The horizon of immunotherapy using CAR-T cells is continuously extending to treat solid tumors beyond the success in the treatment of liquid tumors. Precise in-vitro evaluations of CAR-T cells for their phenotypes, quantity and quality of activation in various tumor microenvironments including different antigen densities, and the resulting effector functions are critical for the successful development of CAR-T therapies and safe translation to clinics. Unfortunately, the development of methods and tools to accommodate these needs have been lagging behind. Here, we developed a novel biomaterial platform, acellular artificial target particles (aaTPs) against CAR-T cells, using magnetic microbeads that are already widely employed in the manufacturing of T cell products. By devising a simple and standardized procedure, we precisely controlled the antigen surface densities presented on the aaTPs for a wide range. By co-incubation of aaTPs with CAR-T cells followed by flow cytometry and cytokine assays, we quantitatively determined the antigen-specific and dose-dependent activation of anti-HER2 CAR-T cells. We also demonstrated that the aaTP can serve as a clean target cell in in-vitro assays to prove the proposed mechanism of action of a next-generation CAR-T product. Overall, the simple, inexpensive, modular and precisely controllable synthetic nature of aaTPs enables the development of clean and standardized in-vitro assays for CAR-T cells, which provides critical advantages over the conventional assays using target cell lines. The design of aaTPs can be extended to include other tumor antigens and relevant surface molecules of physiological target cells. Thus, the aaTP platform has great potential as a standardized tool for the development and evaluation of both conventional and new CAR-T products in the context of approval from regulatory agencies and clinical translation.
KMT2A-rearranged (KMT2A-R) B cell acute lymphoblastic leukemia (ALL) is a high-risk disease in children and adults that is often chemotherapy resistant. To identify non-cytotoxic approaches to therapy, we performed a domain-specific kinome-wide CRISPR screen in KMT2A-R cell lines and patient derived xenograft samples (PDX) and identified dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as a potential target. Pharmacologic inhibition of the KMT2A-fusion transcriptional co-regulator Menin released the KMT2A-fusion complex from the DYRK1A promoter thereby lowering DYRK1A expression levels confirming DYRK1A as a direct target of the KMT2A fusion oncogene. Direct pharmacologic inhibition of DYRK1A decreased cell proliferation of KMT2A-R ALL, thereby confirming the requirement of DYRK1A in this ALL subtype. To further understand the biologic function of DYRK1A in KMT2A-R ALL, we leveraged pharmacologic DYRK1A inhibitors in KMT2A-R PDX and cell line models. DYRK1A inhibition consistently led to upregulation of MYC protein levels, and hyperphosphorylation of ERK, which we confirmed via in vivo treatment experiments. Furthermore, DYRK1A inhibition decreased ALL burden in mice. Our results further demonstrate that DYRK1A inhibition induces the proapoptotic factor BIM, but ERK hyperphosphorylation is the driving event that induces cell cycle arrest. In contrast, combined treatment of KMT2A-R ALL cells in vitro and in vivo with DYRK1A inhibitors and the BCL2 inhibitor, venetoclax, synergistically decreases cell survival and reduced the leukemic burden in mice. Taken together these results demonstrate a unique function of DYRK1A specially in KMT2A-R ALL. Synergistic inhibition of DRYK1A and BCL2 may provide a low-toxic approach to treat this high risk ALL subtype.
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