Allogeneic Immunotherapy for cancer and nonmalignant hematological disorders

在过去的25年里，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。直到最近，这种作用是否能对非血液学“实体”恶性肿瘤产生作用，在很大程度上仍未被探索。大约10年前，我们启动了第一个研究同种异体干细胞移植治疗难治性转移性肾细胞癌（RCC）患者的临床试验。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。我们随后进行了研究，调查那些显示抗rcc作用的免疫重建，试图确定介导这些退化的效应细胞群及其靶抗原。我们实验室已经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细胞克隆能够杀死移植后具有GVT效应的患者的RCC细胞。

The ability of allogeneic lymphocytes to target and eradicate leukemia cells has been established as a true biological entity over the past 25 years. Whether such effects can be generated against non-hematological "solid" malignancies until recently remained largely unexplored. Approximately 10 years ago, we initiated the first clinical trail investigating 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. We have subsequently conducted 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 have recently identified a novel tumor antigen derived from a human endogenous retrovirus over-expressed in RCC cells called HERV-E CT-RCC. This antigen is not expressed on normal tissues and therefore could potentially serve as a target for a future kidney cancer vaccine. Work to identify other antigens derived from this HERV-E that are expressed on common HLA molecules as well as efforts to develop a monoclonal antibody that recognizes HERV-E derived proteins selective it expressed in RCC are ongoing. Finally, we have also sought to characterize the mechanisms accounting for selective expression of the CT-RCC HERV-E in RCC. Recent data shows the VHL tumor suppressor gene regulates expression of this newly identified tumor antigen, potentially accounting for selective expression of the CT-RCC HERV-E in the clear cell variant of RCC. We have also 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 angiogenesis in the first few months of transplantation using VEGFR tyrosine kinase inhibitors. We have also explored whether adoptive transfer of donor NK cells can be used to bolster GVT effects against cancer after allogeneic HCT. Our group has shown that KIR incompatible NK cells are cytotoxic to solid tumor cells in vitro. Using the above mentioned animal model, we have shown that a single infusion of alloreactive NK cells can significantly reduce GVHD and prolong survival in mice with RCC undergoing allogeneic HCT. Based on these findings, we plan to evaluate whether GVT effects against RCC can be enhanced after allogeneic HCT by adoptively infusing donor NK cells. We are also exploring methods to sensitize solid tumors to NK cell attack by altering the phenotype of tumor cells through targeted gene induction. Recently we showed that bortezomib and depsipeptide sensitize tumors to NK cell cytotoxity by enhancing NK-cell mediated TRAIL killing. This sensitization appears to overcome NK inhibition that is mediated through KIR-KIR ligand interactions. We have also 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 were awarded a bench to bedside award in 2006 to scale up our NK cell expansions under GMP to test whether bortezomib could be used to sensitize patient's tumors to adoptive autologous NK cell infusions. We now have developed a method to expand human NK cells by 100 to 1000 fold in bags using GMP conditions. A phase I study entitled Safety and the anti- tumor effects of escalating doses of adoptively infused ex vivo expanded autologous natural killer cells against metastatic cancers or hematological malignancies sensitized to NK TRAIL cytotoxicity with Bortezomib was initiated last year and is currently accruing patients. 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 nonmyeloablative stem cell transplantation. 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, 55 patients with SAA/PNH have been transplanted with a day 100 TRM of 0% and with a sustained donor engraftment rate of 100%. Finally, the inability to find a suitable HLA matched donor limits the application of allogeneic transplantation to only a minority of patients with severe aplastic anemia or RA MDS. For such patients, transplantation using unrelated cord blood (UCB) has been shown to be a reasonable alternative transplant strategy. The major disadvantage of UCB transplantation in adults is the limited number of nucleated cells contained within the cord unit resulting in prolonged neutropenia and failure of engraftment which contributes to infection and TRM. In order to harness the advantage of UCB availability and to overcome the disadvantage of delayed neutrophil recovery, we have recently initiated a clinical trial that evaluates whether co-administration of unrelated umbilical cord blood and a relatively low number of highly purified haploidentical peripheral blood CD34+ cells from a related donor could be used as a method to promote rapid neutrophil recovery. This research protocol is designed to evaluate the safety and effectiveness of co-infusion of unrelated umbilical cord blood and haploidentical CD34+ cells from a related donor following nonmyeloablative conditioning for neutropenic patients with SAA or MDS (RA) that has proven to be refractory to immunosuppressive therapy. Subjects receive a novel non-myeloablative immunosuppressive conditioning regimen of cyclophosphamide, fludarabine, horse ATG and one dose of total body irradiation (200cGy) followed by an infusion of the allografts. The haploidentical stem cell product will be T-cell depleted and enriched for CD34+ cells using the Miltenyi CliniMacs system. To reduce TRM secondary to prolonged neutropenia associated with conventional UCB transplantation, haploidentical CD34+ stem cells will be co-infused with a single UCB unit (serologically matched at d 4/6 HLA loci). The primary endpoint will be cord engraftment (persistent cord derived ANC > 500 cells/ul) by day 42. If successful, this strategy could be used to substantially shorten the prolonged period of neutropenia associated with conventional cord blood transplantation, which potentially could result in a reduction in transplant related morbidity and mortality associated with infection. This study is currently open and is actively accruing patients. Finally, our group is actively involved in preclinical research investigating methods to expand viral reactive T-cells and NK cells from umbilical cord blood units using a device invented in our lab in collaboration with Herb Cullis that selectively accesses cryopreserved cord units.