Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Long-term killing assay of TIM-3.CAR-CIK cells against four primary AML samples compared with NT cells. Blasts survival was assessed by flow cytometry (E:T 1:10 and 1:50, n = 8 donors). See also Figure S2D . (B) Survival of TIM-3 + primary AML blasts (n=3) after 7-day co-culture with TIM-3.CAR-CIK or NT cells (E:T 1:10 and 1:50, n = 4 donors). See also Figure S2E . (C) Recovery of the LSC-enriched CD34 + CD38 - population (n = 9) and ( D ) of GPR56 + blasts (n = 6) after long-term co-culture. (E) Proliferation of TIM-3.CAR-CIK cells assessed by Ki67 staining after 72 hours co-culture with AML blasts (E:T 1:1, n = 7). See also Figure S2G . (F) Cytokine production (IFN-γ, IL-2) after 5 hours co-culture of TIM-3.CAR-CIK or NT cells with primary AML blasts (E:T 1:3, n = 9). See also Figure S2H . Data are presented as individual values and mean ± SD. Statistics were calculated with repeated-measures two-way ANOVA with Bonferroni’s post hoc test. ns, not significant; *p = 0.01, **p < 0.001, ***p = 0.0001 and ****p < 0.0001. See also Figure S2 for TIM-3.CAR validation in KASUMI-3, AML cell line.

Article Snippet: KASUMI-3, KG-1 (AML cell lines) and REH cell lines (ALL cell line) were sourced from American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Co-Culture Assay, Staining, Biomarker Discovery

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) TIM-3 expression on KASUMI-3 cells, primary AML blasts, and healthy immune subsets (CIK cells, monocytes, NK cells) assessed by flow cytometry using QuantiBRITE beads. REH (ALL cell line) served as negative control. (B) Short-term killing assay of TIM-3.CAR-CIK cells against CIK (n = 11) or KASUMI-3 (n = 8) cells compared with NT cells. Target cell lysis was evaluated by flow cytometry (E:T 5:1). (C) Short-term killing assay of TIM-3.CAR-CIK cells against monocytes (n = 8) or NK cells (n = 8) compared with NT (E:T 5:1). KASUMI-3 (n = 4) were included as positive control. (D) Immunoblot analysis of TIM-3 in lysates from monocytes, CIK cells, and KASUMI-3 cells following enzymatic treatment with PNGase F or broad neuraminidase, probed with a commercial anti–TIM-3 antibody (TIM-3-cmAb). GAPDH, loading control. Glycan symbols follow SNFG. (E) TIM-3 immunoprecipitates from monocytes, CIK cells, and KASUMI-3 cells treated with PNGase F or O- glycosidase and analyzed by immunoblot with TIM-3-cmAb and lectin far-western with Aleuria aurantia lectin (AAL; fucosylated epitopes). TGX stain-free total protein signal is shown as a loading/normalization control. (F) KASUMI-3 cells treated with vehicle (mock) or the fucosylation inhibitor 2F-peracetyl-fucose (SGN-2FF), followed by PNGase F or neuraminidase treatment and immunoblot/lectin probing with TIM-3-cmAb and AAL. See also Figure S3A . (G) Short-term killing assay of TIM-3.CAR-CIK cells against untreated or SGN-2FF-treated KASUMI-3 cells at various E:T ratios (5:1, 1:1, 0.5:1, 0.25:1 and 0.125:1, n = 8). (H) Affinity kinetics (left) and binding avidity at 1000 pN force (right) of TIM-3.CAR-CIK cells to untreated or defucosylated KASUMI-3 by LUMICKS analysis (n = 6). Immunoblot experiments (D-F) were repeated in three independent biological replicates with similar results. Data are presented as individual values and mean ± SD. Statistical significance was determined with repeated-measures two-way ANOVA with Bonferroni’s post hoc test (B, C) or using paired t test (G, H). ns, not significant; *p = 0.01, **p < 0.001, ***p = 0.0001 and ****p < 0.0001. Illustrations were created with Biorender.com. See also Figure S3 for loading-matched TIM-3 immunoprecipitation controls.

Article Snippet: KASUMI-3, KG-1 (AML cell lines) and REH cell lines (ALL cell line) were sourced from American Type Culture Collection (ATCC).

Techniques: Expressing, Flow Cytometry, Negative Control, Lysis, Positive Control, Western Blot, Control, Glycoproteomics, Staining, Binding Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Immunoblot profiling of TIM-3 glycoforms in monocytes, CIK cells, and KASUMI-3 lysates using a recombinant scFv-derived monoclonal antibody (TIM-3scFv-mAb) following enzymatic treatment with PNGase F or broad neuraminidase. GAPDH, loading control. (B) TIM-3 immunoprecipitates from healthy monocytes, KASUMI-3 cells, and primary AML blasts treated with neuraminidase and/or PNGase F and analyzed by lectin and antibody probing: Ricinus communis agglutinin I (RCA-I; terminal β-galactose/LacNAc motifs), CA19-9 (sialyl-Lewis A), CSLEX1 (sialyl-Lewis X), and TIM-3scFv-mAb. See also Figure S3B . (C) High-resolution immunoblot of TIM-3 species detected by TIM-3scFv-mAb in CIK cells, primary AML blasts, and KASUMI-3 cells. GAPDH, loading control. See also Figure S3C . (D) RT-qPCR expression profiling of glycosyltransferases (FUT7, FUT8, ST3GAL3, ST3GAL4, ST3GAL6) in monocytes, KASUMI-3 cells, and primary AML blasts. Data are plotted as fold-change relative to monocytes and normalized to 18S RNA; individual points denote biological samples where applicable. (E) Schematic model summarizing a glycoform-biased recognition framework in which AML-associated remodeling of TIM-3 N -glycans contributes to preferential TIM-3.CAR recognition of AML-enriched TIM-3 glycoforms. Representative N -glycan structures are proposed for TIM-3 in AML blasts, monocytes and CIK cells based on enzymatic perturbation and lectin/antibody probing. Sugar moieties drawn with dashed outlines indicate features not directly resolved/assigned. Glycan symbols follow SNFG. Immunoblot and lectin/antibody blot experiments (A-C) were repeated in three independent biological replicates with similar results. Illustrations were created with Biorender.com. See also Figure S3 for additional lectin/antibody probing of TIM-3 glycoforms and terminal galactose exposure.

Article Snippet: KASUMI-3, KG-1 (AML cell lines) and REH cell lines (ALL cell line) were sourced from American Type Culture Collection (ATCC).

Techniques: Western Blot, Recombinant, Derivative Assay, Control, Quantitative RT-PCR, Expressing, Glycoproteomics

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Schematic of the xenograft KASUMI-3 AML model. (B) Representative flow cytometry plots of hCD45 + CD33 + (up) and of hCD45 + TIM-3 + cells (down) in the BM of CTR or TIM-3.CAR treated mice at sacrifice. ( C-E ) Frequencies of hCD33 + and hTIM-3 + cells in the (C) BM, (D), spleen and (E) peripheral blood (PB) at sacrifice. Illustrations were created with Biorender.com. Results represent three independent experiments using TIM-3.CAR-CIK cells generated from 3 different donors. Data are presented as individual values and mean ± SD. Statistical significance was determined by unpaired t test. *p = 0.01, **p < 0.001 and ****p < 0.0001.

Article Snippet: KASUMI-3, KG-1 (AML cell lines) and REH cell lines (ALL cell line) were sourced from American Type Culture Collection (ATCC).

Techniques: Flow Cytometry, Generated

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Schematic of IF-BETTER gate strategy showing dual antigen recognition of CD33 + /TIM-3 + target cell by CD33.CAR/TIM-3.CCR and TIM3.CAR/CD33.CCR-CIK cells. (B) Co-distribution of CD33 and TIM-3 expression (MFI) on bulk AML (top) and LSC-enriched CD34 + CD38 - population (bottom). Each dot represents a distinct patient (n=44 patients). (C) Schematics of next-generation Dual CD33.CAR/TIM-3.CCR and TIM-3.CAR/CD33.CCR constructs. CAR molecules are second-generation, carrying CD28 co-stimulatory domain, while CCR molecules present 4-1BB as co-stimulus. Both constructs were cloned into a pT4-transposon vector. See also Figure S4A, B . (D) Long-term killing assay (E:T 1:10) of all CAR-CIK cells against primary AML blasts (n=8 blasts) compared to NT cells. Blasts survival was determined by flow cytometry (n=13 donors). (E) Recovery of LSC-enriched CD34 + CD38 - population (n=8 patient samples) after 7 days co-culture with all CAR-CIK cells (E:T 1:10), compared to NT cells (n=13). Data are presented as individual values and the mean ± SD. Statistical significance was determined by one-way ANOVA test. ** p<0.01, **** p<0.0001. Illustrations were created with Biorender.com. See also Figure S4 for expression and phenotypic characterization of Dual CAR constructs, Figure S5 for Dual CAR-CIK cell activity against AML cell lines and Figure S6 for Dual CAR-CIK cell off-tumor toxicity against healthy immune and hematopoietic cells.

Article Snippet: KASUMI-3, KG-1 (AML cell lines) and REH cell lines (ALL cell line) were sourced from American Type Culture Collection (ATCC).

Techniques: Expressing, Construct, Clone Assay, Plasmid Preparation, Flow Cytometry, Co-Culture Assay, Activity Assay

Journal: iScience

Article Title: A multimodal atlas for immunotherapeutic targeting of AML surface heterogeneity

doi: 10.1016/j.isci.2026.115337

Figure Lengend Snippet: Systematic ranking of antigens based on expression on AML blasts and non-hematopoietic tissue (A) Antigen intensity threshold for killing by surface targeting modalities. Left: Percent live cells of MOLM13 clones, indicated by their CD33 expression intensities, upon exposure to increasing concentrations of GO. Data are represented as mean ± SD, n = 4. Right: % dead cells following the incubation of MOLM13 clones with CD33-targeting CD33-bbz or CD33-28z CAR-T effector cells for 48 h. Data are represented as mean ± SD, n = 3. Dotted line indicates background target cell viability in the absence of effector cells. ∗∗∗ANOVA p < 0.001. (B) Heatmaps show estimated antigen count for the top 25 most highly expressed across 5,000 blast cells randomly sampled from diagnosis (left) and relapse (right) AML samples. Each column corresponds to an individual blast. Top annotation bar indicates the patient of origin. Higher antigen density for a specific antigen in a single blast is denoted by red shading. ∗Antibodies directed against CD13, CD45, CD47, CD99, and HLA-DR were found to be undersaturated (refer to “ ” in ). (C) Heatmaps showing gene expression of CD33, CLL-1, LAIR1, DEC-205, ITGA4, CD244, ADGRE2, and HER2 in non-hematopoietic cell types using single-cell RNA-seq data downloaded from Tabula Sapiens and GTEx databases. Columns are categorized based on cell types, and the top annotation bar indicates the tissue of origin of the cells. High and low relative expression are indicated by yellow and blue, respectively. Abbreviations: ANOVA, analysis of variance; GO, gene ontology; SD, standard deviation.

Article Snippet: Co-culture experiments of HDR CAR-T and MOLM13 clones and CAR-T and HL-60 (stable AML cell line, ATCC) target cells were conducted to investigate antigen-specific cytolysis at 1:1 E:T ratio.

Techniques: Expressing, Clone Assay, Incubation, Biomarker Discovery, Gene Expression, Single Cell, RNA Sequencing, Standard Deviation

Journal: Journal of Nanobiotechnology

Article Title: Injectable pH-responsive carboxymethyl cellulose hydrogel for sustained delivery of IL-22 in the treatment of alcoholic liver disease

doi: 10.1186/s12951-026-04345-x

Figure Lengend Snippet: Good sustained release, biosafety, degradability, and injectability of the IL-22@CMC. ( a ) ELISA was employed to assess the in vitro drug release behavior of various IL-22@CMC concentrations. ( b ) ELISA measured IL-22 concentration in AML-12 cells. ( c ) ELISA was used to detect the concentration of IL-22 in mice serum. ( d - f ) CCK-8 was used to detect cell activity of AML-12 cells. ( g ) H&E staining of the major organs of mice. ( h ) Representative fluorescence images show the fluorescence distribution observed at designated time points following a single subcutaneous injection of CMC-Rhodamine B in mice. ( i ) Changes in total fluorescence intensity as a function degradation time. ( j ) The injectability of IL-22@CMC (the IL-22@CMC was subjected to a staining treatment). All results of this study were derived from three independent experiments. ** p < 0.01. “ns” indicates no significance. Data are represented as mean ± SD

Article Snippet: The mice hepatocyte cell line AML-12 was maintained at the School of Pharmaceutical Sciences, Anhui Medical University.

Techniques: Enzyme-linked Immunosorbent Assay, In Vitro, Concentration Assay, CCK-8 Assay, Activity Assay, Staining, Fluorescence, Injection, Derivative Assay

Journal: Journal of Nanobiotechnology

Article Title: Injectable pH-responsive carboxymethyl cellulose hydrogel for sustained delivery of IL-22 in the treatment of alcoholic liver disease

doi: 10.1186/s12951-026-04345-x

Figure Lengend Snippet: The expression level of IL-22 was decreased in ALD. ( a , b ) The expression levels of ALT and AST were detected in the serum of normal individuals and ALD patients. ( c , d ) Western blotting was used to determine the expression level of IL-22 in mice liver tissue and AML-12 cells. ( e , f ) Quantifications of IL-22 expression level in mice liver tissues and AML-12 cells. ( g , h ) RT-qPCR was used to detect the mRNA expression level of IL-22 in mice liver tissue and AML-12 cells. ( i ) IHC was used to detect the expression level of IL-22 in mice liver. ( j ) IF was used to detect the expression level of IL-22 in mice liver. All results of this study were derived from three independent experiments. ** p < 0.01, *** p < 0.001. Data are represented as mean ± SD

Article Snippet: The mice hepatocyte cell line AML-12 was maintained at the School of Pharmaceutical Sciences, Anhui Medical University.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Derivative Assay

Journal: Journal of Nanobiotechnology

Article Title: Injectable pH-responsive carboxymethyl cellulose hydrogel for sustained delivery of IL-22 in the treatment of alcoholic liver disease

doi: 10.1186/s12951-026-04345-x

Figure Lengend Snippet: IL-22@CMC could better inhibit EtOH-induced secretion of inflammatory cytokines and lipid accumulation in AML-12 cells. ( a - c ) IF was used to detect the protein expression levels of inflammatory cytokines. ( f - h ) Quantifications of IL-1β, IL-6, and TNF-α fluorescence intensity. ( d , e ) Western blotting was used to detect the protein expression levels of inflammatory cytokines and lipid accumulation. ( i - n ) Quantifications of IL-1β, IL-6, TNF-α, PPAR-α, SREBP-1c, and FASN proteins. All results of this study were derived from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are represented as mean ± SD

Article Snippet: The mice hepatocyte cell line AML-12 was maintained at the School of Pharmaceutical Sciences, Anhui Medical University.

Techniques: Expressing, Fluorescence, Western Blot, Derivative Assay

Journal: Journal of Nanobiotechnology

Article Title: Injectable pH-responsive carboxymethyl cellulose hydrogel for sustained delivery of IL-22 in the treatment of alcoholic liver disease

doi: 10.1186/s12951-026-04345-x

Figure Lengend Snippet: EtOH-induced secretion of inflammatory cytokines and lipid accumulation were inhibited by IL-22@CMC through ERS mediated by the AMPK/SIRT1 signaling pathway. ( a ) KEGG pathway enrichment analysis. ( b ) IF was used to detect the protein expression levels of AMPK phosphorylation and SIRT1 in AML-12 cells. Scale bar, 50 μm. ( c , d ) Quantitative of the fluorescence intensities of SIRT1 and p-AMPK. ( e - j ) Western blotting was used to detect the protein expression levels of inflammatory cytokines and lipid accumulation in AML-12 cells. ( k - p ) Quantifications of inflammatory cytokines and lipid accumulation related proteins. All results of this study were derived from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are represented as mean ± SD

Article Snippet: The mice hepatocyte cell line AML-12 was maintained at the School of Pharmaceutical Sciences, Anhui Medical University.

Techniques: Expressing, Phospho-proteomics, Fluorescence, Western Blot, Derivative Assay