Journal: ImmunoHorizons

Article Title: Chimeric Antigen Cytotoxic Receptors for In Vivo Engineering of Tumor-Targeting NK Cells

doi: 10.4049/immunohorizons.2300099

Figure Lengend Snippet: Dependence of NK-CAR surface expression on signaling adaptors. ( A – C ) NK mRNAs were transfected in Huh7 cells with and without cotransfection with the mRNA for signaling adaptors (A) HER2-NKp30 with and without FcRγ, (B) HER2-NKp46 with and without FcRγ, and (C) HER2-NKp44 with and without DAP12. The percentage of CAR + cells and mean fluorescence intensity of CAR expression are shown. ( D ) The specificity indexes of each CAR-NK construct were calculated as the ratio of the surface expression of CAR in the presence of signaling adaptors to the surface expression in the absence of a signaling adaptor. Representative data of two independent experiments are shown.

Article Snippet: A human HER2/ERBB2 protein-His tag (Sino Biological) conjugated with Alexa Fluor 647 (1:200 dilution) was used to determine the expression of HER2-CAR in transfected cells, and viability was determined by 7-aminoactinomycin D (7-AAD) labeling (1:1000 dilution).

Techniques: Expressing, Transfection, Cotransfection, Fluorescence, Construct

Journal: ImmunoHorizons

Article Title: Chimeric Antigen Cytotoxic Receptors for In Vivo Engineering of Tumor-Targeting NK Cells

doi: 10.4049/immunohorizons.2300099

Figure Lengend Snippet: Antitumor activities of NK-CAR constructs. ( A ) Cell surface expression of NK-CARs in human peripheral blood-activated NK cells electroporated with NK-CAR mRNA detected by surface labeling with dye-conjugated recombinant HER2 protein. ( B ) Quantification of flow cytometry plot shown in (A). ( C ) Tumoricidal activity of HER2-targeting NK-CARs. NK cells were cocultured with HER2 + SKOV3 tumor cells at a 5:1 E:T ratio and incubated for 20 h at 37°C. The graph represents the percentage specific killing of SKOV3 cells by NK-CAR–transfected NK cells compared with mock-transfected NK cells. Data are representative of three independent experiments with two donors. The mean ± SD is plotted on the graph, and statistical significance was determined by an ordinary one-way ANOVA with a Dunnett multiple comparison test between mock-transfected NK cells versus NK-CAR cells. The p values are indicated on the graph. ( D ) NK-CARs induce cytokine (TNF-α, IFN-γ, and GM-CSF) and chemokine (CCL2) production when activated by coculturing with SKOV3 tumor cells. The mean ± SD from three replicates is shown. Statistical significance was determined by two-way ANOVA with a Dunnett multiple comparison test between mock and SKOV3 coculture versus NK-CAR and SKOV3 coculture. The p values are indicated on the graph. Data are representative of two independent experiments.

Article Snippet: A human HER2/ERBB2 protein-His tag (Sino Biological) conjugated with Alexa Fluor 647 (1:200 dilution) was used to determine the expression of HER2-CAR in transfected cells, and viability was determined by 7-aminoactinomycin D (7-AAD) labeling (1:1000 dilution).

Techniques: Construct, Expressing, Labeling, Recombinant, Flow Cytometry, Activity Assay, Incubation, Transfection, Comparison

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) Representation of anti-HER2/CD3 TDB. (B) Binding to human CD8+ cells was analyzed by flow cytometry (n = 1). (C) TDB-induced (1 μg/mL TDBs) TCR signaling pathway activation in splenic CD8+ cells extracted from huCD3 transgenic mice was analyzed by phos–SLP-76 Western blot (see complete unedited blots in the supplemental material; lanes were run on the same gel but were noncontiguous; n = 1). (D) TDB-induced activation of human CD8+cells was analyzed by flow cytometry (n = 1). (E) In vitro killing activity of HER2-amplified SKBR3 (solid lines) and low-HER2–expressing MCF7 (dotted lines) cells was analyzed by Cell Titer Glo viability assay (n = 3). Data presented as mean ± SD.

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Binding Assay, Flow Cytometry, Activation Assay, Transgenic Assay, Western Blot, In Vitro, Activity Assay, Amplification, Expressing, Viability Assay

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) Individual tumor volume response of HER2-amplified KPL4 breast cancer xenografts to HER2–TDB 2 (higher CD3 affinity; groups 4–7) and HER2–TDB 1 (lower CD3 affinity; groups 8–11) in NSG mice supplemented with human PBMCs. Mice with established tumors received a single i.v. dose at day 0 at indicated dose levels. Trellis plots of individual and fitted tumor volumes are presented, with study day on the x axis and tumor volume on the y axis. Each panel in the trellis depicts 1 dose group (panel headers indicate group numbers). Bold, solid black lines indicate the fitted tumor volume for each dose group. Dashed blue lines indicate the fitted tumor volume for the control group (vehicle, histidine buffer). Gray lines indicate the tumor response over time in individual animals present through the course of the study. Red lines indicate the tumor response over time in mice that were removed from the study due to tumor progression beyond prespecified limits. n = 8–9 for each treatment group. (B and C) Tumor-bearing MMTVhuHER2.huCD3.TG mice were treated with a single dose of HER2–TDB 2 (red), HER2–TDB 1 (blue), or vehicle (black) at day 0. (B) Effect of 0.5 mg/kg anti-HER2/CD3 TDB on tumor growth. n = 6–11 for each treatment group. (C) Effect of 0.25 mg/kg (filled symbols) or 0.5 mg/kg (open symbols) dose on T cell activation and proliferation markers on tumor-infiltrating CD8+ cells analyzed by flow cytometry 6 days after dose. Error bars represent mean ± SEM. n = 5–8 for each treatment group. Statistical analysis using unpaired t test with Welch’s correction.

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Amplification, Activation Assay, Flow Cytometry

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) To analyze cytokine release in vitro, human healthy donor whole blood was cocultured with MCF7 cells and HER2–TDB 1 (lower CD3 affinity; blue) or HER2–TDB 2 (higher CD3 affinity; red) for 20 hours, and cytokine levels in media were analyzed by Bio-Plex Pro Human Cytokine Assay. Data presented as mean (n = 3) ± SD. (B and C) Tumor-bearing MMTVhuHER2.huCD3.TG mice were treated with 0.5 mg/kg dose of indicated anti-HER2/CD3 variant (n = 6–7). (B) Serum cytokines were analyzed at indicated time points by Luminex. (C) Body weight change after TDB treatment (n = 6–9). Statistical analysis (unpaired t test with Welch’s correction) of body weights 4 days after treatment are presented in the right panel. Error bars represent mean ± SEM. (D) In vivo cytokine levels from cynomolgus monkeys treated with a single dose of 0.5 mg/kg. Data points represent individual animals (n = 2 for TDB 1; n = 1 for TDB 2).

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: In Vitro, Cytokine Assay, Variant Assay, Luminex, In Vivo

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) SKBR3 cells were treated with HER2 affinity variants of anti-HER2/CD3 TDB. (B) CHO cells were transfected to express cyno-HER2 and treated with HER2–TDB 1 (red) or HER2–TDB 3 (blue). Viability was measured using Cell Titer Glo. Data presented as mean ± SD (n = 3).

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Transfection

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: Cynomolgus monkeys were treated with 0.5 and 1.5 mg/kg of HER2–TDB 1 (lower HER2 affinity; blue and magenta, respectively) or HER2–TDB 3 (higher HER2 affinity; red and green, respectively) on days 0 and 7. (A–C) Peripheral blood was sampled at indicated time points and analyzed for T cell activation (CD69) (A), systemic cytokine levels (B), and number of CD8+ and CD4+ lymphocytes (C). Data are presented as mean ± SEM (A and C) or individual animals (B). Arrowheads indicate time of dosing (A and C) or point out individual animals where dosing was not tolerated (3503 and 4502) or tolerated (4503 and 4504) (B). n = 3–4 for each treatment group.

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Activation Assay

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) Effect of dose fractionation of HER2–TDB 3 on systemic exposure in cynomolgus monkey. Animals in group 1A (0.2 mg/kg on days 0 and 1, 0.4 mg/kg on day 7; n = 1) and group 1B (0.1 mg/kg on days 0 and 1, 0.4 mg/kg on day 7; n = 2) were dosed using dose fractionation. Animals in group 2 (n = 4) received 0.5 mg/kg on days 0 and 7. Blood samples were collected at indicated time points, and human IgG was detected by ELISA. Data are presented as mean ± SD for group 2 and as exposure for individual animal for group 1. PK parameters are presented in Supplemental Table 1. (B) Serum cytokine analysis using Luminex from cynomolgus monkeys dosed using the dose fractionation strategy (n = 3, individual animal data depicted). (C) Individual tumor volume response of HER2-amplified KPL4 breast cancer xenografts to anti-HER2/CD3 TDB in NSG mice supplemented with human PBMCs. Mice were treated with 0.05 mg/kg (once a week x2) dose (blue). Alternatively, the initial dose was fractionated to 2 doses of 0.025 mg/kg administered on days 0 and 1 (red). n = 8–9 for each dose group. Arrows indicate time of dosing.

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Fractionation, Enzyme-linked Immunosorbent Assay, Luminex, Amplification

Journal: JCI Insight

Article Title: Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody

doi: 10.1172/jci.insight.133757

Figure Lengend Snippet: (A) In the 2-tumor model, each NSG mouse was implanted with HER2-amplified KPL4 tumors and HT55 tumors, which express low levels of HER2. Mice were further supplemented with human PBMCs by i.p. injection. HER2 expression was analyzed by Western blot. Lanes were run on the same gel but were noncontiguous (n = 1). (B) Mice with 2 established tumors were treated with single 0.05 mg/kg dose of HER2–TDB 1. n = 5 for each group. Data represented as mean ± SEM.

Article Snippet: Recombinant human (Genentech) and cynomolgus monkey (Sino Biologicals) HER2 extracellular domain (ECD) were used to determine the binding affinity of anti-HER2/CD3 TDB for HER2 ECD by surface plasmon resonance (SPR) technology using a Biacore T200 instrument (GE Healthcare).

Techniques: Amplification, Injection, Expressing, Western Blot

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