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Engineered Toxin Bodies (ETBs) represent a unique therapeutic strategy for fighting cancer by targeting and selectively destroying cancer cells or cancer-promoting immune cells. Featuring an antibody-based targeting domain fused with the cytotoxic, ribosome inactivating Shiga-like toxin A subunit (SLTA), ETBs enable killing of target expressing cells. Although immunotoxins have been explored for decades as promising cancer therapeutics, activation of innate immunity limited this approach. MTEM has developed 1st, 2nd, and 3rd generation ETBs with iterative improvements to reduce immunogenicity and add mechanisms of action. Here, we describe the development of an ex vivo cytokine release assay used to enhance the safety, tolerance, and therapeutic index (TI) of ETBs as cancer therapeutics. MT-3724, a 1st gen CD20 targeting ETB comprising wild type SLTA (WT SLTA) genetically fused to a CD20 targeting scFv, displayed efficacy as monotherapy in relapsed/refractory non-Hodgkin lymphoma. However, a subset of MT-3724-treated patients experienced capillary leak syndrome (CLS), likely driven by innate immune recognition of specific components of WT SLTA. Adverse events were also associated with a particular lot of MT-3724 that contained a higher proportion of aggregated species. Therefore, next gen ETBs were de-immunized by replacing surface exposed residues of WT SLTA while retaining cytotoxicity and were designed to have reduced propensity for aggregation. An ex vivo cytokine release assay, developed to triage candidate ETBs that generate innate immune activation, was validated by probing WT-SLTA and aggregated MT-3724. Cancer free human peripheral blood mononuclear cells (PBMCs) or PBMCs depleted of B cells were treated with positive and negative controls, non-targeted SLTAs, and ETBs. Cytokine concentrations were quantified by Luminex. The positive control TLR4 agonist lipopolysaccharide (LPS) activated robust release of proinflammatory cytokines including IL-1β, TNFα, and IL-6, as well as CCL3, CCL4, GM-CSF, IL-10, IFNγ, and Granzyme B. Non-targeted WT SLTA displayed a similar, but not identical, pattern of cytokine release. Conversely, the de-immunized (DI) SLTA did not activate cytokine or chemokine release, remaining at levels equal to PBS, indicating that scrubbing WT SLTA of immunogenic components can improve the safety profile and TI of our 2nd gen ETBs. Aggregated MT-3724 induced moderate increases in TNFα, IL-6, CCL3, and CCL4 relative to a non-aggregated MT-3724 lot. Removal of B cells from PBMCs did not alter cytokine release patterns of LPS, WT SLTA, or aggregated MT-3724. These data suggest that cytokines were released in response to WT SLTA and protein aggregates in an off-target manner. This assay will be used to evaluate ETB safety by testing candidate ETBs for likelihood of cytokine release and/or innate immune activation prior to selection as candidates for clinical trials. Citation Format: Rachael M. Orlandella, Elizabeth M. Kapeel, Brigitte Brieschke, Garrett L. Robinson, Joseph D. Dekker, Chris B. Moore. Improving immunotoxin-based therapeutics for cancer with de-immunized engineered toxin bodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2579. |
