Webb K S, Ware J L, Parks S F, Briner W H, Paulson D F
Cancer Immunol Immunother. 1983;14(3):155-66. doi: 10.1007/BF00205354.
Mouse monoclonal antibodies alpha Pro3 and alpha Pro5 bind to different epitopes on an antigen (p54) of 54 kD reduced and 175 kD nonreduced MW. p54 antigen has been characterized previously with regard to tissue distribution using alpha Pro3 monoclonal antibody; the p54 antigen is present in substantially greater quantities in malignant prostatic tissue extracts than in benign prostatic and nonmalignant nonurogenital tissue extracts. In this report, we have established that alpha Pro5 and alpha Pro3 monoclonal antibodies exhibit same molecule-different epitope recognition. That both antibodies recognize the same molecular entity has been established by partial physiochemical characterization of the antigen recognized by the two antibodies and by sequential immunoprecipitation experiments. Different determinant recognition was established by lack of competitive surface binding between alpha Pro3 and alpha Pro5 to a prostatic carcinoma cell line. The p54 antigen can be labeled with glucosamine and immunoprecipitated from urea-solubilized membrane proteins; however, p54 cannot be detected by immunoprecipitation in a glycosylated form in spent culture medium removed from glucosamine-labeled cells. Experiments using indirect cellular immunoassays and directly radioiodinated monoclonal antibody have shown that both alpha Pro3 and alpha Pro5 form stable complexes with p54 antigen on the prostatic carcinoma cell surface. To the extent that modulation occurs upon interaction of p54 with alpha Pro3 and alpha Pro5; endocytosis of the immune complex appears to be the primary route of modulation. Furthermore, modulation by endocytosis is more intense when alpha Pro3 and alpha Pro5 are used in combination than when either monoclonal antibody is used alone. Although in vivo biologic behavior does not invariably correlate with in vitro behavior, careful in vitro analysis of monoclonal antibodies with respect to cell surface behavior, nevertheless, should precede in vivo evaluation. The data presented in this report indicate that preliminary in vitro analyses will expedite the effectiveness of in vivo immunotherapeutic trials; preliminary in vitro evaluations are absolutely essential if monoclonal-toxic agent (e.g., ricin A) conjugates, which must be internalized by the tumor cell to achieve cytotoxicity, are employed as immunotherapeutic agents.
小鼠单克隆抗体α Pro3和α Pro5与一种分子量为54 kD（还原型）和175 kD（非还原型）的抗原（p54）上的不同表位结合。先前已使用α Pro3单克隆抗体对p54抗原的组织分布进行了表征；p54抗原在恶性前列腺组织提取物中的含量比在良性前列腺组织和非恶性非泌尿生殖组织提取物中要多得多。在本报告中，我们已证实α Pro5和α Pro3单克隆抗体表现出相同分子 - 不同表位的识别特性。通过对两种抗体所识别抗原的部分物理化学特性分析以及连续免疫沉淀实验，已确定两种抗体识别的是同一分子实体。通过α Pro3和α Pro5对前列腺癌细胞系缺乏竞争性表面结合，确定了不同的决定簇识别特性。p54抗原可用葡糖胺标记，并从尿素溶解的膜蛋白中免疫沉淀出来；然而，在从葡糖胺标记细胞中去除的培养液中，无法通过免疫沉淀检测到糖基化形式的p54。使用间接细胞免疫测定和直接放射性碘化单克隆抗体的实验表明，α Pro3和α Pro5都能与前列腺癌细胞表面的p54抗原形成稳定复合物。就p54与α Pro3和α Pro5相互作用时发生的调节作用而言；免疫复合物的内吞作用似乎是调节的主要途径。此外，当α Pro3和α Pro5联合使用时，通过内吞作用的调节比单独使用任何一种单克隆抗体时更为强烈。虽然体内生物学行为并不总是与体外行为相关，但在进行体内评估之前，对单克隆抗体的细胞表面行为进行仔细的体外分析仍然是必要的。本报告中的数据表明，初步的体外分析将加快体内免疫治疗试验的有效性；如果使用必须被肿瘤细胞内化才能实现细胞毒性的单克隆 - 毒素剂（例如蓖麻毒素A）缀合物作为免疫治疗剂，那么初步的体外评估绝对是必不可少的。
