SPECIFIC MONOCLONAL ANTIBODIES AGAINST GLIOMA AND MEDULLOBLASTOMA

Ib项目的中心主题是世代， 确定型单抗的鉴定及应用 体外和体内抗胶质瘤活性的(MAb)试剂 VIVO设置。 设施Ia1、Ib1和Ic1、IIb和IVb，项目Ib具有AS 具体目的：1.抗表皮生长因子的产生 受体(EGFR)小鼠单抗与A431的比较 鳞状细胞癌细胞系EGFR和胶质瘤表达的EGFR 在体外使用(免疫组织学、生长控制研究)和 体内(定位，模型免疫治疗)应用。使用 EGFR的来源；不同的免疫方案 纯化的A431细胞膜制剂，凝胶分离170kD 将进行EGFR富集带。最优化方法(S) 2.单抗81C6细胞外基质抗原的分析 Tenascin(以前的GMEM)在遗传、表位、分子和 功能水平，以及对Tenascin AS使用的扩展研究 可本地化的目标是最先进和最广泛的目标 Ib项目。鉴于我们已建立的单抗81C6亲和力程序 Tenascin的纯化，我们将用酶法进行亲和纯化 与抗Tenascin试剂反应的裂解的Tenascin片段， 包括多价兔和猕猴血清，小鼠单抗，以及 免疫治疗患者的血清样本。反应性 多肽将接受表位限制分析，氨基酸 测序，并作为免疫原用于直接免疫与 胎儿脑胶质瘤表达受限。来自的表达克隆 使用 项目IIb，限制性表位单抗的区分能力 他们的碎片将在皮下和皮下进行评估 颅内异种移植物的定位研究。3.三个新的胶质瘤- 相关的可定位抗原C12、D12和E9将是 成功后为潜在的体内应用而开发 Tenascin方案：抗原的亲和纯化、生产 第二代单抗和用于定位的单抗片段 学习。4.这个实验室最近获得的数据表明 神经胶质细胞与神经元分化的可能表型标志物 髓母细胞瘤的通路(MAb)；我们建议产生MAb以 由四个已建立的MED细胞系组成的独特的库保持在 与 项目IVb，这些研究将构成完成 髓母细胞瘤的表型研究与之平行 胶质瘤仍在进行中。

The central theme of Project Ib has been the generation, characterization and application of defined monoclonal antibody (Mab) reagents with anti-glioma reactivity in both in vitro and in vivo settings. Interacting with projects Ia, Ic, the core facilities Ia1, Ib1, and Ic1, IIb, and IVb, Project Ib has as specific aims: 1. Generation of anti-epidermal growth factor receptor (EGFR) murine Mabs vs. both the well-characterized A431 squamous cell carcinoma cell line EGFR and glioma-expressed EGFR for use in vitro (immunohistological, growth control studies) and in vivo (localizing, model immunotherapeutic) applications. With project Ic, Mabs will be prepared using the A431 cell line as source of EGFR; different immunization protocols incorporating trypsinized cells, combination protocols of cells and EGF-affinity purified A431 cell membrane preparations, and gel isolated 170 Kd EGFR-enriched bands will be performed. Optimal method(s) identified will be used to produce Mabs to glioma-associated EGFR. 2. Analysis of the Mab 81C6-defined extracellular matrix antigen tenascin (formerly GMEM) at the genetic, epitopic, molecular, and functional levels, and extended studies of the use of tenascin as a localizable target is the most advanced and extensive aim of Project Ib. Given our established procedures for Mab 81C6 affinity purification of tenascin, we will affinity purify enzymatically cleaved tenascin fragments reactive with anti-tenascin reagents, including polyvalent rabbit and macaque sera, murine Mabs, and serum samples obtained from immunotherapy patients. Reactive polypeptides will undergo epitope restriction analysis, amino acid sequencing, and serve as immunogens for directed immunization vs. epitopes of evolutionarily conserved, organotypic, and potentially fetal brain-glioma restricted expression. Expression clones from a lambdal gt 11 library produced by Dr. Mario Bourdon also will be screened and used to define the genomic profile of tenascin. With Project IIb, the differential capacities of restricted epitope Mabs and their fragments will be assessed in both subcutaneous and intracranial xenograft localization studies. 3. Three new glioma- associated, localizable antigens, C12, D12, and E9 will be developed for potential in vivo application following successful tenascin protocols: affinity purification of antigen, production of second generation Mabs, and Mab fragments for localization studies. 4. Recent data obtained in this laboratory suggest possible phenotypic markers for glial vs. neuronal differentiation pathways in medulloblastoma (Mab); we propose to generate Mabs to the unique bank of four established MED cell lines maintained at Duke, and a fifth available in xenograft form. In conjunction with project IVb, these studies will form the basis for complete phenotypic investigation of medulloblastoma parallel to that ongoing in glioma.