Vooijs W C, Post J, Wijdenes J, Schuurman H J, Bolognesi A, Polito L, Stirpe F, Bast E J, de Gast G C
Department of Hematology, University Hospital, Utrecht, The Netherlands.
Cancer Immunol Immunother. 1996 Jul;42(6):319-28. doi: 10.1007/s002620050289.
Immunotherapy based on the delivery of toxic agents to the tumor site using monoclonal antibodies (mAb) may be a promising modality in the treatment of hematological malignancies. In the selection of mAb, both for ex vivo but even more for in vivo therapy, not only their reactivity to the neoplastic cells should be considered, but also reactivity to other body constituents. Here we describe the screening of two human plasma-cell-reactive mAb B-B2 and B-B4, which may be used for immunotherapy of multiple myeloma. Cross-reactivity of B-B2 and B-B4 was determined by immunohistochemistry on a series of tissues. This revealed for both B-B2 and B-B4 a strong staining of epithelial cells in various organs, e.g. lung, liver, skin, kidney and gut, while only a weak and diffuse staining was seen with endothelial cells. In bone marrow reactivity was only found with plasma cells and not with hemopoietic precursors (CD34+ cells). Immunotoxins from B-B2 and B-B4 were constructed by coupling them to the plant-derived ribosome-inactivating protein saporin. Both B-B2 and B-B4 immunotoxins appeared to be efficient in specific inhibition of protein synthesis in plasma cell lines (IC50 respectively 1 nM and 0.1 nm). The immunotoxins were also tested on epithelial cell line A431, on liver cell line HepG2 and on human umbilical vein endothelial cells. The epithelial cell line A431 was reactive with both B-B2 and B-B4, but was only inhibited by B-B4 immunotoxin. Cell line HepG2 was reactive with both mAb, but was not inhibited by either immunotoxin. The endothelial cells showed no reactivity with B-B2 and B-B4 and were not inhibited by either immunotoxin. Bone marrow treated with B-B2 and B-B4 immunotoxin did not show a decrease in colonies of hemopoietic precursor cells. Incubation of multiple-myeloma-derived bone marrow with these immunotoxin resulted in a clear decrease of the number of plasma cells. From these data we conclude that B-B2 and B-B4 immunotoxin can be used for ex vivo bone marrow purging. Discrepancies were found between immunohistochemistry, binding assays and cytotoxicity assays with the mAb and the immunotoxin, which underlines the necessity for these various assays as a preclinical screening.
基于使用单克隆抗体（mAb）将毒性剂递送至肿瘤部位的免疫疗法可能是治疗血液系统恶性肿瘤的一种有前景的方法。在选择mAb时，无论是用于体外治疗还是更用于体内治疗，不仅应考虑其对肿瘤细胞的反应性，还应考虑其对身体其他成分的反应性。在此，我们描述了两种人浆细胞反应性mAb B-B2和B-B4的筛选，它们可用于多发性骨髓瘤的免疫治疗。通过对一系列组织进行免疫组织化学测定B-B2和B-B4的交叉反应性。这显示B-B2和B-B4在各种器官（如肺、肝、皮肤、肾和肠道）的上皮细胞中均有强染色，而在内皮细胞中仅见弱而弥漫的染色。在骨髓中，仅在浆细胞中发现反应性，而在造血前体细胞（CD34+细胞）中未发现反应性。通过将B-B2和B-B4与植物来源的核糖体失活蛋白皂草素偶联构建免疫毒素。B-B2和B-B4免疫毒素似乎都能有效特异性抑制浆细胞系中的蛋白质合成（IC50分别为1 nM和0.1 nM）。还对上皮细胞系A431、肝细胞系HepG2和人脐静脉内皮细胞进行了免疫毒素测试。上皮细胞系A431与B-B2和B-B4均有反应，但仅被B-B4免疫毒素抑制。细胞系HepG2与两种mAb均有反应，但未被任何一种免疫毒素抑制。内皮细胞与B-B2和B-B4无反应，也未被任何一种免疫毒素抑制。用B-B2和B-B4免疫毒素处理的骨髓中造血前体细胞集落未减少。用这些免疫毒素孵育多发性骨髓瘤来源的骨髓导致浆细胞数量明显减少。从这些数据我们得出结论，B-B2和B-B4免疫毒素可用于体外骨髓净化。在免疫组织化学、结合测定以及mAb和免疫毒素的细胞毒性测定之间发现了差异，这强调了这些各种测定作为临床前筛选的必要性。
