EAGER: Biomanufacturing: Physiologically-inspired Large Scale Manufacturing and Potency-Biomarker identification for Chimeric Antigen Receptor (CAR)-T cells

提出了一种新颖的、受生物学启发的策略，通过模仿淋巴结的环境和条件来增强治疗性T细胞的扩张，这些细胞通常在体内扩张。这种T细胞在合成淋巴结样壁龛内扩张的想法是独特的，在那里细胞彼此交流，并与“自我”交流，但坚定地植根于生理原理。我们的方法可以极大地增强T细胞的扩展潜力，使更多的T细胞可用于治疗，还可以降低总体制造成本，使治疗更实惠，更广泛。此外，在将这些人造T细胞注入患者体内之前，对它们的功效和效力进行预测是至关重要的。将对制造的T细胞进行全面的表征，以开发强大的相关标记物，这些标记物可以预测细胞的效力，并最终用作输注前确定这些细胞质量的常规分析工具。这个EAGER项目将研究人类CAR-T细胞在模拟淋巴结（LN）内T细胞壁龛的合成3D微环境中的扩增。将使用抗cd3 /抗cd28功能化的微载体，人类CAR-T细胞将在模拟ln的3D环境中培养，其中T细胞保持高密度，细胞间密切接触，并允许有效的旁分泌/自分泌信号传导。这些参数在当前的T细胞制造概念中是不存在的，因为T细胞在局部分泌大量的白细胞介素（il）以促进快速、大规模的扩张，因此这些参数是至关重要的。因此，这种方法还可以减少培养基和IL的需求，从而显著降低成本。我们将评估低氧张力、不同细胞播种密度和抗cd3 /抗cd28配体密度对扩增效率和T细胞质量的影响。此外，LN-like生态位将与搅拌槽或灌注生物反应器相结合，以确定动态培养和流动灌注是否能提高膨胀效果、产品质量、可扩展性和成本效益。

1547638 Roy, KrishnenduA novel, biologically-inspired strategy is proposed to enhance the expansion of therapeutic T cells by mimicking the environment and conditions of lymph nodes, where these cells normally expand in the body. This idea of T cell expansion inside synthetic lymph-node-like niches, where cells communicate with each other and with 'self' is unique, but firmly rooted on physiological principles. Our approach could vastly enhance the expansion potential of T cells making larger numbers of them available for therapy and also reduce overall manufacturing cost, making the treatment more affordable and widely available. Further, predictively measuring the efficacy and potency of these manufactured T cells, before infusing them in patients, is of critical importance. A comprehensive characterization of manufactured T cells will be carried out to develop robust correlative markers that can predict cell potency and can eventually be used as a routine analysis tool to determine the quality of these cells before infusion.This EAGER project will investigate human CAR-T cell expansion within synthetic 3D microenvironments that mimic the T cell niches within lymph nodes (LN). Anti-CD3/anti-CD28-functionalized microcarriers will be used and human CAR-T cells will be cultured in LN-mimicking 3D niches where T cells remain at high density with close cell-cell contact, and allow efficient paracrine/autocrine signaling. These parameters, absent from the current T-cell manufacturing concepts, are critical since T cells secrete large amounts of interleukins (ILs) locally to promote rapid, large scale expansion. Thus, this approach could also reduce culture media and IL requirements, thereby significantly reducing cost. The effect of low O2 tension will be evaluated together with various cell-seeding densities and anti-CD3/anti-CD28 ligand densities on expansion efficacy and T cell quality. Further, the LN-like niche will be combined with stirred tank or perfusion bioreactors, to determine whether dynamic culture and flow perfusion improve expansion efficacy, product quality, scalability and cost effectiveness.