Allogeneic Immunotherapy

在过去的十（10）年中，异体淋巴细胞靶向和根除白血病细胞的能力已经被确立为一个真正的生物实体。对于非血液学“实体”恶性肿瘤是否能产生这种效果仍未研究。我们在难治性转移性肾细胞癌（RCC）患者中启动了一项使用非骨髓清除方法的异体干细胞移植的临床试验。同种异体移植物抗rcc效应的明确证据已被证实，在一些患者中观察到的大转移完全消退。最近我们在一个结肠癌患者身上发现了类似的效果。我们随后启动了研究，调查那些显示抗rcc作用的免疫重建，试图确定介导这些回归的效应细胞群及其靶抗原。今年，我们证实了少量组织相容性抗原在肾癌细胞表面的表达。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RCC细胞。我们还能够在两个应答的患者中扩增t细胞克隆，识别肿瘤细胞特异性或广泛表达的抗原，这些抗原存在于患者的造血细胞和RCC细胞上。这些观察结果首次揭示了免疫机制介导非清髓性同种异体移植后转移性癌症的消退。我们也在寻求从健康的异体供体（体外）中开发“肿瘤靶向”t细胞克隆的方法，用于移植后的过继输注。扩增这些t细胞群的方法包括使用异体树突状细胞作为刺激物（由供体单核细胞产生），转染了编码癌症相关基因（即HTERT， PRAME, G250）的腺病毒，或电融合到患者肿瘤细胞中。使用一种新型的电融合装置，我们最近将树突/肿瘤融合的效率从2-3%提高到15 - 20%。这些“融合体”在体外刺激肿瘤特异性t细胞的能力将很快得到测试。这些树突状细胞扩大来自健康供体的肿瘤特异性t细胞群的能力是研究的中心焦点，并可能导致未来的靶向免疫治疗方案。这些研究是与国家癌症研究所泌尿肿瘤科和外科肿瘤科合作进行的。最近，我们在携带转移性RCC的宿主中开发了一种同种异体SCT小鼠模型，在该模型中，与自体移植相比，可重复的GVT效应延长了动物的生存期。移植后肿瘤疫苗接种研究将在该模型中进行，并研究PDGF、VEGF和EGF-R酪氨酸激酶抑制剂在移植后最初几个月抑制血管生成的影响。

The ability of allogeneic lymphocytes to target and eradicate leukemia cells has been established as a true biological entity over the past ten (10) 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. 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. This year we were able to confirm 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. 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. We are also pursing methods to develop 'tumor targeted' T-cell clones from healthy allogeneic donors (in vitro) for adoptive infusion posttransplant. Methods to expand these T-cell populations involve the use of allogeneic dendritic cells as stimulators (generated from donor monocytes) transfected with adenovirus encoding cancer associated genes (i.e., HTERT, PRAME, G250), or electrically fused to the patients tumor cells. Using a novel electrofusion device, we have recently improved our efficiency of dendritic/tumor fusions from 2-3% to consistently in the 15 to 20% range. The in vitro ability of these 'fusions' to stimulate tumor specific T-cell in vitro will shortly be tested. The ability of these dendritic cells to expand tumor specific T-cell populations from healthy donors is a central focus of investigation and potentially could lead to future targeted immunotherapy regimens. These studies are being conducted in collaboration with the Urology Oncology Branch and the Surgical Oncology Branch of the National Cancer Institute. 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 will be 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. Acute GVHD remains a major toxicity of allogeneic stem cell transplantation. Steroid refractory GVHD has a nearly uniform abysmal prognosis. This year, our group demonstrated that monoclonal antibodies to the alpha chain of the IL-2 receptor (anti-Tac) and to TNF-alpha can dramatically improve the outcome in patients with steroid refractory acute GVHD. One of the key componenets to the successful treatment of acute GVHD is early diagnosis and treatment. 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 2 investigational methods to rapidly and specifically diagnose acute GVHD. The first 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. The second method involves assesing the percentage of interferon-gamma positive T-cells in post-transplant patient PBL samples following co-culture with patient CD-40 ligand expanded B-cells. Preliminary testing of this approach is just now being initiated. Finally, we are conducting research to characterize genetic expression profile in T-lymphocytes collected from patients with acute GVHD. The technique involves collecting PBL from patients at the onset of acute GVHD, isolating pure T-cell population by FACS negative depletion, and analysis of gene expression profiles using using high-density cDNA microarrays. Our hope is to characterize potentially novel gene pathways involved in mediating GVHD so that targeted therapies for this disorder can be developed and tested. 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, 22 patients with SAA/PNH have been transplanted with a day 200 TRM of 0%.