Development of cancer immunogene therapy with gene-modified adenoviral vector re-targeted by tumor-specific peptides

CD40L是B细胞恶性肿瘤包括B-慢性淋巴细胞白血病（B- cll）免疫治疗的一个很好的候选分子，因为它可以增加恶性细胞呈递肿瘤抗原的能力。然而，操纵人类CD40L （hCD40L）分子表达的努力由于缺乏一致的基因转移到恶性靶细胞的相关问题而失败。我们已经开发了一种新的，高度可重复性的方法来诱导hCD40L表面在恶性B细胞上的表达。10个B-CLL样本与MRC-5成纤维细胞共培养，之前用编码hcd40l基因的腺病毒载体转导。共培养后，恶性细胞表面表达高水平的hCD40L、B7-1、B7-2和ICAM-1。hCD40L的转移不通过膜融合介导。转移的hCD40L功能完整，表达该分子的B-CLL细胞诱导自体外周血T淋巴细胞产生干扰素g增加。这种…More方法不使用任何直接的基因转移到原发性白血病细胞，可以很容易地扩大生产临床B-CLL疫苗。另一方面，为了提供体外基因治疗的细胞结合配体和体内药物和基因治疗的靶向B-CLL配体，我们通过筛选患者原代B-CLL细胞，从肽呈递噬菌体文库中选择了44个20-mer肽。选取29个肽进行细胞结合测定。选择的肽中有14个结合B- cll、B、T和单核细胞，6个仅结合B- cll和B细胞，1个仅结合B细胞。然而，8个选择的肽是B-CLL特异性的。当肽在噬菌体环境之外进行测试时，合成肽1-5能够在功能上重新靶向腺病毒载体，以增加体外基因传递到原代B-CLL细胞。这项工作还强调了患者肿瘤异质性对靶向肽鉴定及其在基因治疗载体靶向中的功能应用的重要性。我们正在计划这些方法的临床应用。少

CD4O ligand (CD40L) is a good candidate molecule for the immunotherapy of B cell malignancies including B-chronic lymphocytic leukemia (B-CLL), because it may increase the capacity of the malignant cells to present tumor antigens. However, efforts to manipulate expression of the human CD40L (hCD40L) molecule have foundered on problems associated with lack of consistent gene transfer into the malignant target cells. We have developed a new, highly reproducible method for inducing hCD40L surface expression on malignant B cells. Ten B-CLL samples were cocultured with MRC-5 fibroblasts previously transduced with an adenoviral vector encoding the hCD40Lgene. The malignant cells expressed high levels of surface hCD40L, B7-1, B7-2, and ICAM-1 after coculture. hCD40L transfer was not mediated, by membrane fusion. The transferred hCD40L was functionally intact and B-CLL cells expressing this molecule induced increased interferon-g production from autologous peripheral blood T lymphocytes. This … More approach does not use any direct gene transfer to primary leukemia cells and can readily be scaled up for prod uction of clinical B-CLL vaccines.On the other hand, to provide cell-binding ligands for ex vivo gene therapy and B-CLL-targeting ligands for in vivo drug and gene therapy, we have selected 44 20-mer peptides from peptide-presenting phage libraries by panning against patient primary B-CLL cells. 29 of the selected peptides were assayed for cell binding. Fourteen of the selected peptides bound B-CLL, B, T, and monocyte cells, six bound only B-CLL and B cells, and one peptide bound only B cells. However, eight of the selected peptides were B-CLL specific. When peptides were tested out of the context of phage, synthetic peptide 1-5 was able to functionally re-target adenoviral vectors for increased ex vivo gene delivery to primary B-CLL cells. This work also emphasizes the importance of patient to patient tumor heterogeneity for the identification of targeting peptides and their functional application for gene therapy vector targeting. We are now planning the clinical application with these methods. Less