human cd3 epsilon  (AMS Biotechnology)

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Novel tri-specific tribodies induce strong T cell activation and anti-tumor effects in vitro and in vivo.

doi: 10.1186/s13046-022-02474-3

Figure Lengend Snippet: Fig. 1 Schematical representation of 53X tribodies. A Scheme of constructs for 53X tribodies. SP, human interleukin (IL-2) signal peptide; VH_5T4, VL_5T4, amino acid sequences coding for the immunoglobulin heavy and light chain variable regions from Tb535H with specificity for human 5T4; CH1, CL, amino acid sequences coding for the human immunoglobulin heavy chain constant region 1 and the kappa light chain constant region, respectively; VH_CD3, VL_CD3, amino acid sequences coding for the variable heavy and light chain regions from humanized OKT3 building a scFv with specificity for human CD3; L1, L2, aminoacid sequences with flexible linker, GPGGGSPG, and GGGGSGGGGSGGGGS [(GGGS)3], respectively. VH_C, VL_C, amino acid sequences coding for the variable heavy and light chain from PD-1_1, PD-L1_1, 10_12, LAG3_1, scFvs with specificities for human PD-1, PD-L1 or LAG-3, respectively. The scFv from Palivizumab was used in an additional tribody for isotype control; 6xHis, amino acid sequences coding for a hexahistidine tag. B Scheme of assembled tribody proteins for Tb535H [(5T4)2 × CD3], 53D [5T4 x CD3 x PD-1], 53L1 [5T4 x CD3 x PD-L1], 53L10 [5T4 x CD3 x PD-L1], 53G [5T4 x CD3 x LAG-3] and 53P [5T4 x CD3 x isotype control], respectively

Article Snippet: Human 5T4/His was purchased from ACROBiosystems TPG- (Newark, Delaware USA), human CD3 epsilon and CD3 delta heterodimeric protein was purchased from AMSBIO (Milton, Abingdon UK).

Techniques: Construct, Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Novel tri-specific tribodies induce strong T cell activation and anti-tumor effects in vitro and in vivo.

doi: 10.1186/s13046-022-02474-3

Figure Lengend Snippet: Fig. 3 Binding affinity of parental Tb535H and novel 53X tribodies to recombinant CD3 protein by ELISA. A Binding curves by ELISA assays of tribodies (0 – 500 nM) to immobilized recombinant human CD3ε/δ heterodimer. Binding values were reported as the mean of determinations obtained in three independent experiments. B Table reporting the EC50 (nM) values for CD3 binding of each indicated tribody. Standard Deviations were ≤ 3—10%

Article Snippet: Human 5T4/His was purchased from ACROBiosystems TPG- (Newark, Delaware USA), human CD3 epsilon and CD3 delta heterodimeric protein was purchased from AMSBIO (Milton, Abingdon UK).

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Novel tri-specific tribodies induce strong T cell activation and anti-tumor effects in vitro and in vivo.

doi: 10.1186/s13046-022-02474-3

Figure Lengend Snippet: Fig. 6 T cell activation bioassays in the presence of 5T4-expressing target cells. A Schematic representation of TCR/CD3 activation in the presence of 5T4-expressing cells by T cell activation bioassay (NFAT). B Genetically engineered Jurkat T cells which expresses a luciferase reporter driven by a NFAT-response element (NFAT-RE) (“TCR/CD3 effector cells”) were incubated with increasing concentrations of the indicated tribodies in the presence of CHO-K1-5T4 cells. After 4 h incubation at 37℃, Bio-Glo™ reagent was added and luminescence was measured by a luminometer. Luminecence values were reported as the mean of determinations obtained in three independent experiments. C Table reporting the EC50 (nM) values for 5T4-mediated TCR/CD3 activation of each indicated construct

Article Snippet: Human 5T4/His was purchased from ACROBiosystems TPG- (Newark, Delaware USA), human CD3 epsilon and CD3 delta heterodimeric protein was purchased from AMSBIO (Milton, Abingdon UK).

Techniques: Activation Assay, Expressing, Bioassay, Luciferase, Incubation, Construct

Journal: mAbs

Article Title: Manufacturability and functionality assessment of different formats of T-cell engaging bispecific antibodies.

doi: 10.1080/19420862.2023.2231129

Figure Lengend Snippet: Figure 1. Schematic diagram of the eight T-bsAb formats tested in the current study. Formats tested in this study include “1 + 1”, “2 + 2” and “2 + 1” mixed-valency bispecific antibodies enabled by either a homodimeric Fc or a heterodimeric Fc using the ‘Knob in hole’ technology. One arm of the scFv2 only contains the hinge, CH2, and CH3 domains. Green arms target HER2, and Blue arms target CD3.

Article Snippet: The antigens used in this binding assay were purchased from ACROBiosystems: Human CD3 epsilon CD3 delta Heterodimer Protein, His Tag-Free (Cat# CDD-H52W1), Human HER2/ErbB 2 Protein, His Tag (Cat# HE2-H5225).

Techniques:

Journal: mAbs

Article Title: Manufacturability and functionality assessment of different formats of T-cell engaging bispecific antibodies.

doi: 10.1080/19420862.2023.2231129

Figure Lengend Snippet: Figure 5. Flow cytometric analysis of antigen binding capacities of eight different HER2×CD3 T-bsAbs. The binding capacities of various HER2×CD3 T-bsAbs for antigens CD3 (a) and HER2 (b) antigens were determined by a competitive binding assay using flow cytometry. Jurkat T or SK-OV-3 cells were stained with diluted anti- human CD3 (clone UCHT1) or HER2 (Trastuzumab) antibodies conjugated with APC in the presence of various serially diluted T-bsAbs. The intensities of APC fluorescence were assayed on an LSR II (BD Pharmingen) and used for calculating the IC50 with a four-parameter analysis model of GraphPad Prism 6. The smallest and highest mean fluorescence intensity (MFI) for each data set was normalized as 0% and 100%, respectively.

Article Snippet: The antigens used in this binding assay were purchased from ACROBiosystems: Human CD3 epsilon CD3 delta Heterodimer Protein, His Tag-Free (Cat# CDD-H52W1), Human HER2/ErbB 2 Protein, His Tag (Cat# HE2-H5225).

Techniques: Binding Assay, Competitive Binding Assay, Flow Cytometry, Staining, Fluorescence

Journal: mAbs

Article Title: Manufacturability and functionality assessment of different formats of T-cell engaging bispecific antibodies.

doi: 10.1080/19420862.2023.2231129

Figure Lengend Snippet: Figure 7. Tumor cell-killing and cytokine production by T cells induced by eight different HER2×CD3 T-bsAbs. (a) Analysis of T-bsAb-mediated killing of SK-OV-3 cells by T cells. Purified CD3+ human T cells were co-cultured with Her2+ SK-OV3-luciferase cells at an E:T ratio of 1:1 in the presence of the indicated concentration of various HER2×CD3 T-bsAb for 48 hours. The luciferase activity was used as the surrogate for cell viability and determined by measuring the luminescence in triplicates after adding the substrate luciferin. The EC50 of the tumor cell-killing of the T-bsAb was calculated with a four-parameter logistic sigmoidal dose-response curve using GraphPad Prism. (b,c) Production of IL-2 (b) and INFγ (c) by T cells upon stimulation of T-bsAbs. Human T cells were co-cultured with HER2+ SK-OV-3-luc cells in the presence of the indicated concentrations of various HER2×CD3 T-bsAb for 48 h. IL-2 and IFNγ in the culture supernatant were determined using ELISA in duplicates. The data shown are representative of more than three independent experiments.

Article Snippet: The antigens used in this binding assay were purchased from ACROBiosystems: Human CD3 epsilon CD3 delta Heterodimer Protein, His Tag-Free (Cat# CDD-H52W1), Human HER2/ErbB 2 Protein, His Tag (Cat# HE2-H5225).

Techniques: Purification, Cell Culture, Luciferase, Concentration Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunobiology

Article Title: Preclinical characterization of a Fab-like CD3/CLDN18.2 XFab® bispecific antibody against solid tumors.

doi: 10.1016/j.imbio.2022.152283

Figure Lengend Snippet: Fig. 1. Generation of a Fab-like CD3/ CLDN18.2 XFab® bispecific antibody. (A) Schematic of the CD3/CLDN18.2 XFab® bispecific antibody (ZWB67). The anti- hCD3e variable heavy chain and variable light chains were fused with anti-hCLDN18.2 Fab frameworks by an IgG1 hinge with a deletion mutation (C266 deletion) and located at the C-terminus of the Fab frame works. (B) The size and purity of purified ZWB67 were confirmed using SDS-PAGE analysis. M, molecular weight markers; 1, non-reducing (NR); 2, reducing (R). The experiment was repeated once with the same design, and data were reproducible. Here, representative experiments were shown.

Article Snippet: For binding assays, human CD3 epsilon protein was purchased from ACROBiosystems (Beijing, China).

Techniques: Mutagenesis, Purification, SDS Page, Molecular Weight

Journal: Immunobiology

Article Title: Preclinical characterization of a Fab-like CD3/CLDN18.2 XFab® bispecific antibody against solid tumors.

doi: 10.1016/j.imbio.2022.152283

Figure Lengend Snippet: Fig. 6. ZWB67-induced anti-tumor activity in an in vivo xenograft model. (A) ZWB67 inhibits tumor growth in humanized CD3 EDG mice bearing MC-38-hCLDN18.2 subcutaneous xenografts. Tumor growth is presented as mean ± SEM, and the P-value was calculated using a repeated-measures two-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison. The black arrow indicates the day of ZWB67 administration; *vehicle vs ZWB67 0.3 mg/kg; $vehicle vs ZWB67 1.0 mg/ kg; #ZWB67 0.3 mg/kg vs ZWB67 1.0 mg/kg. (B) ZWB67 prolongs the survival of humanized CD3 EDG mice bearing MC-38-hCLDN18.2 xenografts. Survival curves were obtained for the treatment groups A. The survival rate was analyzed using the log-rank (Mantel-Cox) test. Against the vehicle group, *P < 0.05; **P < 0.01; ***P < 0.001; against the ZWB67 0.3 mg/kg group, #P < 0.05; ##P < 0.01; ###P < 0.001. (C) Mean body weight loss was not observed in any of the mice. The experiment was repeated once with the same design, and data were reproducible. Here, representative experiments were shown.

Article Snippet: For binding assays, human CD3 epsilon protein was purchased from ACROBiosystems (Beijing, China).

Techniques: Activity Assay, In Vivo, Comparison