Mezzanzanica D, Canevari S, Ménard S, Pupa S M, Tagliabue E, Lanzavecchia A, Colnaghi M I
Division of Experimental Oncology E, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy.
Int J Cancer. 1988 Apr 15;41(4):609-15. doi: 10.1002/ijc.2910410422.
In the perspective of in vivo therapeutic applications, the monoclonal antibody (MAb) MOv18 was selected for its restricted reactivity with human ovarian carcinoma. Using the pH 2.8 desorption assay, we found that the antigen recognized by MOv18 had a high stability on the cell membrane and poor internalization. Therefore, a therapeutic approach which does not require internalization, i.e., the re-targeting of cytotoxic T lymphocytes (CTL) by bispecific MAbs, was investigated. MOv18 and anti-CD3 MAbs were used to produce bispecific reagents, obtained either by chemical cross-linkage (hetero-conjugates) or by somatic hybridization techniques (hybrid MAbs). The maintenance of the binding reactivity and specificity of the bispecific MAbs was analyzed by solid-phase radioimmunoassay, immunofluorescence and cross-competition tests on the relevant target cells (ovarian carcinoma cell line OVCA 432 for MOv18 and PHA-stimulated peripheral blood mononuclear cells for anti-CD3 MAbs), and on 2 irrelevant tumor cell lines. Bv a 51Cr-release assay the bispecific MAbs were found to efficiently promote, at picomolar concentration, cell lysis by CTL clones, but the specificity pattern was wider than that predicted by the binding studies. The F(ab')2 fragment of one hybrid MAb mediated a lysis which was just as efficient as the entire MAb on the relevant target cells and allowed specific lysis to be distinguished from Fc-receptor-mediated lysis. Human immunoglobulins were unable to compete with the Fc receptor binding of the hybrid MAbs and therefore, in the perspective of in vivo applications, Fc fragment removal seems to be an essential step. Analysis of the bispecific reagents indicated that hybrid MAbs are superior to the heteroconjugate as far as storage stability is concerned.
从体内治疗应用的角度来看，单克隆抗体（MAb）MOv18因其与人卵巢癌的反应性受限而被选中。使用pH 2.8解吸试验，我们发现MOv18识别的抗原在细胞膜上具有高稳定性且内化能力差。因此，研究了一种不需要内化的治疗方法，即通过双特异性单克隆抗体对细胞毒性T淋巴细胞（CTL）进行重新靶向。使用MOv18和抗CD3单克隆抗体来制备双特异性试剂，这些试剂可以通过化学交联（异源缀合物）或体细胞杂交技术（杂交单克隆抗体）获得。通过固相放射免疫测定、免疫荧光以及对相关靶细胞（针对MOv18的卵巢癌细胞系OVCA 432和针对抗CD3单克隆抗体的PHA刺激的外周血单核细胞）以及两种无关肿瘤细胞系进行交叉竞争试验，分析双特异性单克隆抗体的结合反应性和特异性的维持情况。通过51Cr释放试验发现，双特异性单克隆抗体在皮摩尔浓度下能有效促进CTL克隆介导的细胞裂解，但特异性模式比结合研究预测的更广泛。一种杂交单克隆抗体的F(ab')2片段介导的裂解在相关靶细胞上与完整单克隆抗体一样有效，并且能够将特异性裂解与Fc受体介导的裂解区分开来。人免疫球蛋白无法与杂交单克隆抗体的Fc受体结合竞争，因此，从体内应用的角度来看，去除Fc片段似乎是一个关键步骤。对双特异性试剂的分析表明，就储存稳定性而言，杂交单克隆抗体优于异源缀合物。
