ht1080 cd20 cells  (ATCC)

Journal: Science Advances

Article Title: Rapid, site-specific labeling of “off-the-shelf” and native serum autoantibodies with T cell–redirecting domains

doi: 10.1126/sciadv.abn4613

Figure Lengend Snippet: ( A ) An anti-CD3 scFv was fused with a pAbBD. Two hours of irradiation with nondamaging long-wavelength ultraviolet (UV) light induces covalent attachment of the fusion protein to the IgG Fc region. ( B ) Six human antibodies—rituximab, cetuximab, trastuzumab, IgG2, IgG3, and IgG4—and the resulting BiAbs produced by photocrosslinking with pAbBD–anti-CD3 fusion protein were analyzed on a reducing SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Unbound, excess pAbBD-scFv was removed via filtration. The bands represent IgG heavy chains (HC) before and after photocrosslinking. ( C to E ) HER2 + T617, epidermal growth factor receptor–positive (EGFR + ) MDA-MB-468, and CD20 + HT1080 cell lines were seeded, fixed, and blocked with normal goat serum. Binding curves of photocrosslinked (C) anti-HER2 (trastuzumab) × anti-CD3, (D) anti-EGFR (cetuximab) × anti-CD3, and (E) anti-CD20 (rituximab) × anti-CD3 BiAbs and the respective monospecific antibodies from which they were derived were obtained after incubation with a fluorescent secondary antibody. Fluorescence intensity was measured using a plate reader. The dissociation constant ( K d ) values were (A) 0.9 nM for the BiAb and 1.16 nM for monoclonal antibody, (B) 0.60 nM for the BiAb and 0.49 nM for the monoclonal antibody, and (C) 8.2 nM for the BiAb and 12 nM for the monoclonal antibody. All coefficient of determination ( R 2 ) values are greater than 0.9. ( F ) Human T cells were incubated with serial dilutions of anti-EGFR × anti-CD3 scFv BiAb, free anti-CD3 scFv, and positive control OKT3, and binding was measured via flow cytometry. The K d values were found to be 1.89, 35, and 34 nM for OKT3, CD3 scFv, and BiAb with R 2 values of 0.99, 0.985, and 0.99, respectively. AU, arbitrary units.

Article Snippet: The relative binding affinity of BiAbs versus the corresponding unconjugated monoclonal antibodies were assessed using HER + T617 cells (provided by M. Greene, University of Pennsylvania), CD20 + HT1080 cells (transduced with human CD20 Full-length containing lentivector from G&P Bioscience), and EGFR + MDA-MB-468 [American Type Culture Collection (ATCC)] cells.

Techniques: Irradiation, Produced, Polyacrylamide Gel Electrophoresis, SDS Page, Filtration, Binding Assay, Derivative Assay, Incubation, Fluorescence, Positive Control, Flow Cytometry

Journal: Science Advances

Article Title: Rapid, site-specific labeling of “off-the-shelf” and native serum autoantibodies with T cell–redirecting domains

doi: 10.1126/sciadv.abn4613

Figure Lengend Snippet: ( A to C ) T cell–mediated cytolysis of HER2 + T617, EGFR + MDA-MB-468, and CD20 + HT1080 cell lines was monitored for 72 hours as a function of BiAb dose using an xCELLigence real-time cell analysis (RTCA) instrument. Increasing concentrations of anti-HER2 (trastuzumab) × anti-CD3, anti-EGFR (cetuximab) × anti-CD3, and anti-CD20 (rituximab) × anti-CD3 BiAbs were analyzed (circles), as well as mixtures of the respective unconjugated antibodies and anti-CD3 scFv (triangles), and nontargeted BiAbs (squares). The nontargeted BiAbs in (A) and (C) consisted of an anti-EGFR × anti-CD3 BiAb and were tested on EGFR-negative cell lines (HER2 + /EGFR − and CD20 + /EGFR − , respectively), while in (B), an anti-Her2 × anti-CD3 BiAb was tested on an EGFR + /HER2 − cell line. Here, BiAbs were prepared using the pAbBD–anti-CD3 scFv. Equivalent studies using the nanobody format are presented in fig. S4. All assays were performed with human T cells at a 10:1 E:T ratio. BiAbs EC 50 values were (A) 0.038 nM, (B) 0.05 nM, and (C) 0.007 nM. All R 2 values were greater than 0.9. ( D and E ) Kinetics of T cell–mediated cytolysis of EGFR + tumor cells for (D) increasing concentrations of anti-EGFR (cetuximab) × anti-CD3 BiAb or (E) a mixture of anti-EGFR cetuximab and anti-CD3 scFv. All assays were performed with human T cells using an E:T of 10:1. ( F ) T cell–mediated cytolysis of EGFR + cells with increasing E:T ratios, 12 hours after treatment with 0.1 nM EGFR-targeted BiAbs produced with pAbBD–anti-CD3 scFv or 0.1 nM cetuximab mixed with 0.2 nM free anti-CD3 scFv.

Article Snippet: The relative binding affinity of BiAbs versus the corresponding unconjugated monoclonal antibodies were assessed using HER + T617 cells (provided by M. Greene, University of Pennsylvania), CD20 + HT1080 cells (transduced with human CD20 Full-length containing lentivector from G&P Bioscience), and EGFR + MDA-MB-468 [American Type Culture Collection (ATCC)] cells.

Techniques: Cell Analysis, Produced