human cd20 negative cell line k562  (ATCC)

Journal: Cancers

Article Title: Facile Generation of Potent Bispecific Fab via Sortase A and Click Chemistry for Cancer Immunotherapy

doi: 10.3390/cancers13184540

Figure Lengend Snippet: In vitro efficacy of BiFabs. ( a ) The binding abilities of Fabs and BiFab with CD20-positive Ramos and Jurkat cells. ( b ) The in vitro efficacy of the BiFab CD20/CD3 on T cell activation. After CD20-positive B cell depletion, fresh PBMCs were treated with serial concentrations of BiFab CD20/CD3 in the presence of target tumor cells at an E:T ratio of 5:1 for 48 h. The expression levels of CD69 and CD25 on T cells, two biomarkers for T cell activation, were evaluated after immuno-staining via flow cytometry. ( c ) Evaluation of the binding abilities of BiFab Her2/CD3 with CD3-positive Jurkat cells and HER2-positive SK-OV-3 cells by flow cytometry. ( d ) The quantification of interferon-γ release from T cells activated by BiFab CD20/CD3 . Fresh PBMCs were incubated with Daudi or Raji cells at an E:T ratio for 5:1 for 48 h. The secreted interferon-γ from T cells was quantified by ELISA Kit. ( e ) BiFab CD20/CD3 mediated T cell proliferation in the presence of CD20-negative K562 cells or CD20-positive Ramos cells at an E:T ratio of 5:1 for 48 h. ( f ) After treatment with various concentrations of BiFab CD20/CD3 with an E:T ratio of 5:1 for 48 h, T cell proliferation was analyzed by flow cytometry.

Article Snippet: The human CD20-positive cell lines Ramos, Raji, Daudi and the human CD20-negative cell line K562 were purchased from the American Type Culture Collection (ATCC, San Francisco, CA, USA), and were cultured in 1640 medium (Gibco) with 10% fetal bovine serum (FBS, Gibco).

Techniques: In Vitro, Binding Assay, Activation Assay, Expressing, Immunostaining, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Facile Generation of Potent Bispecific Fab via Sortase A and Click Chemistry for Cancer Immunotherapy

doi: 10.3390/cancers13184540

Figure Lengend Snippet: The in vitro and in vivo antitumor activities of BiFabs. ( a ) The in vitro efficacy of BiFab CD20/CD3 . Target cells (Ramos and Daudi) and active T cells (E:T = 2:1) were incubated with serial diluted BiFab CD20/CD3 for 24 h (data shown as mean ± SD, n = 3). LDH release was determined by ELSIA kit and used to calculate cell viability. ( b ) The in vitro cytotoxicity of BiFab CD20/CD3 was analyzed by Annexin V/PI apoptosis detection kit, by using the same condition as described in ( a ). ( c ) Study on potential Fc-related cytotoxicity of BiFab CD20/CD3 . The K562 cells and PBMCs were co-cultured with serial concentrations of non-binding IgG-based bispecific antibody or BiFab. The apoptosis rate was determined by Annexin V-Cy5 Apoptosis Detection Kit. ( d ) The in vitro cytotoxicity of BiFab Her2/CD3 . Target tumor cells (SK-OV-3 or MDA-MB-468) and PBMC (E:T = 4:1) were incubated with serial concentrations of BiFab Her2/CD3 for 72 h, and the LDH release in the supernatant was determined by LDH detection kit. All data were shown as mean ± SD, n = 3. ( e ) The in vivo antitumor activities of BiFab CD20/CD3 in mouse xenograft model. Mice were inoculated subcutaneously with 2.5 × 10 6 Ramos cells in the presence of 1 × 10 7 fresh human PBMC from healthy donors at an E:T ratio of 4:1. All samples were administered intravenously via the tail vein at following dosages, 1 mg/kg of Fab CD3 and 1 mg/kg or 3 mg/kg of BiFab CD20/CD3 at every two days for four times.

Article Snippet: The human CD20-positive cell lines Ramos, Raji, Daudi and the human CD20-negative cell line K562 were purchased from the American Type Culture Collection (ATCC, San Francisco, CA, USA), and were cultured in 1640 medium (Gibco) with 10% fetal bovine serum (FBS, Gibco).

Techniques: In Vitro, In Vivo, Incubation, Cell Culture, Binding Assay