited engraftment ( n = 5 mice). Data represents mean ± SEM. ** p < 0.01; *** p < 0.005; **** p < 0.001. A non-parametric t-test was used for statistical analysis between groups, and for ( k ), a Two-way ANOVA followed by post-hoc testing was applied. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: In Vivo, Expressing, Imaging, Control, Isolation, Functional Assay, Comparison

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Pathway analysis of proteins involved in AKT3 interaction, modifications or regulation of its expression with emphasis on FOXO4. b Relative mRNA expression levels (normalized to beta actin; ACTB) of key genes show upregulation of FOXO4 mRNA in b20/19-AKT3 PROTAC CAR T ( n = 6 biologically independent samples). c Flow cytometry histograms of total FOXO4 and phosphorylated FOXO4 (p-FOXO4) in CAR T cells after TR with RajiCD19 −/− cells. d Histogram analysis of the flow cytometry plots ( n = 10 biologically independent samples). e Bar graph shows the percentage of CD8 + CAR T cells expressing different phenotypes. Pie charts illustrate the proportional distribution of these subsets across conditions ( n = 5 biologically independent samples). f Survival of CAR T cells over 15 days under various conditions ( n = 4 biologically independent samples). g Violin plots showing the percentage of mTOR activity (% mTOR activity) in various conditions, with shRNA based FOXO4 knockdown elevated mTOR activity ( n = 6 biologically independent samples). h Bar plots show the percentage of MFI of autophagy from autophagic flux assay ( n = 8 data points from three independent experiments). i Dot plot showing ECAR in NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 conditions, with FOXO4 knockdown increasing shift from OXPHOS to glycolysis ( n = 12 data points from three independent experiments). j Similarly, OCR with FOXO4 knockdown decreases mitochondrial respiration. Individual data points are shown for each condition ( n = 12 data points from three independent experiments). k Box-and-whisker plot showing percentage of expression of CD19 (yellow), CD20 (blue), and CD22 (purple) across 129 ALL patient samples, with varying expression levels for each marker ( n = 63 patient samples). l Bar graph showing the number of patient samples categorized as Negative/Dim, Moderate, or Bright for CD19, CD20, and CD22 expression. m Schematic illustration of K562 WT and CD20 expressing K562 stable cells transduced with different MOIs to obtain three populations: CD20 L (low), CD20 M (medium), and CD20 H (high), which were further FACS sorted. Created in BioRender. Chauhan, V. (2025) https://BioRender.com/uj3gas6 . n Violin plots showing the percentage of CD20 expression (% CD20 expression) in the sorted CD20-expressing K562 cell populations, confirming distinct expression levels ( n = 10 data flow cytometry points from three independent experiments). o Representative super-resolution microscopy images of differential CD20 surface expression in K562 cells. Images show CD20 (red) in K562-CD20 L (low), K562-CD20 M (medium), and K562-CD20 H (high) cell. p–r Survival curves of K562 cells expressing varying CD20 expression levels under CAR T cell treatments. The line graph shows the percentage of CD20 + cell survival when treated with Rituximab-based monospecific CAR (Rtx-m20, dark green), in-house humanized anti-CD20 CAR (AB21-m20, green) ( n = 4 biologically independent samples). s Survival of CAR T cells with varying CD20-targeting CAR constructs over 15 days ( n = 5). Data represents mean ± SEM. **** p < 0.001. A nonparametric t-test was used for statistical analysis between groups. For e , f and s , a Two-way ANOVA followed by post-hoc testing was applied. Scale bar: 5 μm. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: Expressing, Flow Cytometry, Activity Assay, shRNA, Knockdown, Flux Assay, Whisker Assay, Marker, Transduction, Super-Resolution Microscopy, Construct

Journal: Nature Communications

Article Title: AI-guided CAR designs and targeted pathway modulation to enhance multi-antigen CAR T cell durability and overcome antigen escape

doi: 10.1038/s41467-025-68272-5

Figure Lengend Snippet: a Schematic of the strategy for trispecific CAR T cells, integrating b20/19-AKT3 PROTAC with a secretory BiTE module consisting of nanobodies targeting CD3 and CD22 (nbCD3/22). b Correlation of expression of nbCD3, nbCD22, CD19 CAR, and CD20 CAR at various MOIs. The cells were treated with Brefeldin, and data were obtained using intracellular flow cytometry ( n = 7 data points from three independent experiments). c Experimental setup for T cell activation, using Jurkat-GFP cells and Dynabeads (db) coated with CD3 to assess secreted nbCD3/22 functionality via flow cytometry. d Dose-dependent T cell activation (CD69 expression) in response to culture supernatants (used at various ratios with culture media) with nbCD3/22, using db coated with CD3 for validation ( n = 6 data points from three independent experiments). e Line graph of HEK293T synNotch reporter assay showing dose-dependent inhibition of CD22-CAR signaling by nbCD22 in CAR T cell supernatants, confirming BiTE functionality under two condition 1 and condition 2. f Experimental timelines for in vitro T cell engineering, transduction, and co-culture with Raji cells (WT or knockout for CD19, CD20, or CD22). Anti-tumor assays were performed on days 9, 11, and 13. g , h Functional assay of CAR T cells against Raji cells (WT or knockout for CD19, CD20, or CD22) demonstrates that b20/19AKT3 PROTAC CAR T cells co-expressing nbCD3/22 exhibit stronger antitumor activity compared to b20/19-AKT3 PROTAC or mCD19 CAR T cells at Day 7 and Day 14. Data represent mean ± SEM. **** p < 0.001; ns: not significant. A nonparametric t-test was used for statistical analysis between groups. Source data are provided as a file.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Miltenyi Biotec, #130-129-550) and CD22 (Miltenyi Biotec, #130-126-727).

Techniques: Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery, Reporter Assay, Inhibition, In Vitro, Transduction, Co-Culture Assay, Knock-Out, Functional Assay, Activity Assay

Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: The cells were then washed twice and stained with phycoerythrin (PE) streptavidin (BD bioscience, USA) for 15 min. CART-22 cells and CART-22/19 cells were washed once and incubated with CD22 Fc Alexa Fluor® 647 Protein (R&D Systems, USA) for 15 min. To detect in vitro cytotoxicity of CART-19 cells, transduced and untransduced T cells were co-cultured with Nalm-6 cells (total 1 × 106 cells) at E: T ratios (0.2:1, 0.5:1, 1:1, 5:1) for 6, 24 and 72 h. Cells were pipetted to incubate with antibodies for 30 min at room temperature in the dark and washed twice with PBS.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay

Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) t-distributed stochastic neighbor embedding (t-SNE) visualization of snRNA-seq data (Smart-seq) from multiple cortical areas of human brain colored by cell type. Data from Allen Brain Atlas (19). (B) t-SNE visualization of snRNA-seq data from multiple cortical areas of human brain colored by CD22 expression. (C) Representative image of human brain tissue probed for MOG (green), CD22 (magenta), and AIF1 (red) transcripts by multiplexed fluorescent RNAscope. Clustered puncta within 4′,6-diamidino-2-phenylindole–positive nuclei suggest true signal. (D) Schematic of FACS analysis of various cell types from fresh human primary cortical tissue. (E) Flow cytometry analysis of surface CD22 protein expression in CD45+ microglia (pink), MAP2+ neurons (orange), O4+MBP− OPCs (blue) and O4+MBP+ oligodendrocytes (purple) from fresh human primary cortical tissue (PCW 22). PE quantification beads are shown in gray. (F) Quantification of CD22-PE molecules bound to the surface of various human brain cell types calculated using PE bead standards (n = 2 biological replicates; PCWs 20 to 22; O4+MBP+ cells only detected at PCW 22). (G) t-SNE visualization of snRNA-seq data from multiple cortical areas of human brain colored by ST6GAL1 expression. (H) Flow cytometry analysis of human iMGLs stained with fluorophore-conjugated CD22 lacking its sialic acid–binding domain (sCD22-Δ, gray) or the full-length CD22 ECD (sCD22-ECD, red). In one condition, cells were pretreated with sialidase before sCD22-ECD staining (blue).

Article Snippet: For CD22-IGF2R kinetic analysis, anti-human Fc capture biosensors were loaded with recombinant human CD22 Fc-fusion protein (R&D Systems) and associated into recombinant His-tagged human IGF2R analyte at seven different concentrations (1000 nM, serially diluted 1:3).

Techniques: Expressing, RNAscope, Flow Cytometry, Staining, Binding Assay

Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) Schematic of human primary cortical oligodendrocyte isolation and differentiation protocol [adapted from (64)]. IHC, immunohistochemistry. (B) Representative flow cytometry of O4+ cells after 12 days in culture (d.p.c.) at the onset of antibody treatment, showing a mixed population of premyelinating O4+MBP−CD22− cells (blue) and a subpopulation of myelinating O4+MBP+CD22+ oligodendrocytes (red). (C) Representative bright-field images of isotype-, anti-CD22−, and anti-MOG–treated oligodendrocytes on day 15 after isolation (day 3 after treatment). Confluence mask overlaid in purple. Scale bar, 100 μm. (D) Quantification of confluence in isotype (gray)–, anti-CD22 (green)–, and anti-MOG (purple)–treated cells assessed by time-lapse microscopy over 3 days (n = 3 from two separate primary tissue samples, means ± SEM). (E) Representative immunofluorescence images of isotype-, anti-CD22−, and anti-MOG–treated oligodendrocytes on day 15 after isolation (day 3 after treatment), stained for MBP (green), and OLIG2 (red). Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (F) Quantification of MBP+ cells among OLIG2+ nuclei in isotype (gray)–, anti-CD22 (green)–, and anti-MOG (purple)–treated cells (n = 3 from two separate primary tissue samples, one-way ANOVA, means ± SEM).

Article Snippet: For CD22-IGF2R kinetic analysis, anti-human Fc capture biosensors were loaded with recombinant human CD22 Fc-fusion protein (R&D Systems) and associated into recombinant His-tagged human IGF2R analyte at seven different concentrations (1000 nM, serially diluted 1:3).

Techniques: Isolation, Immunohistochemistry, Flow Cytometry, Time-lapse Microscopy, Immunofluorescence, Staining

Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) Schematic of CRISPR-Cas9 screen for genetic modifiers of sCD22 binding. (B) Volcano plot of hits from CRISPR-Cas9 screen, highlighting KOs that inhibit CD22 binding (blue) and promote CD22 binding (red). (C) Flow cytometry analysis of CD22 ligand expression on U937 cells infected with a safe-targeting sgRNA (control, red) or an IGF2R-targeting sgRNA (purple). Isotype control–stained WT cells are shown in gray. AF647, Alexa Fluor 647. (D) Schematic of affinity purification LC-MS screen for direct binding partners of sCD22. (E) Volcano plot of hits from affinity purification LC-MS screen, highlighting proteins enriched in the CD22-bound fraction (red). (F) Kinetics of the CD22-IGF2R interaction determined by biolayer interferometry. Red line shows nonlinear fit of association-dissociation curve. Kd, dissociation constant.

Article Snippet: For CD22-IGF2R kinetic analysis, anti-human Fc capture biosensors were loaded with recombinant human CD22 Fc-fusion protein (R&D Systems) and associated into recombinant His-tagged human IGF2R analyte at seven different concentrations (1000 nM, serially diluted 1:3).

Techniques: CRISPR, Binding Assay, Flow Cytometry, Expressing, Infection, Control, Staining, Affinity Purification, Liquid Chromatography with Mass Spectroscopy

Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) Schematic of mAb generation and screening pipeline. (B) Screening results of 38 mAb clones for binding to CD22 (first column) and blocking of sCD22 to IGF2R on cell surface (second and third columns are two independent experiments). Three clones with adequate binding and potent blocking are highlighted (M22, M28, and M42). (C) Association-dissociation curves of antibody candidates binding to CD22 determined by biolayer interferometry. (D) Dose-response curves of CD22-IGF2R blockade by antibody candidates determined by flow cytometry. IC50, median inhibitory concentration. (E) Time-lapse fluorescence microscopy analysis of NPC2 trafficking to lysosomes in U937 cells treated with sCD22-Δ (gray), sCD22-ECD and an isotype control antibody (purple), or sCD22-ECD and clone M42 (green) (n = 2, ANOVA, means ± SEM). (F) Schematic of pipeline to generate isogenic WT and I1061T mutant iMGLs from iPSCs edited by CRISPR-Cas9–directed homologous recombination. After introduction of donor single-stranded DNA (ssDNA) by electroporation, a homozygous T3182C nucleotide substitution was confirmed by Sanger sequencing. NPC1 reduction was confirmed by Western blot. Mutant and isogenic control iPSCs were subsequently directed toward a hematopoietic lineage and differentiated into microglia-like cells. (G) Western blot quantification of NPC1 expression normalized to a loading control (β-actin) in WT and I1061T mutant iPSCs (n = 3, t test, means ± SEM). (H) Representative images of WT and I1061T mutant iMGLs stained for Filipin III (red, unesterified cholesterol) and IBA1 (green, microglia marker). Scale bar, 20 μm. (I) Quantification of Filipin-positive area normalized to total IBA1-positive area in WT (gray) and I1061T mutant (blue) iMGLs (n = 5 biological replicates, t test, means ± SEM). (J) Schematic of human in vitro model of microglia in NPC. Three components (iPSC-derived microglia, I1061T patient mutation, and NPC patient CSF) were combined to test the proof-of-principal in vitro efficacy of anti-CD22 in NPC. (K) Representative images of I1061T mutant iMGLs treated with NPC CSF and an isotype control antibody stained for Filipin III (red, unesterified cholesterol), LAMP2 (gray, lysosome marker), and IBA1 (green, microglia marker). Scale bars, 20 μm. (L) Representative images of I1061T mutant iMGLs treated with NPC CSF and anti-CD22 stained for Filipin III (red, unesterified cholesterol), LAMP2 (gray, lysosome marker), and IBA1 (green, microglia marker). Scale bars, 20 μm. (M) Quantification of Filipin-positive area normalized to total IBA1-positive area in isotype (gray)– and anti-CD22 (green)–treated iMGLs (n = 7 biological replicates, paired t test, means ± SEM; lines connect wells treated with the same patient’s CSF). (N) Quantification of LAMP2-positive area normalized to total IBA1-positive area in isotype (gray)– and anti-CD22 (green)–treated iMGLs (n = 7 biological replicates, paired t test, means ± SEM; lines connect wells treated with the same patient’s CSF). (O) Heatmap of normalized counts (z score) for differentially expressed genes in WT and I1061T mutant iMGLs treated with NPC CSF and isotype or anti-CD22. (P) Gene Ontology (GO) biological process enrichment analysis of differentially expressed genes between anti-CD22− and isotype–treated I1061T iMGLs. Up- or down-regulation is represented on the color scale, and the number of genes differentially expressed is indicated for each term. IRE1, inositol-requiring enzyme 1; IFN-γ, interferon-γ; UPR, unfolded protein response. (Q) GO cellular component enrichment analysis of differentially expressed genes between anti-CD22− and isotype–treated I1061T iMGLs. Up- or down-regulation is represented on the color scale, and the number of genes differentially expressed is indicated for each term. MHC-II, major histocompatibility complex class II; ER, endoplasmic reticulum.

Article Snippet: For CD22-IGF2R kinetic analysis, anti-human Fc capture biosensors were loaded with recombinant human CD22 Fc-fusion protein (R&D Systems) and associated into recombinant His-tagged human IGF2R analyte at seven different concentrations (1000 nM, serially diluted 1:3).

Techniques: Clone Assay, Binding Assay, Blocking Assay, Flow Cytometry, Concentration Assay, Fluorescence, Microscopy, Control, Mutagenesis, CRISPR, Homologous Recombination, Electroporation, Sequencing, Western Blot, Expressing, Staining, Marker, In Vitro, Derivative Assay, Immunopeptidomics

Journal: bioRxiv

Article Title: Efficient and robust NK-Cell transduction with Baboon Envelope pseudotyped lentivector: a major tool for immunotherapy

doi: 10.1101/625285

Figure Lengend Snippet: (A) Schematic representation of the different LVs used for NK-cell transduction using BaEV-LV (relative scale according to size in base pairs). (B) Percentage of transduced NKAES cells using BaEV-LVs coding for an anti-CD22 CAR (n=9). (C) Flow cytometry plot representative of CAR-CD22 expression after NK-cell transduction with BaEV-LVs. (D) Cytotoxic assays against either parental (n=7) or CD19/22 KO -RS4;11 cells (B-ALL; n=2) using CAR-CD22-NK-cells (p<0.0001; 2-way ANOVA test with Bonferroni correction). (E) Cytotoxic assays against parental (n=7) or CD19/CD22 KO RS4;11 B-ALL cells (n=2) using NK-cells transduced with a dual CAR (3 rd construct illustrated in A) (p<0.0001; 2-way ANOVA test with Bonferroni correction). Data are presented as the mean ± SEM. *p<0.05; **p<0.001, ***p<0.0001

Article Snippet: To detect CAR-expression, cells were incubated with 2 µl Siglec2(CD22)-Fc chimera (50 mg/ml, R&D) for 30 minutes at 4°C, washed and stained with anti-Fc-PE (Jackson Immune), anti-CD56-APC and anti-CD3-FITC (Biolegend).

Techniques: Transduction, Flow Cytometry, Expressing, Construct

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 1A: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 indicated Nalm6 leukemia on day -3, followed by 5e6 CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day 0, as well on days 5 and 11 post-CAR. 1B: Quantification of bioluminescence data in A. 1C: ELISA measuring Granzyme B in supernatant after 16 hour co-culture of CD22-CAR T cells with the indicated leukemia. 1D: Degranulation as measured by CD107a expression after 4 hour co-culture assay. 1E: Activation as measured by CD69 expression after 6 hour co-culture assay. 1F: Activation as measured by CD25 expression after 24 hour co-culture assay. All in vitro assays performed with n=3 technical replicates, 1 experiment. In vivo assay performed with n=5 mice per group, 1 experiment. Data represent mean +/-SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: In Vivo, Injection, Imaging, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Expressing, Co-culture Assay, Activation Assay, In Vitro

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 2A: Flow cytometry plots showing IL-2 by IFNg production after 6 hour coculture of the indicated CD22-CAR T cell with the indicated leukemia. 2B: Quantification of cytokine data in A. 2C: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 indicated Nalm6 leukemia on day -3, followed by 4e6 CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR injection. Mice were monitored for survival. 2D: Quantification of bioluminescence data against WT leukemia from C. 2E: Survival of mice bearing WT leukemia. 2D: Quantification of bioluminescence data against CD22 Lo leukemia from C. 2E: Survival of mice bearing CD22 Lo leukemia. All in vitro assays performed with n=3 technical replicates, and are representative of two experiments with two independent donors. In vivo assay performed with n=5 mice per group, 1 experiment. Data represent mean +/-SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: Flow Cytometry, In Vivo, Injection, Imaging, In Vitro

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: S1A: Cell-based direct antigen-binding affinity titration assay. Indicated CARs were stained with indicated concentrations of fluorophore-conjugated CD22 Protein Fc. MFI of CAR+ Populations were measured and normalized to peak protein binding for each individual CAR. Data represents one experiment with one replicate per concentration.

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: Binding Assay, Titration, Staining, Protein Binding, Concentration Assay

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: Figures S2A to S2F quantify indicated metrics by flow cytometry after coculture of indicated CD22-CAR with indicated leukemia after 6 hour coculture. S2A: %+ and MFI for IFNg production against WT leukemia. S2B: %+ and MFI for IL2 production against WT leukemia. S2C: %+ of cells making IFNg and IL-2 against WT leukemia. S2D: %+ and MFI for IFNg production against CD22 Lo leukemia. S2E: %+ and MFI for IL2 production against CD22 Lo leukemia. S2F: %+ of cells making IFNg and IL-2 against CD22 Lo leukemia. Figures S2G to S2J quantify CAR and leukemia counts relative to a starting 5:1 ratio of leukemia and CAR to fluorescent counting beads. Aliquots were taken from each condition and analyzed by flow cytometry at each of the indicated time points. S2G: Quantification of CAR Count/Bead Count ratio against WT leukemia. S2H: Quantification of Leukemia Count/Bead Count ratio for WT leukemia. S2I: Quantification of CAR Count/Bead Count ratio against CD22 Lo leukemia. S2J: Quantification of Leukemia Count/Bead Count ratio for CD22 Lo leukemia. All in vitro assays performed with n=3 technical replicates. are representative of two experiments with two independent donors. are representative of one experiment. Data represent mean +/- SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: Flow Cytometry, In Vitro

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 3A: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 WT Nalm6 leukemia on day-3, followed by 2e6 of indicated CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR. 3B: Quantification of average bioluminescence data for each group in A. 3C: Quantification of individual bioluminescence data for each group in A. 3D: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 WT Nalm6 leukemia on day -3, followed by 4e6 of indicated CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR. 3E: Quantification of average bioluminescence data for each group in D. 3F: Survival of mice treated with 4e6 of the indicated CAR T cells. In vivo assay performed with n=5 mice per group, 1 experiment (3A to 3C) or 3 experiments with independent donors (3D to 3F). 3D to 3E are representative data from one experiment. 3F is pooled data, SA-SL (n=15), HA-SL (n=15), HA-LL (n=15). Data represent mean +/- SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: In Vivo, Injection, Imaging

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 4A: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 CD22 Lo Nalm6 leukemia on day -3, followed by 4e6 of indicated CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR. 4B: Quantification of average bioluminescence data for each group in A. 5C: Quantification of individual bioluminescence data for each group in A. For 4D to 4E, bone marrow was analyzed by flow cytometry at day 18 post-CAR for indicated cell population. 4D: % CAR+ of live marrow. 4E: % leukemia of live marrow. 4F: Survival of mice treated with 4e6 of indicated CAR T cells. In vivo assay performed with n=5 mice per group, 4 experiments with independent donors. Data in 4A to 4C is representative data from one experiment. Survival is pooled from 3 experiments with independent donors: Mock (n=10), SA-SL (n=15), HA-SL (n=10), HA-LL (n=15). Data represent mean +/- SD. * p<0.05, ** p<0.01, *** p<0.001, ****

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: In Vivo, Injection, Imaging, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors

doi: 10.1074/jbc.M117.795047

Figure Lengend Snippet: Binding of IgG1-Fc variants to glycan receptors. A , mutants lacking the Asn-297 glycan are severely restricted in their capacity to bind DC-SIGN by ELISA. The addition of an N -linked sugar at position 221 results in proteins with a reduced capacity to bind DC-SIGN compared with their equivalent variants in which Asn-221 is absent. B , the hypersialylated D221N mutants bind Siglec-1. No binding was observed with the N297A/N563A glycan-deficient mutant ( error bars represent standard deviations around the mean value, n = 2 independent experiments).

Article Snippet: The same ELISA protocol used to detect DC-SIGN binding was used for human Siglec-1, Siglec-4, and Siglec-3 (Sino Biologicals).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors

doi: 10.1074/jbc.M117.795047

Figure Lengend Snippet: Model showing the contribution of different N -linked glycan and cysteine residues on Fc stoichiometry. The presence of Cys-575 allows optimal disulfide bonding between tail pieces of monomeric-Fcs. The tail piece glycan Asn-563 controls the number of monomeric tails that fit into the central corona (five to six in the case of hexa-Fc) while still allowing Cys-309 interdisulfide bridge formation. Cys-575 allows disulfide bonding between tail pieces of different monomers, but the absence of the Asn-563 glycan (the N563A mutant) allows many more tail pieces (up to twelve in the case of dodecamers) to fit into the central corona while still allowing disulfide bond formation through Cys-309 and/or Cys-575. The absence of Cys-575 prevents disulfide bonding between tail pieces, thereby generating sialylated monomers at Asn-563. The additional Asn-563 tail piece glycan in these monomers must explain the increased binding seen to Siglec-1 ( , A and B , and inset in this figure). The bulkier Asn-563 glycan with its predicted overall negative charge may lead to repulsion between two monomers, thus preventing disulfide bond formation between two Cys-309 residues in each monomeric Fc. The loss of both Asn-563 and Cys-575 (the N563A/C575A mutant) means that the observed laddered multimers must arise through Cys-309–mediated disulfide bonding in the Cγ2 domain. The presence of monomers, dimers, trimers, tetramers, pentamers, hexamers, and other intermediates in this mutant ( C ) suggests that these structures arise through a different mechanism, most likely via the sequential addition of 25-kDa half-mer Fc units at Cys-309. The lack of observable ladders with the L448STOP mutant implies that other amino acids in the tail piece are involved in bringing about monomer interactions that then facilitate disulfide bonding through either Cys-309 and/or Cys-575. Monomers with glycans located at both the N- and C-terminal ends of the Fc (Asn-221 and Asn-563) may allow for binding to receptors in cis as shown ( inset ).

Article Snippet: The same ELISA protocol used to detect DC-SIGN binding was used for human Siglec-1, Siglec-4, and Siglec-3 (Sino Biologicals).

Techniques: Mutagenesis, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors

doi: 10.1074/jbc.M117.795047

Figure Lengend Snippet: Binding of monomeric IgG1-Fc glycan variants to sialic acid-binding immunoglobulin-type lectins (Siglecs) with specificity for α2,3-linked sialic acid. A , the C575A monomer binds Siglec-1. B , the D221N/C575A monomer binds Siglec-1 and Siglec-4. ELISA as described under “Experimental procedures” with receptors coated down at 2 μg/ml and Fc-fragments at 20 μg/ml in TMS buffer ( error bars represent standard deviations around the mean value, n = 2 independent experiments).

Article Snippet: The same ELISA protocol used to detect DC-SIGN binding was used for human Siglec-1, Siglec-4, and Siglec-3 (Sino Biologicals).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay