Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet: Rational design and implementation of ADA1 in CAR T cells (A) Four different versions of ADA1 were designed for use in CAR T cell therapy. (B) Human PBMCs ( n = 3) were stimulated with 100 μM ADO, 5 μg/mL coated caveolin-1 protein (CD26), 1 μg/mL coated OKT3 mAb (CD3), combined CD3/ADO, or combined CD3/CD26. After 48 h, ecto-ADA1 was measured by ADA activity assay. (C) HER2-MR-CAR T cells ( n = 3) were stimulated with 1 μg/mL coated OKT3 mAb (CD3), 10 μg/mL coated mAb 134-2C2 (CD26), or combined CD3/CD26. After 48 h, cell culture medium was subjected to ADA1 ELISA. p = 0.0352 for CD3/CD26 vs. CD3/CD28. (D) HER2-MR-CAR T cells ( n = 3), HER2-CAR T cells, or NT cells were cultured at indicated density for 24 h. After incubation, IFN-γ was measured using ELISA. The experiments were conducted in triplicate. ∗ p value for HER2-MR-CAR T cells + A549 vs. HER2-CAR T cells + A549. (E) mRNA sequencing ( n = 3) was used to analyze inflammatory cytokines, granzyme A, and granzyme B. (F) RT-qPCR was used to measure IFN-γ ( n = 3). (G) mA.26.HER2 ( n = 3) or NT cells were cultured either with or without A549 tumor cells, while mA.26.GPC3 or NT cells were cultured either with or without Huh7 cells. 24 h later, IFN-γ were measured using ELISA. p = 0.0000008 and p = 0.000236 for mA.26.HER2 and mA.26.GPC3 vs. NT. p = 0.0000003 for mA.26.HER2 vs. NT (A549). p = 0.0000001 for mA.26.GPC3 vs. NT (Huh7). Error bars represent SEM. p values were determined by two-tailed t test.

Article Snippet: Alexa flour 647 conjugated HER2 protein (Biotechne, AFR1129) was used to detect HER2 expression.

Techniques: Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Sequencing, Quantitative RT-PCR, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet: Overexpression of CD26 resisted TGF-β1 suppression and promoted CAR T cell mobility and proliferation (A) Rv-CD26-transduced T cells were cultured in the presence of TGF-β1 for 48 h, and CD26 expression was detected by flow cytometry. The experiments were conducted in duplicate. (B and C) CCR2 and CCR5 expression was determined by flow cytometry. The experiments were conducted in duplicate. (D) The heatmap shows the expression levels of chemokine receptor genes. Triplicate samples ( n = 3) were used for each group and are represented on the x axis. (E and F) Rv-CD26-transduced T cells ( n = 3) were subjected to a fluorescent migration assay and Transwell migration assay. (G) PBMCs ( N = 2) were transduced with retroviral vectors expressing HER2-CAR (HER2-CAR), ADA1.CD3scFv and HER2-CAR (ADA1.CD3scFv-HER2-CAR), CD26 and HER2-CAR (CD26-HER2-CAR), or ADA1.CD3scFv/CD26 and HER2-CAR (HER2-MRCAR) and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The T cells were then cultured for 4 days and subjected to flow analysis. (H) GPC3-MR-CAR T cells and GPC3-CAR T cells were expanded in vitro , and the cell numbers were determined at different time points using a hemocytometer. The results are presented as a growth curve. The figure indicates the p values for GPC3-MR-CAR vs. GPC3-CAR. The experiments were conducted in triplicate. Error bars represent SEM. p values were determined by two-tailed t test.

Article Snippet: Alexa flour 647 conjugated HER2 protein (Biotechne, AFR1129) was used to detect HER2 expression.

Techniques: Over Expression, Cell Culture, Expressing, Flow Cytometry, Migration, Transwell Migration Assay, Transduction, Labeling, In Vitro, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet: ADA1.CD3scFv enhances CAR T cell expansion preferentially without impacting tumor cells (A and B) Luciferase reporter Jurkat-Dual or Jurkat-NFAT cells either expressing CD26 or lacking CD26 were transduced with overexpressing vectors of ADA1 or ADA1.CD3scFv and cultured for 24 h. After incubation, the cells ( n = 3) were subjected to an ADA activity assay. CD26-positive Jurkat T cells transduced with ADA1.CD3scFv overexpressing vector were used as the maximum value to calculate the percentage. The data are presented as the percentage of ADA1 or ADA1.CD3scFv binding with the Jurkat T cells. The figure indicates the p values for the binding of ADA1 with CD26 + Jurkat T cells vs. the binding of ADA1.CD3scFv with CD26 + Jurkat T cells. (C) Jurkat T cells ( n = 3) were transduced with retroviral vector expressing ADA1, ADA1.CD3scFv, CD26, ADA1, and CD26 or ADA1.CD3scFv and CD26, respectively. Jurkat T cells were labeled with CFSE, and cell proliferation was measured by flow analysis. The experiments were conducted in duplicate. (D) The conditioned medium collected from the culture of HER2-MR-CAR T cells, HER2-CAR T cells, or NT human T cells was added to A549 cell cultures ( n = 3) and incubated for 24 or 48 h. The numbers of tumor cells were quantified daily using a hemocytometer. p = 0.000002 for ADA1 vs. ADA1.CD3scFv at both 24 and 48 h. (E and F) HER2-MR-CAR T cells or HER2-CAR T cells were co-cultured with A549 tumor cells in a Transwell plate for 72 h. Similarly, GPC3-MR-CAR T cells or GPC3-CAR T cells were co-cultured with Huh7 tumor cells. The CAR T cells and tumor cells were counted using a hemocytometer and are presented. (G) 293T cells were transduced with retroviral vectors expressing either ADA1 or ADA1.CD3scFv. After 48 h of culture, the cell culture medium was collected and added to the culture of CD26-negative or CD26-positive Jurkat-NFAT T cells for 24 h. The Jurkat-NFAT cells were then subjected to luciferase activity assay. p = 0.0034 for ADA1 vs. ADA1.CD3scFv in CD26-negative Jurkat T cells. p = 0.0000003 for ADA1.CD3scFv in CD26-positive Jurkat T cells vs. CD26-negative Jurkat T cells. The experiments were conducted in triplicate. Error bars represent SEM. p values were determined by two-tailed t test.

Article Snippet: Alexa flour 647 conjugated HER2 protein (Biotechne, AFR1129) was used to detect HER2 expression.

Techniques: Luciferase, Expressing, Transduction, Cell Culture, Incubation, Activity Assay, Plasmid Preparation, Binding Assay, Labeling, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet: MR-CAR T cells displayed enhanced antitumor cytotoxicity in vitro (A–F) Human PBMCs were transduced with either HER2-MR-CAR or HER2-CAR and expanded in vitro . The cytotoxic activity of HER2-MR-CAR and HER2-CAR T cells against Calu3 and A549 cells was evaluated using LDH assay. The figure indicates the p values for HER2-MR-CAR vs. HER2-CAR. (G–J) The cytotoxic activity of GPC3-MR-CAR and GPC3-CAR T cells against HepG2 and Huh7 cells was evaluated using LDH assay. The figure indicates the p values for GPC3-MR-CAR vs. GPC3-CAR. The experiments were conducted in triplicate. Error bars represent SEM. p values were determined by two-tailed t test.

Article Snippet: Alexa flour 647 conjugated HER2 protein (Biotechne, AFR1129) was used to detect HER2 expression.

Techniques: In Vitro, Transduction, Activity Assay, Lactate Dehydrogenase Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet: MR-CAR T cells demonstrated antitumor activity in xenograft mouse models (A–C) A549 tumor-bearing mice ( n = 5) were treated with a single dose of either 5 × 10 6 or 1 × 10 7 or two doses of 2 × 10 6 (at 1 week intervals) HER2-MR-CAR T cells, HER2-CAR T cells, or PBS. Tumor size and mouse body weight were monitored every 2–3 days. Data represent mean ± SD ( n = 5). (D–F) At week 0, mice ( n = 10) were intercostally injected with 2 × 10 6 A549-luc tumor cells. One week after tumor implantation, the mice were administered either 2 × 10 6 T cells or PBS through the tail vein. Tumor development was monitored weekly using bioluminescence in vivo imaging (D). Mean photon count with SDs of mice groups is shown at the indicated time points (E). p = 0.04513, 0.01437, or 0.002137 for MR-CAR vs. ADA1.CD3scFv-HER2-CAR at weeks 9, 10, or 11 individually. Mouse survival was monitored (F). p = 0.0177 for HER2-MR-CAR vs. HER2-CAR in survival. (G and H) A murine HCC xenograft model was established in NSG mice ( n = 5) by subcutaneous inoculation of 2 × 10 6 Huh7 tumor cells on the right flank. When the average tumor size reached 4–6 mm in diameter, experimental mice were treated with a single dose of 2 × 10 6 GPC3-MR-CAR T cells, 2 × 10 6 GPC3-CAR T cells, or PBS. Tumor size and mouse body weight were monitored every 2–3 days. Data represent mean ± SD ( n = 5). p = 0.0209 and p = 0.00191 for GPC3-MR-CAR T cells vs. GPC3-CAR T cells on days 9 and 13 respectively. No difference in body weight was observed among groups. Error bars represent SEM. p values were determined by two-tailed t test.

Article Snippet: Alexa flour 647 conjugated HER2 protein (Biotechne, AFR1129) was used to detect HER2 expression.

Techniques: Activity Assay, Injection, Tumor Implantation, In Vivo Imaging, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet: MR-CAR T cells retained their capability to proliferate, migrate, and lyse tumor cells in the tumor microenvironment (TME) NSG mice were subcutaneously inoculated with 2 × 10 6 A549 tumor cells on the right flank. When the average tumor size reached 200–300 mm 3 , experimental mice ( n = 10) were treated with a single dose of 1 × 10 7 HER2-MR-CAR T cells, HER2-CAR T cells, or NT T cells. (A) Seven days after treatment, tumor tissues were dissected, and a single-cell suspension was subjected to an ADA activity assay to measure inosine concentrations. ∗ p = 0.00847 for HER2-MR-CAR vs. HER2-CAR. (B) After 7 days of treatment, tumor-infiltrating CD3 + cells were sorted using flow cytometry. The number of sorted cells was quantified and is presented ( n = 3). ∗ p = 0.000177 for HER2-MR-CAR vs. HER2-CAR. (C and D) Single-cell suspensions from tumor tissues were stained and analyzed by flow cytometry. (E and F) Sorted CD3 + cells were co-cultured with A549 tumor cells at an effector-to-target (E:T) ratio of 1:1 overnight. IFN-γ was determined by ELISA. To determine the tumor-killing capacity, an LDH assay was performed. (G) Sorted CD3 + cells were co-cultured with A549 tumor cells in a Transwell culture plate to assess the migration capacity of the CD3 + cells. ∗ p = 0.00053 for HER2-MR-CAR vs. HER2-CAR. (H) Sorted CD3 + cells were labeled with CFSE and then co-cultured with A549 tumor cells at an E:T ratio of 1:1 for 48 h, followed by flow analysis to determine their proliferation. Error bars represent SEM. p values were determined by two-tailed t test.

Article Snippet: Alexa flour 647 conjugated HER2 protein (Biotechne, AFR1129) was used to detect HER2 expression.

Techniques: Suspension, Activity Assay, Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Migration, Labeling, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

doi: 10.1016/j.xcrm.2024.101530

Figure Lengend Snippet:

Article Snippet: Alexa flour 647 conjugated HER2 protein (Biotechne, AFR1129) was used to detect HER2 expression.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, LDH Cytotoxicity WST Assay, Chemotaxis Assay, Modification, Membrane, Software

Journal: bioRxiv

Article Title: CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

doi: 10.1101/2024.02.06.579138

Figure Lengend Snippet: A-B) Nanoparticle Tracking Analysis (NTA) of particles secreted by transfected HEK293T cells. Representative size distribution profiles of Mock, CD81-GFP, anti-HER2 samples. The black curve indicates the mean of three measurements, with SE in red. Mode and Mean diameters, and particle concentration are plotted. Error bars include at least three biological replicates. Significance * is P<0.05, *** P<0.001, vs Mock condition. C) Representative Cryo-EM images of Mock, CD81-GFP and antiHER2 EV samples confirming the vesicular structure and size heterogeneity of recovered vesicles. The indicated scale bar is 100 nm. D) Plot of the observed diameter of vesicles in Cryo-EM images (n=35 for Mock, n=99 for CD81-GFP, n=74 for antiHER2) and lamellarity, expressed as percentage of unilamellar and multilamellar vesicles over the observed bulk EV populations.

Article Snippet: The resulting product was pre-incubated with 30 nM HER2-DDK (TP322909, OriGene) for 30 min before addition to the other components: 10 nM tGFP Ab (TA150041, OriGene), anti-FLAG donor and protein G coated acceptor beads (10 ng/μl final concentration; AS103D and AL102C, PerkinElmer).

Techniques: Transfection, Concentration Assay, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

doi: 10.1101/2024.02.06.579138

Figure Lengend Snippet: A) Representative immunoblotting of cell and EV lysates (1 μg proteins/well). EVs are positive to transmembrane (CD9) and cytosolic proteins (SYNTENIN, TSG101), while negative to CALNEXIN, and with low detectable levels of GAPDH compared to cell lysates. B) . Dot plots of imaging flow cytometry to detect GFP-positive EVs. The green fluorescent signal (Ch 2, 488 nm laser) was detected as sub-gating of EVs labeled with Cell Mask Deep Red (CMDR, in orange, Ch 11, 635 nm) to side-scatter (Ch 6). Non-fluorescent, calibrator SpeedBeads, Amnis (1 µm) were continuously run during acquisitions. The graph shows the quantification of double-positive particles. Mean and error bars derive from three independent experiments. C) Sandwich designed for the AlphaLISA competitive assay. CD81-GFP and antiHER2 EVs were tested for competition with HER2-DDK. Image created with BioRender.com. The graph shows the measured alpha counts normalized to the GFP-positive EV population as calculated by NTA and imaging flow cytometry. Mean and SD derive from three independent experiments (significance is **** P<0.0001). D) Representative western blotting of recombinant EVs immunoprecipitation with HER2-DDK or anti-GFP antibody in serum-free DMEM. GFP-positive fusion proteins are enclosed in the yellow box above the antibody heavy chains (black arrow), SYNTENIN, and HER2-DDK. Controls of beads flow through with HER2-DDK (*) or anti-GFP antibody (**) are shown on the right, indicating saturation of the beads’ surface to avoid non-specific binding. The graph shows the densitometric quantification of antiHER2 EVs captured by both HER2-DDK and anti-GFP Ab, with a competition effect of Trastuzumab. Mean and SD refer to two independent experiments.

Article Snippet: The resulting product was pre-incubated with 30 nM HER2-DDK (TP322909, OriGene) for 30 min before addition to the other components: 10 nM tGFP Ab (TA150041, OriGene), anti-FLAG donor and protein G coated acceptor beads (10 ng/μl final concentration; AS103D and AL102C, PerkinElmer).

Techniques: Western Blot, Imaging, Flow Cytometry, Labeling, Recombinant, Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

doi: 10.1101/2024.02.06.579138

Figure Lengend Snippet: A) Immunofluorescence of MDA-MB-231 and SK-BR-3 breast cancer cell lines as HER2 negative or positive cells, respectively. HER2 receptor is in red (Alexa Fluor 633), nuclei are shown in cyan (Hoechst). The indicated scale bar is 50 μm. B) Left: HER2 protein detection by Dot blot in lysates from wild-type or transfected (OE) MDA-MB-231 cells. Right: Immunoblot for checking the selection of SK-BR-3 cells with HER abrogation ( ERBB2 -KO, or KO). C) Representative confocal time lapse of recombinant EVs incubated with live cells (time points are indicated). GFP-EVs are shown in green, lysosomes are shown in magenta (Lysotracker red), and nuclei in cyan (Hoechst). The white squares highlight the co-localization between EVs and lysosomes (white arrowhead). The indicated scale bar is 20 μm. D) Representative confocal image of fixed SK-BR-3 cells recognizing HER2 (Alexa Fluor 633) and nuclei (Hoechst) after 4 hr incubation with CD81-GFP EVs (green spots). White arrows or the arrowhead indicate different localization of EVs upon cell interaction. Indicated scale bar is 20 μm. E-F) Quantification of recombinant EVs with recipient breast cancer cell lines. MDA-MB-231 (WT and HER2 OE) and SK-BR-3 (WT and KO) were incubated with EVs for 4 hours, then washed with PBS before fixation and HER2 immunofluorescence. Fourteen Z-stacks were acquired within around 11 μm of total Z-size and the Maximum Intensity Projections have been analyzed with an automated pipeline (using CellProfilerTM 4.0.7). Graphs report Mean and SD of the spot distribution from three independent experiments (* if P<0.05, ** if P<0.01, *** if P<0.001).

Article Snippet: The resulting product was pre-incubated with 30 nM HER2-DDK (TP322909, OriGene) for 30 min before addition to the other components: 10 nM tGFP Ab (TA150041, OriGene), anti-FLAG donor and protein G coated acceptor beads (10 ng/μl final concentration; AS103D and AL102C, PerkinElmer).

Techniques: Immunofluorescence, Dot Blot, Transfection, Western Blot, Selection, Recombinant, Incubation, IF-P