Ex vivo generation of tumor-reactive T cells for adoptive cell transfer using an immune enhanced, patient derived tumor organoid-on-a-chip (iTOC)

项目摘要 免疫性T细胞疗法（ACT）是一种免疫疗法，涉及宿主免疫细胞的再增殖。 系统与离体扩增的肿瘤反应性T细胞的选择性群体。肿瘤反应性T细胞可以是 从肿瘤中分离（肿瘤浸润淋巴细胞，TIL）或通过基因工程（TCR或CAR）产生 T细胞）。通过在抑制性肿瘤微环境之外离体扩增和活化细胞， 将它们输回宿主体内，可以诱导成功的反应率。然而，许多肿瘤具有高 基因组异质性，这可能导致肿瘤内一些细胞的新抗原表达减少， 允许免疫逃逸，限制检查点抑制剂或靶向免疫缺陷的基于T细胞的疗法的功效。 单一抗原（CAR-T，TCR）。更强大的T细胞反应，靶向多样化和更多数量的肿瘤 新抗原可能导致更有效的免疫疗法。TIL疗法的支持者认为这些T细胞 可能是一种更有效的ACT方法，因为它们能够识别更多数量的肿瘤新抗原 因为它们在病人的肿瘤中被发现。可在高达72%的患者中诱导缓解率。然而，在这方面， 一些患者不能进行手术以获得TIL，其他患者不能等待所需的4-6周， 将TIL扩增到足以用于治疗的数量，而其他患者可能根本没有足够的TIL。最后，虽然 在外周血中发现肿瘤反应性T细胞，先前尝试分离，增加肿瘤抗原 识别，并扩大这些T细胞的数量足以治疗一直是徒劳的。然而，外围 如果人们能够富集T细胞以识别肿瘤抗原，那么血液可以作为潜在的无限的T细胞来源。 为了克服这些限制，我们将采用生物工程免疫增强肿瘤芯片平台 在一个实施方案中，本发明提供了一种用于富集肿瘤抗原识别并扩增来自外周血的肿瘤反应性T细胞的iTOC（iTOC）。在iTOC中 在微流体平台上，肿瘤细胞和自体淋巴结来源的免疫细胞组合以形成 免疫增强的患者肿瘤类器官，由此患者肿瘤类器官的异质性和患者肿瘤类器官之间的相互作用， 肿瘤、基质和免疫细胞保持完整。我们假设来自外周血的T细胞的循环 通过iTOC将导致肿瘤反应性T细胞的富集群体，其能够安装免疫调节剂。 比TIL或未循环的外周血T细胞更强的免疫应答。目标1将决定效果 iTOC灌注对T细胞活力、增殖和表型的影响。目标2将确定iTOC灌注 与TIL和非循环外周血相比， 血T细胞这些研究将确定iTOC改善患者肿瘤特异性适应性免疫应答的功效。 通过按需产生适于过继性T细胞转移的产物来产生免疫应答。虽然我们的测试 载体是黑色素瘤，功能性离体和体外免疫系统如iTOC，将用于指导临床 作为研究复杂的肿瘤免疫微环境的重要研究工具。

PROJECT SUMMARY Adoptive T cell therapy (ACT) is a form of immunotherapy that involves the repopulation of the host immune system with a selective population of ex vivo expanded, tumor reactive T cells. Tumor reactive T cells can be isolated from the tumor (tumor infiltrating lymphocytes, TIL) or created through genetic engineering (TCR or CAR T cells). By expanding and activating the cells ex vivo, outside of the suppressive tumor microenvironment, and infusing them back into the host, successful response rates can be induced. However, many tumors have high genomic heterogeneity, which can result in decreased neoantigen expression by some cells within the tumor, allowing for immune-escape, limiting the efficacy of checkpoint inhibitors or T cell-based therapies that target a single antigen (CAR-T, TCR). A more robust T cell response that targets a diverse and greater number of tumor neoantigens may lead to more effective immunotherapies. Proponents of TIL therapy contend that these T cells may be a more effective approach to ACT due their ability to recognize a greater number of tumor neoantigens as they are found within a patient’s tumor. Response rates can be induced in up to 72% of patients. However, some patients are unable to undergo surgery to obtain TIL, others are unable to wait the 4-6 weeks required to expand TIL to numbers sufficient for therapy, and other patients may not have sufficient TIL at all. Lastly, while tumor reactive T cells are found in peripheral blood, previous attempts to isolate, increase tumor antigen recognition, and expand these T cells to numbers sufficient for therapy have been futile. However, peripheral blood could serve as a potentially limitless source of T cells, if one can enrich them for tumor antigen recognition. To overcome these limitations, we will employ a bioengineered immune-enhanced tumor-on-a-chip platform (iTOC) to enrich tumor antigen recognition and expand tumor-reactive T cells from peripheral blood. In the iTOC microfluidic platform, tumor cells and autologous lymph node-derived immune cells are combined to form immune-enhanced patient tumor organoids whereby the heterogeneity of and the interplay between the patient’s tumor, stroma, and immune cells remain intact. We hypothesize that circulation of T cells from peripheral blood through the iTOC will result in an enriched population of tumor reactive T cells that are capable of mounting a more robust immune response than TIL or uncirculated peripheral blood T cells. Aim 1 will determine the effect of iTOC perfusion on T cell viability, proliferation, and phenotype. Aim 2 will determine how iTOC perfusion impacts T cell receptor diversity and tumor-specific effector function compared to TIL and uncirculated peripheral blood T cells. These studies will establish the efficacy of the iTOC to improve a patient’s tumor-specific adaptive immune response through on demand generation of a product suitable for adoptive T cell transfer. While our test vehicle is melanoma, functional ex vivo and in vitro immune systems like the iTOC, will be used to guide clinical therapy and serve as important research tools to study the complex tumor-immune microenvironment.