Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Incorporation of bacterial immunoevasins to protect cell therapies from host antibody-mediated immune rejection.

doi: 10.1016/j.ymthe.2021.06.022

Figure Lengend Snippet: Figure 6. Shield cells survive antibody-mediated cytotoxicity from CAR T cell patient sera, shown to have CAR-binding IgG (A) Serum samples from 12 ALL patients treated with 2 infusions of 1928z CAR T cells were analyzed for binding to the 1928z CAR by ELISA. For patients 1 and 4, two samples at different time points (“a” and “b”) were tested after the second CAR T cell infusion. Details regarding the time points can be found in Table S1. Lysates from WT HEK293t and 1928z-expressing HEK cells were used as the target cell antigen. A binding ratio of 1928z to WT HEK cells was used to exclude non-specific binding. Samples are numbered for each unique patient and labeled a and b to designate samples obtained from the same patient at different intervals after CAR T cell infusion. (B) Patient sera were used as primary antibodies in a WB assay. WT and 1928z HEK lysates were run on SDS-PAGE gel and then incubated with diluted patient sera overnight. Anti-human HRP-labeled secondary was used for detection. (C) CDC assay to test the cytotoxicity of the patient serum samples. WT and 1928z HEK producer cells were used as targets, incubated with CAR T cell patient sera and rabbit complement, and cell number was measured by Cell Titer-Glo. (D) Patient sera 1a and 4a inhibited the binding of CAR anti- idiotype AF647-antibody as measured by flow cytometry. (E) Supernatant fluid from experiment in (D) was analyzed by WB, revealing cleavage of IgG in CAR T cell patient sera by the Shield cells (arrow shows Fc fragment). (F) CDC assay to test cytotoxicity against Shield HEK cells. Cells were incubated with diluted patient sera 1a and 4a for 2 h and then incubated with rabbit complement. Cell number was measured by Cell Titer-Glo. Data represent mean ± SD of triplicate samples. All experiments were repeated at least twice. Representative data are shown. *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001; Student’s t test.

Article Snippet: Briefly, 2–3 105 CAR T cells and Shield CAR T cells were seeded in a 96 U-bottom plate with different concentrations of rabbit anti-mouse Fab-specific antibody (Jackson ImmunoResearch) in complete RPMI medium.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Labeling, SDS Page, Incubation, CDC Assay, Cytometry