Basic study of anti-CD3 X anti-tumor bispecific antibody therapy using helper/killer cells for malignant hemangioentothelioma.

LAK过继免疫治疗是公认的治疗恶性血管内皮瘤(MHE)的唯一有效方法。为了推进这一治疗，我们回顾了利用辅助/杀伤细胞进行双特异性抗体(Ab)治疗的基础研究。我们建立了人恶性内皮细胞系(ISO-Has)作为肿瘤抗原(Ag)，以制备单抗(McAb)，并作为细胞毒检测的靶细胞。它起源于头皮上的MHE，表达多种癌基因和突变的隐性癌基因。应用ISO-HAS，我们获得了一些对ISO-HAS和正常内皮细胞(EC)都有反应的抗体，但没有针对ISO-HAS的特异性抗体。在rIL-2和抗CD3抗体的双重刺激下，我们培养出了CD4~+杀伤细胞。需要用固定化抗CD3单抗间断刺激才能大量培养。采用RT-PCR方法观察IL-2和γ-干扰素在CD4~+杀伤细胞中的表达。这些细胞在高E/T比(100/1)下对ISO-HAS显示出约70%的细胞毒作用。我们的结果表明，MHE的肿瘤特异性抗原太弱，不能制造单抗。此外，为了在低E/T比值下表现出高的细胞毒活性，CD4~+杀伤细胞需要一种桥联抗体来与肿瘤细胞接触。因此，我们现在正在用抗CD3和CD31的ABS制作一种双特异性抗体，它保存在MHE和正常的EC上。我们将提交最终报告，包括我们使用这种双特异性抗体的研究结果。

LAK adoptive immunotherapy is recognized as the one and the only effective therapy for malignant hemangioendothelioma (MHE) that is an extremely fatal neoplasm. To advance this therapy, we examined basic studies of bispecific antibody (Ab) therapy using helper/killer cells.We established a human malignant endothelial cell line (ISO-HAS) as tumor antigen (Ag) to make monoclonal antibody (monoAb) and as target cell for cytotoxic assay. It was derived from MHE arising on the scalp and expressed several oncogenes and mutated suppessive oncogenes. Using ISO-HAS,we obtained some Abs responsive to both ISO-HAS and normal endothelial cells (EC), but no specific Ab against ISO-HAS.CD4^+T cells were separated from PBMC by magnetic cell separation system (MACS). We cultured CD4^+killer cells by bi-stimulation of rIL-2 and anti-CD3 Ab. Discontinuous stimulation by immobilized anti-CD3 Ab was needed to culture them massively. By RT-PCR methods, we observed the expression of mRNA of IL-2 and gamma IFN in CD4^+killer cells. These cells alone showed about 70% cytotoxicity against ISO-HAS in high E/T ratio (100/1).Our results suggested that tumor-specific Ag of MHE is too weak to make a monoAb. Moreover, to show high cytotoxic activity in low E/T ratio, CD4^+killer cells need a bridging Ab to make contact with tumor cells. Therefore, we are now making a bispecific Ab using ABs to CD3 and CD31 which is preserved on MHE as well as normal EC.We will present the final report including the results of our studies using this bispecific Ab.