Journal: eBioMedicine

Article Title: Tandem CD19/CD22 CAR T-cells as potential therapy for children and young adults with high-risk r/r B-ALL

doi: 10.1016/j.ebiom.2025.105872

Figure Lengend Snippet: Tandem anti-CD19/CD22 CAR T-cell therapy response. A) Schematic diagram of the tandem anti-CD19/CD22 CAR structure. B) Flow chart of the study. C) Swimmer plot showing clinical responses after tandem anti-CD19/CD22 CAR T-cell product infusion. D) PET-TC imaging of patient P9 before (A.1 and A.2) and 28 days after (B.1 and B.2) tandem anti-CD19/CD22 CAR T-cell infusion. E) Event free survival (EFS) Kaplan–Meier curve in all patients (n = 10). F) Overall survival (OS) Kaplan–Meier curve in all patients (n = 10). For E-F, black dots on the curve represent censored observations. HSCT, haematopoietic stem cell transplantation. MRD, minimal residual disease. EMR, extramedullary relapse. PD, progression of disease. ∗ For P6, 6 reinfusions were performed (every two weeks), the last with 3 doses. ∗∗P4 joined a clinical trial with carfilzomib after relapse, with PD shortly after.

Article Snippet: The CD19 (130-129-550) and CD22 (130-126-727) CAR detection reagents, anti-CD19-Viogreen (130-113-649), anti-CD3-Viogreen (130-113-142), and anti-biotin-PE (130-110-951) were from Miltenyi Biotec.

Techniques: Imaging, Transplantation Assay

Journal: eBioMedicine

Article Title: Tandem CD19/CD22 CAR T-cells as potential therapy for children and young adults with high-risk r/r B-ALL

doi: 10.1016/j.ebiom.2025.105872

Figure Lengend Snippet: Tandem anti-CD19/CD22 CAR T-cell product immunophenotype and analysis. A) Tandem anti-CD19/CD22 CAR T-cell expansion in all products manufactured in the CliniMACS Prodigy closed system. B) CD19-CAR and CD22-CAR expression in all products manufactured by flow cytometry. C) CD4 + and CD8 + cell populations in all 10 products manufactured. D) PD-1 receptor expression on product cells. In C and D, graphs show box and whisker plots (vertical bars, min to max points; box, first to third quartile, with median as horizontal bar). E) Memory subpopulations determined by flow cytometry. Central memory cells (CD45RA − CD27 + ), effector memory cells (CD45RA − CD27 − ), naïve cells (CD45RA + CD27 + ) and TEMRA cells (CD45RA + CD27 − ) are represented. F) Specific-lysis by tandem anti-CD19/CD22 CAR T-cells against SEM cell line determined by 4-h Europium-BATDA assay at different E:T ratios. G) Degranulation assay against SEM cell line determined by CD107a expression after 4 h of co-culture at 1:1 or 1:2 E:T ratios (see Methods). A–D and F and G , in green, living patients; in purple, relapsed patient; in black, patients who died.

Article Snippet: The CD19 (130-129-550) and CD22 (130-126-727) CAR detection reagents, anti-CD19-Viogreen (130-113-649), anti-CD3-Viogreen (130-113-142), and anti-biotin-PE (130-110-951) were from Miltenyi Biotec.

Techniques: Expressing, Flow Cytometry, Whisker Assay, Lysis, Degranulation Assay, Co-Culture Assay

Journal: eBioMedicine

Article Title: Tandem CD19/CD22 CAR T-cells as potential therapy for children and young adults with high-risk r/r B-ALL

doi: 10.1016/j.ebiom.2025.105872

Figure Lengend Snippet: Tandem anti-CD19/CD22 CAR T-cell persistence in patients after product infusion. A) Tandem anti-CD19/CD22 CAR expression was determined with anti-CD19 CAR gated on CD3 + cells. Left panel, absolute numbers of peripheral blood CAR T-cells per μl in infused patients detected by flow cytometry. Right panel, percentage of CAR + cells within the T cell compartment. B) Copies per ml as detected using real-time qPCR (see Methods) (16). C) IL-6 levels from serum after CAR T-cell infusion. D) Peak IL-6 levels were upregulated in patients with ICANS or severe CRS. Box and whisker plot shows all points (vertical bars, min to max points; box, first to third quartile, with median as horizontal bar). # No CRS group included patients with mild phenotype (I-II grade); CRS group included patients with III-IV grade. In green, living patients; in purple, relapsed patient; in black, patients who died.

Article Snippet: The CD19 (130-129-550) and CD22 (130-126-727) CAR detection reagents, anti-CD19-Viogreen (130-113-649), anti-CD3-Viogreen (130-113-142), and anti-biotin-PE (130-110-951) were from Miltenyi Biotec.

Techniques: Expressing, Flow Cytometry, Whisker Assay

Journal: bioRxiv

Article Title: MiniCARbids: Minimalistic human binding domains specifically tailored to CAR T applications

doi: 10.1101/2025.09.09.675083

Figure Lengend Snippet: (A) The K D values of CD22-miniCARbids were determined by titrations of soluble CD22-miniCARbids on NALM6 cells. (B) A representative example of titrations of miniCARbids 22_1611 and 22_1317 on NALM6 cells is shown. The binding intensity was assessed via anti-His-tag staining by flow cytometry. Data were fitted with a 1:1 binding model (solid lines) for the calculation of the respective K D values illustrated in (A) (average ± SD, n=3 or 4, biological replicates). (C) Thermostability of CD22-miniCARbids and their parental protein 5UMR was assessed using DSC (average ± SD of 3 independent measurements, technical replicates). (D) Aggregation properties of CD22-miniCARbids were assessed using SEC-HPLC. One representative analysis (n=3, technical replicates) of CD22-miniCARbids and their parental protein 5UMR is shown. (E) Binding specificity was assessed by incubating NALM6, Raji or Jurkat (CD22-negative) cells with 250 nM CD22-miniCARbid, followed by flow cytometric analysis (one of three biological replicates is shown).

Article Snippet: Selection campaigns started with magnetic bead selections using Dynabeads Biotin Binder (Thermo Fisher Scientific) as described previously., Yeast display selections for miniCARbids against CD22 were based on a soluble, biotinylated CD22 protein (AcroBiosystems, SI2-H82E3).

Techniques: Binding Assay, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: MiniCARbids: Minimalistic human binding domains specifically tailored to CAR T applications

doi: 10.1101/2025.09.09.675083

Figure Lengend Snippet: (A) CAR architecture used for the in vitro assessment of CAR activity. (B) Expression of CARs based on ten CD22-specific miniCARbids and scFvs HA22, m971-1xG 4 S and m971-4xG 4 S as benchmarks in Jurkat Nur77 reporter cells was assessed via anti-MAP-tag staining by flow cytometry (average ± SD, n=3, biological replicates). (C) Activation of CD22-specific CARs in Jurkat Nur77 reporter cells in the presence or absence of a 2-fold excess of NALM6 target cells was assessed via the expression of mKO2 by flow cytometry (average ± SD, n=3, biological replicates). (D) Cytotoxicity of CD22-specific CAR T cells and mock T cells (no CAR) against Raji cells (E:T 2:1, average ± SD, n=4, biological replicates). (E and F) Release of IFN-γ (E) and IL-2 (F) analyzed via ELISA. The cytokines were analyzed in the supernatants of co-cultures with Raji cells (E:T 2:1, average ± SD, n=4, biological replicates). (G) Cytotoxicity of CD22-specific CAR T cells and mock T cells (no CAR) against NALM6 cells (E:T 2:1, average ± SD, n=4, biological replicates). (H and I) Release of IFN-γ (H) and IL-2 (I) analyzed via ELISA. The cytokines were analyzed in the supernatants of co-cultures with NALM6 cells (E:T 2:1, average ± SD, n=4, biological replicates). Statistical analysis was performed using a repeated measure One-Way ANOVA with a Tukey post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001). The statistical analysis for the cytokine concentration was performed using log-transformed values. Parts of this figure were created with BioRender.com.

Article Snippet: Selection campaigns started with magnetic bead selections using Dynabeads Biotin Binder (Thermo Fisher Scientific) as described previously., Yeast display selections for miniCARbids against CD22 were based on a soluble, biotinylated CD22 protein (AcroBiosystems, SI2-H82E3).

Techniques: In Vitro, Activity Assay, Expressing, Staining, Flow Cytometry, Activation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transformation Assay

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD22 , REA340 , 50 , 130-124-223 , FITC , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Deep spatial profiling of human palatine tonsil tissues. (A) Hematoxylin and eosin (H&E) staining after 92 MICS cycles, including the marked epithelium, germinal center (GC), and T cell zone of the lymphoid follicle. MICS DAPI and stroma staining depicting the composition and structure of the tonsil. Markers: Collagen III, collagen IV, fibronectin (all extracellular matrix (ECM), cytokeratin (epithelium), podoplanin (lymphatic vessels), CD105/SM Actin (blood vessels). (B) Immune cell content of a human palatine tonsil comprising T cells (CD3), B cells (CD19/CD20), plasma cells (PCs) (CD38/CD138), NK cells (CD56), granulocytes (CD15/CD66b), mast cells (CD117), macrophages (MΦ) (CD163/CD169/CD206), myeloid dendritic cells (mDCs) (CD11c), and plasmacytoid dendritic cells (pDCs) (CD123). (C) Detailed view on the T cell zone, mainly composed of CD4 + helper T cells (T h ) and CD8 + cytotoxic T cells (T c ), mDCs (CD11c), and PCs (CD38/CD138). (D) Detailed view on the GC-mantle zone border, showing different B cells (CD11b, CD21, CD22), mDCs (CD11c), and PCs (CD38/CD138). (E) Cell annotations of three different tonsil samples plus respective bar graphs of gated cell populations, comparing the cell content between the three tonsil samples. Depicted markers and annotated cell types as indicated by the color code. ROI sizes: 976 x 640 µm, zoomed-in subregions in (C, D) : 334 µm x 219 µm. Scale bar: 100 µm.

Article Snippet: CD22 , REA340 , 50 , 130-124-223 , FITC , Miltenyi Biotec.

Techniques: Staining, Clinical Proteomics

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail does not alter tyrosine phosphorylation of CD22 or ERK phosphorylation. Splenocytes from IgM and IgMG transgenic mice were stimulated with 50 μg/ml anti-IgM F(ab′) 2 . Total cellular proteins (A) or immunoprecipitated CD22 (B) were fractionated by SDS-PAGE and Western blotted with antiphosphotyrosine antibody (A and B, top) or anti-CD22 antibody (B, bottom). The ratios of phosphorylated CD22 to total CD22 are indicated. (C and D) Mean fluorescence intensity (MFI) of permeabilized B220 + CD21 medium CD23 + follicular B cells stained by flow cytometry for phosphorylated ERK either (C) at the indicated times after stimulation with 50 μg/ml anti-IgM F(ab′) 2 or (D) in unstimulated (−) versus stimulated (+) cells after 2 min in the presence of MEK inhibitors PD98059, U0126, or DMSO as diluent controls. Data are representative of two experiments.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Phospho-proteomics, Transgenic Assay, Immunoprecipitation, SDS Page, Western Blot, Fluorescence, Staining, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail evokes an augmented Ca 2+ response independent of CD22. RBC-depleted splenocytes from mice of the indicated genotypes were labeled with 1 μM Indo-1 for 30 min at 37°C. (A and B) The cells were counterstained with antibodies against B220 and CD21 for the final 10 min of Indo-1 loading. The stained cells were at first acquired for 30 s and then stimulated with the indicated concentrations of anti-IgM F(ab′) 2 antibody (A, 5 μg/ml; and B, 0.5 μg/ml). Lines show the mean Indo-1 ratio in B220 + CD21 medium cells. Data from one out of three independent experiments are shown. (C and D) Indo-1–loaded cells stained with antibody against B220 were simulated with the indicated concentrations of HEL conjugated to PE (HEL-PE; C, 5 μg/ml; and D, 0.5 μg/ml), and the Indo-1 ratio was measured on gated HEL + B220 + cells.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Labeling, Staining

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail increases antibody production independent of CD22. 5 × 10 5 HEL-binding splenic B cells from IgMG or IgM transgenic donors of the indicated CD22 genotypes were adoptively transferred into nonirradiated C57BL/6 mice, and the recipient mice were immunized with HEL in CFA. The concentration of anti-HEL IgM a antibody 10 d after immunization was measured in the serum of individual recipient mice (circles) by ELISA. Data are representative of three separate experiments. Significant differences were determined by the Mann-Whitney test. *, P < 0.05; **, P < 0.01.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Binding Assay, Transgenic Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: CD22 deficiency suppresses IgM but not IgMG marginal zone B cell differentiation. Splenocytes were stained with HEL and combinations of antibodies against CD21/CD23/HyHEL9/B220 (A and B) or CD21/CD1d/HyHEL9/B220 (C and D). (A and C) The displayed profiles are gated on B220 + HEL-binding cells, and the percentage of B220 + HEL-binding cells in each window is shown. (B and D) Percentages of HEL-binding B cells in the marginal zone subset in individual mice (circles). Significant differences, as determined by the Student's t test, are indicated.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Cell Differentiation, Staining, Binding Assay