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与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较高的临床活性，但对其他表达cd22的恶性肿瘤患者的活性较低。我们最近证明，免疫毒素的细胞毒性活性取决于细胞暴露的时间；因此，当临床使用的大剂量连续给药时，它们在体内的疗效大大增加。紫杉醇本身在体内对淋巴瘤的抑制作用很小，但它能使免疫毒素丸剂的抗淋巴瘤活性提高2倍，连续给药免疫毒素的抗淋巴瘤活性可达100倍。在此基础上，我们新开发了靶向cd22的“十二指肠毒素”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系具有增强的活性。在体内，大剂量的LMIT-2-DM1比免疫毒素或仅使用dm1的对照更有效。持续输注LMIT-2-DM1的疗效尚未得到检验。在此建议的支持下，我们的目标是生产一个放大的LMIT-2-DM1来测试腹腔内植入渗透泵持续输注的效果。此外，多重毒素的体外细胞死亡机制将被生化确定，多重毒素将在使用淋巴瘤细胞系和来自患者的原发性ALL细胞的其他异种移植模型中进行测试。目前，紫杉醇增强体内免疫毒素的机制尚不清楚。由于多毒素同时将两种药物递送到靶细胞，基于RNA深度测序方法的小鼠骨髓分选细胞被用于阐明体内特异性药物协同作用背后的异常机制。靶向cd22的多重毒素将两种高度协同的药物直接递送到癌细胞，而不影响健康组织，这可能成为复发/难治性b细胞恶性肿瘤患者的一种强有力的治疗选择。

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.