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Cells were incubated overnight at 37C, and then the supernatant was analyzed by ELISA. activity in the presence of anti-cell IgGin vivo. This technology may be useful for repeated human infusions of engineered cells, more complex engineered cells, and expanding widespread use of off-the-shelf allogeneic cellular therapies. Keywords:CAR T cells, cell therapies, immunogenicity, humoral response, anti-CAR IgG, IdeS == Graphical abstract == Cellular therapies are engineered using foreign and synthetic protein sequences. Utilizing the IgG protease IdeS, we programmed CAR T Floxuridine cells to defeat humoral immune attacks. This technology may be useful for repeated human infusions of engineered cells, more complex engineered cells, and expanding widespread use of off-the-shelf allogeneic cellular therapies. == Introduction == Chimeric antigen receptor (CAR) T cell therapies have been approved for the treatment of acute lymphoblastic leukemia (ALL) and diffuse large B cell Floxuridine lymphoma (DLBCL) with CD19-targeting CARs.1,2,3More complex CAR T cell therapies have been engineered to secrete cytokines, single-chain variable fragments (scFv), and bispecific T cell engagers to potentiate their activity.4,5,6,7CRISPR-Cas9 technology also has been used for site-specific integration of the CAR.8,9,10,11Off-the-shelf allogeneic CAR T and natural killer (NK) cells are currently in clinical development.12,13,14However, major challenges remain in the application of these technologies to other cancers. In addition, genetically modified cells rely on the use of foreign biological material or unnatural amino acid sequences, resulting in immunogenicity.15,16 Humoral responses and anaphylaxis to engineered cells have been reported in human trials.17,18,19,20,21,22Such antibodies Floxuridine may lead to limited persistence, poor engraftment, and reduced functionality of the CAR T cells.17,20,23Whereas humanizing CARs renders them less immunogenic,24not only are the antibody constructs potentially immunogenic but so are the artificial linkers used in the fusion sequences, as well as viral products used throughout the transduction process, which may be recognized as foreign.15,16 Although the immune response to engineered cells in the host can be both cellular and humoral, reported efforts have been directed at defining and possibly reducing the T cell response13but not the immunoglobulin (Ig) response. To investigate the scope of this issue, in this work we first screened sera from 12 patients who received two or more rounds of 1928 CAR T cells at Memorial Sloan Kettering Cancer Center (MSKCC) and confirmed that host IgG responses were prevalent. Disease progression and outcomes for these patients were published recently.25Our hypothesis was that anti-CAR IgG antibodies could prevent the expansion and function of the re-infusions Floxuridine of CAR T cells. We therefore designed an innovative approach based on the microbial immunoevasin IgG-degrading enzyme ofStreptococcus pyogenes(IdeS)26for arming engineered cells with an effective means to circumvent the host humoral immune response. IdeS is a cysteine protease with high specificity for human IgG that has already been safely used in highly sensitized patients to treat renal allograft rejection.26,27,28The CAR T cells expressing IdeS, termed Shield CAR T cells, retained their original immune functions, cleaved IgGin vitroandin vivo, covered themselves in a protective coating of non-functional F(ab)2, thereby preventing new attack by other IgG, and have improved persistence and efficacy over traditional CAR T cell therapies in the presence of antibodies directed to their cell surfaces. == Results == == Murine CAR T cells induce a host IgG response in a syngeneic mouse model == To better understand the syngeneic anti-CAR responses observed in human trials,17,18,19,20we recapitulated a humoral response to a murine CAR in a syngeneic C57BL/6J mouse model in which weekly infusions of littermate-derived mouse m4H1128 CAR T cells led to the development of anti-CAR IgG responses (Figure 1A). m4H1128 CAR T cells have been previously established and tested in syngeneic systems targeting MUC16+ tumors.6The 4H11 scFv is murine, as are FAS the signaling domains. When we tested the mouse sera, we found that IgGs bound to a HEK293t-derived cell line that expressed the m4H1128 CAR construct (Figure 1B). Time-dependent.