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: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: Amino acid sequence of the dimeric HER2-binding Affibody (Z HER2 ) 2:L -Cys. The protein construct is designed to be made up of two domains with three-helix folds connected by a 15-residue linker containing a unique cysteine, underlined in the sequence, for simple labeling of the construct with thiol-reactive compounds. The dimer has three N-terminal glycines and carries a C-terminal sortase A recognition motif (LPETG) to make the construct a substrate for sortase A-mediated cyclization. The hexahistidine tag in the linear construct is used for IMAC purification of the Escherichia coli -expressed construct, but is cleaved off during the cyclization reaction. Helical regions in a crystal structure of the parental monomeric Z HER2:342 Affibody are indicated by helices 1–3 and 4–6, respectively .

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: Sequencing, Binding Assay, Construct, Labeling, Purification

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: ( A ) Homology model of (Z HER2 ) 2:L -Cys generated with the SWISS-MODEL protein modelling server based on a crystal structure of two tandem B-domains connected by a conserved linker (PDB code: 4NPF ). Residues 1–3, i.e., the three N-terminal glycines, and C-terminal residues 135–154, containing the sortase A recognition site and the His6-tag, were not included in this homology model. The unique cysteine, C 70 , is predicted to be situated in the linker region between domains 1 and 2. ( B ) Schematic structure of the cyclic Z HER2 -dimer, (Z HER2 ) 2:C . Sortase A-mediated cyclization of the dimeric protein results in the formation of a native peptide bond between Gly 1 and Thr 146 . ( C ) Schematic illustrations of three different approaches to intramolecular crosslinking or backbone cyclization of Affibody molecules previously published by our group and others. The HER2-binding Z HER2:CL was synthesized using solid phase peptide synthesis (SPPS) and has an intramolecular thioether bond going from a cysteine residue in the loop between helices 1 and 2 and the chloroacetyl-modified side chain of the C-terminal lysine residue . Z min is a truncated version of the Z-domain, in which the two IgG-binding helices 1 and 2 are joined by a peptide bond. Z min was prepared using SPPS and backbone-cyclized using natural chemical ligation . Lasso is a recombinantly expressed Z-domain dimer. The two IgG-binding Z-domains are joined by flexible linkers and the construct is backbone-cyclized using split-intein technology . The schematic drawings of Z HER2:CL , Z min and lasso are based on information found in references [ , , ].

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: Generated, Binding Assay, Synthesized, Modification, Ligation, Construct

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: SDS-PAGE analysis of sortase A-mediated cyclization of the Z HER2 -dimer. M: Molecular weight marker. Lane 1: Sortase A 3 *, lane 2: (Z HER2 ) 2:L , and lane 3: unpurified sample taken after a 20 h sortase A-mediated cyclization reaction.

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: SDS Page, Molecular Weight, Marker

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: Theoretical and observed molecular weights of proteins in this study.

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques:

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: ( A ) Circular dichroism (CD) expressed as molar ellipticity of linear (Z HER2 ) 2:L (blue) and (Z HER2 ) 2:C (red) before (solid line) and after (dashed line) refolding after thermal melt at 90 °C. Inserted is a close-up of the CD spectra in the 205–230 nm region. ( B ) Fraction folded protein as a function of temperature for (Z HER2 ) 2:L (blue) and (Z HER2 ) 2:C (red). The melting temperature, T m , of the linear dimer was 65 °C and it was elevated to 68 °C in the cyclic dimer.

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques:

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: Evaluation of kinetic titration series of ( A ) the linear dimer (Z HER2 ) 2:L and ( B ) the cyclic dimer (Z HER2 ) 2:C binding to immobilized HER2-Fc.

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: Titration, Binding Assay

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: In vitro digestion of (Z HER2 ) 2:L and (Z HER2 ) 2:C . ( A ) Digestion with trypsin (0.9 µg/mL) plus chymotrypsin (0.4 µg/mL). ( B ) Digestion with carboxypeptidase A (0.5 µM).

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: In Vitro

Journal: Molecules

Article Title: Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

doi: 10.3390/molecules26102874

Figure Lengend Snippet: Fluorescent microscopy images of either SKOV-3 cells or MCF-7 cells with DAPI-stained nuclei (blue) treated with 2.4 nM of (Z HER2 ) 2:C -DL594 (red). ( A ) SKOV-3 cells treated with (Z HER2 ) 2:C -DL594 as a positive control in the first row. In the second row, SKOV-3 cells pre-incubated with unlabeled (Z HER2 ) 2:C (1.2 µM) before addition of (Z HER2 ) 2:C -DL594. ( B ) MCF-7 cells treated with (Z HER2 ) 2:C -DL594. ( C ) SKOV-3 cells treated with (Z HER2 ) 2:C -DL594 as a positive control in the first row. The second row presents SKOV-3 cells pre-incubated with trastuzumab (Herceptin; 1 mg/mL) before the addition of (Z HER2 ) 2:C -DL594.

Article Snippet: A carboxymethylated, dextran-coated CM5 chip was activated with EDC/NHS and HER2-Fc (Her2/ERBB2 Protein, Human, Recombinant (hFc Tag); Sino Biological Inc., Beijing, China) was immobilized on the surface with an immobilization level of 760 RU.

Techniques: Microscopy, Staining, Positive Control, Incubation