anti tim 3  (R&D Systems)

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) Frequency (left) and mean fluorescence intensity (MFI; right) of TIM-3 expression on bulk AML blasts compared with residual healthy bone marrow (BM) cells from the same patient (n=25). (B) TIM-3 expression in the LSC-enriched CD34 + CD38 - population (n=17). (C) Schematic of the third-generation TIM-3.CAR containing CD28-OX40 costimulatory domains cloned into the pT4-transposon vector. (D) Transduction efficiency of TIM-3.CAR-CIK cells compared to non-transduced (NT) CIK cells (mean 71.24±16.99, n = 13). See also Figure S1A . (E) Fold increase of TIM-3.CAR-CIK cells compared to CD33.CAR-CIK and NT cells at day 21 of culture (n=9). (F) Viability of TIM-3.CAR-CIK cells, CD33.CAR-CIK and NT cells at days 4, 6 and 8 post-transduction, assessed by flow cytometry. See also Figure S1B . (G) Frequency of CD3 + TIM-3 + cells within NT, TIM3.CAR- and CD33.CAR-CIK populations over the first 8 days of CIK differentiation (n=7 donors for TIM-3.CAR-CIK and NT cells, n=4 donors for CD33.CAR-CIK cells). See also Figure S1C . Data are shown as individual values with mean ± standard deviation (SD). Significance was assessed using paired t test (A, D) or repeated-measures two-way ANOVA with Bonferroni’s post hoc test (E-G). ns, not significant, *** p = 0.0001 and **** p < 0.0001. See also Figure S1 for longitudinal TIM-3 and TIM-3.CAR expression and CD4 + and CD8 + subset analysis.

Article Snippet: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

Techniques: Fluorescence, Expressing, Clone Assay, Plasmid Preparation, Transduction, Flow Cytometry, Standard Deviation

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: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

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: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

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: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

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: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

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: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

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

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, F ) Schematic of KG-1 TIM-3 + low (A) and high (F) burden in vivo experimental design. ( B, G ) Representative FACS plots of hCD45 + CD33 + cells in the BM of CTR and CAR-treated mice in low-(B) and high-burden (G) settings. ( C, H ) Summary of BM hCD33 + cell percentages for all groups. ( D, I ) Analysis of TIM-3 expression on residual hCD33 + cells in BM of CTR and treated mice. ( E, J ) Leukemic burden in spleen (left) and PB (right) evaluated by flow cytometry, in low- (E) and (J) high-burden models. Illustrations were created with Biorender.com. Results represent two independent experiments using CAR-CIK cells generated from 2 donors. Data are presented as individual values and the mean ± SD. Statistical significance was determined by one-way ANOVA. **p < 0.001, ***p = 0.0001 and ****p < 0.0001.

Article Snippet: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

Techniques: In Vivo, Expressing, Flow Cytometry, Generated