Allogeneic Immunotherapy

在过去的十（20）年中，同种异体淋巴细胞靶向和根除白血病细胞的能力已被确立为一种真正的生物实体。这种效果是否可以对非血液学“实体”恶性肿瘤产生仍然没有探索。我们在难治性转移性肾细胞癌（RCC）患者中开展了一项使用非清髓性异基因干细胞移植方法的临床试验。同种异体移植物抗RCC效应的有力证据已被证实，在一些患者中观察到的大转移完全消退。最近，我们证明了类似的效果，发生在结肠癌和前列腺癌患者。随后，我们开始了研究免疫重建在那些表现出抗RCC效果，试图确定介导这些回归的效应细胞群体以及它们的靶抗原。我们的实验室已经证实在肾癌细胞表面表达次要组织相容性抗原。我们证明了对次要抗原具有特异性的细胞毒性T细胞克隆能够杀死移植后具有GVT效应的患者中的RCC细胞。我们还能够在两个应答患者中扩增T细胞克隆，其识别存在于患者造血细胞和RCC细胞上的肿瘤细胞特异性或广泛表达的抗原。这些观察结果提供了第一次深入了解免疫机制介导的非清髓性异基因移植后转移性癌症的消退。使用c-DNA表达克隆，我们目前正在鉴定从共振患者中分离和扩增的RCC特异性T细胞克隆的靶抗原。如果发现这种抗原在相当大比例的RCC细胞上表达，它可能成为未来肾癌疫苗的靶点。最近，我们已经开发了一种小鼠模型的同种异体SCT在承载转移性RCC的主机，其中发生可重复的GVT效应延长动物的生存期相比，自体移植的受体。在该模型中进行移植后肿瘤疫苗接种研究，以及在移植的前几个月使用PDGF、VEGF和EGF-R酪氨酸激酶抑制剂对抑制血管生成的影响的研究。我们的小组最近表明，KIR不相容的NK细胞在体外对实体瘤细胞具有细胞毒性。最近，我们已经开发了一种方法，用于在未来基于NK细胞的过继免疫治疗试验中过继输注从健康供体扩增> 4 log NK细胞。我们还在探索通过靶向基因诱导改变肿瘤细胞表型来使实体瘤对NK细胞攻击敏感的方法。 急性GVHD是异基因造血干细胞移植的主要毒性反应。不幸的是，GVHD的诊断目前是基于从受累器官获得的活检标本中观察到的组织病理学变化。GVHD的组织确认的周转时间为48小时是典型的。我们目前正在评估研究方法，以快速和特异性诊断急性GVHD。该技术涉及使用SELDI的血清蛋白模式识别。该测定需要血清样品，周转时间为2-3分钟。在我们的第一个队列中，20例急性GVHD患者和60例移植后GVHD阴性对照，该检测具有100%的灵敏度和特异性诊断GVHD。目前正在进行确认队列研究。 我们的研究小组继续探索同种异体SCT在非恶性疾病如PNH或ATGF难治性重型再生障碍性贫血患者中的应用。我们最近也发现PNH可以通过非骨髓抑制性干细胞移植治愈。我们实验室进行的体外研究表明，PNH细胞对同种异体免疫攻击的敏感性与正常GPI阳性免疫细胞相同。目前，27例SAA/PNH患者已接受移植，200天TRM为0%。

The ability of allogeneic lymphocytes to target and eradicate leukemia cells has been established as a true biological entity over the past ten (20) years. Whether such effects can be generated against non-hematological "solid" malignancies remains unexplored. We initiated a clinical trail using a non-myleoablative approach of allogeneic stem cell transplantation in patients with treatment refractory metastatic renal cell carcinoma (RCC). Definitive evidence for an allogeneic graft-versus-RCC effect has been demonstrated with complete regression of large metastasis observed in some patients. Recently we demonstrated a similar effect to have occured in a patient with colon carcinoma and prostate cancer. We have subsequently initiated studies investigating immune reconstitution in those demonstrating an anti-RCC effect in attempts to identify both the effector cell populations mediating these regressions as well as their target antigens. Our laboratory has confirmed the expression of minor histocompatibility antigens on the surface of kidney cancer cells. We demonstrated that cytotoxic T-cell clones with specificity for minor antigens are capable of killing RCC cells in patients having a GVT effect post transplant. We also were able to expand T-cell clones in two responding patients that recognized either tumor cells specifically or broadly expressed antigens present on both patient hematopoietic cells and RCC cells. These observations provide the first insight into the immune mechanisms mediating the regression of metastatic cancer following non-myeloablative allogeneic transplantation. Using c-DNA expression cloning, we are currently in the process of identifying the target antigen of an RCC specific T-Cell clone isolated and expanded from on resonding patient. If it turns out this antigen is expressed on a significant % of RCC cell, it could potentially serve as a target for a future kidney cancer vaccine. Recently we have developed a murine model of allogeneic SCT in hosts bearing metastatic RCC, in which reproducible GVT effects occur extending animal survival compared to recipients of autologous transplants. Post transplant tumor vaccination studies are being conducted in this model, as well as investigations into the impact of inhibiting angiogeneisis in the first few months of transplantation using PDGF, VEGF, and EGF-R tyrosine kinase inhibitors. Our group has recently shown that KIR incompatible NK cells are cytotoxic to solid tumor cells in vitro. Recently, we have developed a method to expand by > 4 logs NK cells from healthy donors for adoptive infusion in future NK-Cell based adoptive immunotherapy trials. We are also exploring methods to sensitize solid tumors to NK cell attack by altering the phenotype of tumor cells through targeted gene induction. Acute GVHD remains a major toxicity of allogeneic stem cell transplantation. Unfortunately, the diagnosis of GVHD is currently based on histopathic changes observed in biopsy specimens obtained from of involved organs. A turnover time of 48 hours for tissue confirmation of GVHD is typical. We are currently evaluating investigational methods to rapidly and specifically diagnose acute GVHD. The technique involves serum protein pattern recognition using SELDI. The assay requies a sample of serum and has a turn-over time of 2-3 minutes. In our first cohort of 20 patients with acute GVHD and 60 post-transplant GVHD negative controls, the assay had 100% sensitivity and specificity for the diagnosis of GVHD. A confirmation cohort is currently being pursued. Our group continues to explore the use of allogeneic SCT in patients with nonmalignant diseases such as PNH or ATGF-Refractory severe aplastic anemia. We have also recently shown that PNH can be cured following nonmyeloabaltive stem cell transplanation. In vitro studies conducted in our laboratory have shown PNH cells are equally sensitive to allogeneic immune attack as normal GPI-positive immune cells. At present, 27 patients with SAA/PNH have been transplanted with a day 200 TRM of 0%.