Immunotherapy with Dendritic-Allogeneic Tumor Cells

描述（由申请人提供）：树突状细胞（dc）诱导原代T细胞反应，并被广泛评估为癌症免疫治疗中抗原（Ag）递送的载体。一种策略是利用DCs与肿瘤细胞融合产生的杂交细胞。从技术上讲，利用聚乙二醇（PEG）或病毒等化学聚变原进行核聚变一直受到效率低、毒性强和可重复性差的困扰。我们最近开发了一种电融合方案，用它可以产生高融合率的异核体。在临床前研究中，融合细胞刺激IFN？CD4和CD8免疫T细胞分泌，对于免疫治疗，单次接种可导致肿瘤消退。基于这些观察，我们最近设计了一项临床试验，通过疫苗融合自体dc和异体黑色素瘤细胞系（PMCV或CanvaxinTU）治疗转移性黑色素瘤。在这个方案中，3个选择的异体肿瘤细胞提供了肿瘤相关Ags的来源。然而，很少有实验室分析集中于确定这种方法的免疫原性和潜在的治疗效果。由于同种异体肿瘤细胞对组织不相容的同种异体抗原的免疫反应，对肿瘤抗原的免疫反应可能下调或上调。异体肿瘤疫苗的作用机制有待进一步研究。本研究将利用两种动物肿瘤系统进行同种异体疫苗的研制。第一个系统将利用β -半乳糖苷酶（β -gal）作为替代肿瘤排斥Ag，该抗原已被引入B16/F10 （H-2b）黑色素瘤和4T1 （H-2d）乳腺癌。第二个系统由三种不同单倍型的小鼠黑色素瘤[B16 (H-2b), K1735 （H-2k）和cloudman S91 (H-2d)]组成，但共享许多天然肿瘤相关的Ags。这些异基因肿瘤将与同基因dc电熔。我们将分析它们的分子特性、免疫原性和治疗同基因肿瘤的效果。具体目的是：1)确定电融合产生的同种dc -异体肿瘤（alloo - dc -tumor）杂种的免疫原性和治疗效果；2)表征和分析肿瘤相关抗原和同种异体组织相容性抗原的免疫反应；3)主动免疫治疗晚期肿瘤；4)用同种异体dc -肿瘤杂交细胞免疫小鼠产生肿瘤免疫T细胞进行过继免疫治疗。

DESCRIPTION (provided by applicant): Dendritic cells (DCs) induce primary T cell responses and are being extensively evaluated as vehicles for antigen (Ag) delivery in cancer immunotherapy. One strategy employs the use of hybrid cells generated by fusing DCs with tumor cells. Technically, fusion using chemical fusogens such as polyethylene glycol (PEG) or viruses has been plagued by low efficiency, toxicity and poor reproducibility. We recently developed an electrofusion protocol with which heterokaryons are generated with high fusion rates. In pre-clinical studies, fusion cells stimulated IFN? secretion from both CD4 and CD8 immune T cells, and for immunotherapy, a single vaccination resulted in tumor regression. Based on these observations, we have recently designed a clinical trial for the treatment of metastatic melanoma by vaccination with fusion of autologous DCs and allogeneic melanoma cell lines (PMCV or CanvaxinTU). In this protocol, 3 selected allogeneic tumor cells provide a source of tumor-associated Ags. However, little laboratory analysis has focused on defining the immunogenicity and potential therapeutic effects of this approach. Because of the immune response to histo-incompatible allo-Ags on allogeneic tumor cells, immune responses to tumor Ags may be subjected to down- or up-regulation. The mechanisms of allogeneic tumor vaccine need to be analyzed. In this proposal, two animal tumor systems will be used for the development of allogeneic vaccine. The first system will utilize beta-galactosidase (beta-gal) as a surrogate tumor rejection Ag that has been introduced into the B16/F10 (H-2b) melanoma and 4T1 (H-2d) breast carcinoma. The second system consists of three murine melanomas [B16 (H-2b), K1735 (H-2k) and cloudman S91 (H-2d)] of different haplotypes but sharing a number of natural tumor-associated Ags. These allogeneic tumors will be electrofused with syngeneic DCs. Their molecular characteristics, immunogenicities, and therapeutic effects will be analyzed against syngeneic tumors. The specific aims are: 1) To determine the immunogenicity and therapeutic efficacy of syngeneic DC-allogeneic tumor (allo-DC-tumor) hybrids generated by electrofusion; 2) To characterize and analyze immune responses to tumor-associated Ags and to allogeneic histocompatibility Ags; 3) Active immunotherapy against advanced tumors; and 4) To generate tumor-immune T cells from mice immunized with allo-DC-tumor hybrid cells for adoptive immunotherapy.