Antibody-targeted duotoxins combining Pseudomonas exotoxin and mertansine (DM1) for the treatment of B-cell malignancies

免疫毒素是抗体片段和假单胞菌外毒素的融合蛋白。CD22靶向免疫毒素对毛细胞白血病具有很高的临床活性，但在其他CD22表达的恶性肿瘤患者中活性较低。我们最近证明，免疫毒素的细胞毒活性依赖于细胞暴露的时间；因此，当临床使用的团注剂量被换成连续给药时，它们在体内的有效性显著增加。紫杉醇本身对淋巴瘤的体内疗效很小，它将免疫毒素的剂量提高一倍，并连续给予免疫毒素，最高可提高100倍。基于这些结果，我们新近开发了以CD22为靶点的双毒素LMIT-2-DM1，它将假单胞菌外毒素(PE)和紫杉醇样小分子DM1结合在一个抗体片段上。LMIT-2-DM1在体外对选定的细胞株具有更好的活性。在体内，LMIT-2-DM1团注剂量比免疫毒素或仅DM1对照更有效。LMIT-2-DM1持续输注的有效性尚未得到测试。在这项提议的支持下，我们的目标是生产一种放大的LMIT-2-DM1，以测试通过腹膜内植入渗透泵持续输注的疗效。此外，将从生化角度建立多毒素的体外细胞死亡机制，并将使用淋巴瘤细胞系和患者的原代ALL母细胞在额外的异种移植模型中测试多毒素。目前，紫杉醇在体内增强免疫毒素的机制尚不清楚。由于十二毒素同时将两种药物输送到靶细胞，因此基于小鼠骨髓分选细胞的RNA深度测序方法被应用于阐明体内特殊药物协同作用背后的机制。CD22靶向杜毒素将两种高度协同的药物直接输送到癌细胞，同时健康组织幸免于难，可能成为复发/难治性B细胞恶性肿瘤患者的有效治疗选择。

Immunotoxins are fusion proteins of an antibody fragment and the Pseudomonas exotoxin. CD22-targeted immunotoxins have a high clinical activity against hairy cell leukemia but are substantially less active in patients with other CD22-expressing malignancies. We recently demonstrated that the cytotoxic activity of immunotoxins depends on the time that cells are exposed; thus, their efficacy in vivo increases substantially when the clinically used bolus dose is exchanged for continuous administration. Paclitaxel, which by itself has very little in vivo efficacy against lymphoma, enhances the anti-lymphoma activity of bolus doses of immunotoxins two-fold and continuously administered immunotoxin up to 100-fold. Based on these results we newly developed the CD22-targeted “Duotoxin” LMIT-2-DM1 which combines Pseudomonas exotoxin (PE) and the paclitaxel-like small molecule DM1 on one antibody fragment. LMIT-2-DM1 has improved activity against selected cell lines in vitro. In vivo, bolus doses of LMIT-2-DM1 are more efficacious than the immunotoxin or the DM1-only control. The efficacy of continuous infusion of LMIT-2-DM1 has not been tested yet.Supported by this proposal we aim to produce an up-scaled LMIT-2-DM1 to test the efficacy of continuous infusion by intraperitoneally implanted osmotic pumps. Furthermore, the in vitro cell death mechanism of duotoxins will be biochemically established and the duotoxins will be tested in additional xenograft models using lymphoma cell lines and primary ALL blasts from patients. Currently, the mechanism by which paclitaxel enhances immunotoxins in vivo is not understood. Because duotoxins simultaneously deliver both drugs to the target cells, RNA deep sequencing based approaches on sorted cells from the murine bone marrow are applied to clarify the mechanism behind the exceptional in vivo-specific drug synergy.The CD22-targeted duotoxins delivering two highly synergistic drugs directly to the cancer cells while healthy tissue is spared may become a powerful treatment option for patients with relapsed/refractory B-cell malignancies.