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%.
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