Strategies to enhance cytotoxicity of tumor-infiltrating γδ T-cell subsets against autologous tumor cells

人肿瘤浸润性γδ T细胞（γδ TIL）的定位、分布、细胞毒性和激活状态及其对肿瘤患者生存的影响是改进γδ T细胞免疫治疗的重要研究方向。上皮性卵巢癌（EOC）的高复发率-即使在成功的初始手术和多化疗治疗后-反映了一个重要的问题。在姑息治疗的情况下，化疗标准方案往往没有持久的疗效。肿瘤内细胞毒性T细胞的存在与良好的临床结果相关。最近，我们证明了在新分离的EOC组织中，v - γ2,3,4 γδ t细胞浸润增加，共表达Vδ1-或v δ3链，强烈产生细胞毒性介质，如颗粒酶A/B。此外，我们发现了tnf相关凋亡诱导配体（TRAIL）受体4的新作用，增强抗肿瘤Vδ1 t细胞的细胞毒性。由于中枢记忆和效应记忆Vδ1 TIL在EOC组织中增加，它们很可能在肿瘤部位被激活。在FOR2799中，将研究v - γ2、3、4和v - γ9 γδ t细胞克隆位点特异性的鉴定，以及v - γ 9v δ1和非v δ1/Vδ2 TIL的亲丁酸蛋白依赖性激活。我们之前的研究结果表明，双特异性T细胞接合剂（bsTCE） [(HER) 2v γ - 9]增强了v γ - 9 T细胞对表达人表皮生长因子受体(HER)-2的自体EOC细胞的细胞毒性。此外，bsTCE部分克服了阻止γδ t细胞活化的固有肿瘤抵抗机制。初步结果显示，[(HER) 2v γ -9]在我们的新型患者来源的体外肿瘤组织模型（包括免疫抑制肿瘤微环境（TME））中具有30-90%的抗肿瘤功效。由于v - γ2,3,4 TIL在EOC组织中积累，我们产生了bsTCE [(HER) 2v γ2,3,4]，它允许与[(HER) 2v γ9]联合靶向EOC患者中高达78%的γδ TIL。[(HER) 2xv γ2,3,4]对γδ T细胞介导的自体EOC细胞裂解的影响和TME的影响将在NSG小鼠模型和患者源性离体肿瘤组织模型中进行研究。后者在试图减少临床前和临床研究之间的转化差距时非常有用。bsTCE在患者源性离体肿瘤组织模型中的应用，也可以有针对性地研究v - γ2、3、4和v - γ9 TIL的效应功能。由于我们的bsTCE不会像重复应用磷酸化抗原那样诱导γδ t细胞死亡，这是bsTCE应用的决定性优势。我们期望我们的bstce反映出未来基于γδ T细胞的免疫治疗的新工具。

Localization, distribution, cytotoxicity and activation state of human tumor-infiltrating γδ T cells (γδ TIL) and their impact on survival of cancer patients are of great interest for an improvement of γδ T cell-based immunotherapy. High recurrence rates in epithelial ovarian cancer (EOC) - even after successful initial surgical and poly-chemotherapeutic treatment - reflects a significant problem. Chemotherapeutic standard regimens are often not of long-lasting efficacy in the palliative situation. The presence of intra-tumoral cytotoxic T cells has been correlated with a favorable clinical outcome.Recently, we demonstrated an increased Vγ2,3,4 γδ T-cell infiltration co-expressing Vδ1- or Vδ3-chains in freshly isolated EOC tissue which strongly produce cytotoxic mediators such as granzyme A/B. Further, we discovered a novel role of TNF-related apoptosis-inducing ligand (TRAIL)-receptor 4 that enhance anti-tumoral Vδ1 T-cell cytotoxicity. Since central and effector memory Vδ1 TIL are increased within the EOC tissue, it is likely that they are activated at the tumor-site. The identification of site-specific occurrence of Vγ2,3,4 and Vγ9 γδ T-cell clones and a butyrophilin-dependent activation of Vγ9Vδ1and non-Vδ1/Vδ2 TIL will be investigated within the FOR2799.Our previous results demonstrated that the bispecific T cell engager (bsTCE) [(HER)2xVγ9] enhanced Vγ9 T-cell cytotoxicity against autologous EOC cells expressing the human epidermal growth factor receptor (HER)-2. Further, bsTCE partially overcomes intrinsic tumor resistance mechanisms which prevent γδ T-cell activation. Preliminary results revealed a 30-90% anti-tumor efficacy of [(HER)2xVγ9] in our novel patient-derived ex vivo tumor tissue model including an immunosuppressive tumor microenvironment (TME). Since Vγ2,3,4 TIL accumulate in the EOC tissue, we generated the bsTCE [(HER)2xVγ2,3,4], which allows in combination with [(HER)2xVγ9] to target up to 78% of the γδ TIL in EOC patients. The efficacy of [(HER)2xVγ2,3,4] on γδ T cell-mediated lysis of autologous EOC cells and the impact of the TME will be examined in the NSG mouse model and the patient-derived ex vivo tumor tissue model. The latter is of great use when trying to reduce the translational gap between preclinical and clinical research. The use of the bsTCE in the patient-derived ex vivo tumor tissue model also allows the targeted investigation of the effector function of Vγ2,3,4 and Vγ9 TIL. Since our bsTCEs do not induce γδ T-cell death like repetitive application of phosphorylated antigens does, this is a decisive advantage of bsTCE application. We expect that our bsTCEs reflect a novel tool for a future γδ T cell-based immunotherapy.