Bioengineering a Unique Deimmunized Bispecific Targeted Toxin That Simultaneously Recognizes Human CD22 and CD19 Receptors in a Mouse Model of B-Cell Metastases
Bioengineering a Unique Deimmunized Bispecific Targeted Toxin That Simultaneously Recognizes Human CD22 and CD19 Receptors in a Mouse Model of B-Cell Metastases
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DOI：
10.1158/1535-7163.mct-10-0203
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发表时间：
2010-06-01
影响因子：
5.7
通讯作者：
Frankel, Arthur E.
中科院分区：
文献类型：
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作者：
Vallera, Daniel A.;Oh, Seunguk;Frankel, Arthur E.
需要一种高效且免疫原性降低的药物来开发一种靶向生物药物，当全身注射时可以穿透恶性B细胞。因此，一种新型的去免疫双特异性配体定向毒素通过双高亲和力单链Fvs （scFv）与带有KDEL cooh末端的PE38拼接而成。目的是通过诱变降低毒素的免疫原性，测量突变药物引起抗毒素抗体反应的能力，并证明突变药物对体内系统性b细胞淋巴瘤有效。将人抗cd22 scFv和抗cd19 scFv克隆到具有截断假单胞菌外毒素（PE38）的同一单链分子上，以制造药物。位点特异性诱变用于突变7个关键表位毒素区域的氨基酸，这些区域决定了b细胞产生中和抗毒素抗体。生物测定法测定突变是否降低效力，elisa法测定抗毒素抗体是否降低。最后，使用了一种功能强大的转基因荧光素酶异种移植模型，该模型可以实时成像，以确定对系统性恶性人b细胞淋巴瘤Raji-luc的影响。B系急性淋巴细胞白血病、B细胞慢性淋巴细胞白血病和B淋巴瘤患者CD22和CD19表达较高。2219KDEL7mut对小鼠的系统性Raji-luc有效，并能阻止转移扩散。诱变使中和抗毒素抗体减少了80%，而体外或体内活性没有明显损失。由于2219KDEL7mut的免疫原性显著降低，且药物在体内非常有效，我们现在可以在未来的临床试验中使用靶向毒素进行多种药物治疗。巨蟹座；9 (6);1872 - 83。AACR (C) 2010。
A drug of high potency and reduced immunogenicity is needed to develop a targeted biological drug that when injected systemically can penetrate to malignant B cells. Therefore, a novel deimmunized bispecific ligand-directed toxin targeted by dual high-affinity single-chain Fvs (scFv) spliced to PE38 with a KDEL COOH-terminus was genetically engineered. The aims were to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit antitoxin antibody responses, and show that mutated drug was effective against systemic B-cell lymphoma in vivo. Both human anti-CD22 scFv and anti-CD19 scFv were cloned onto the same single-chain molecule with truncated pseudomonas exotoxin (PE38) to create the drug. Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISAs were done to determine whether antitoxin antibodies were reduced. Finally, a powerful genetically altered luciferase xenograft model was used that could be imaged in real time to determine the effect on systemic malignant human B-cell lymphoma, Raji-luc. Patient B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B lymphoma were high in CD22 and CD19 expression. 2219KDEL7mut was significantly effective against systemic Raji-luc in mice and prevented metastatic spread. Mutagenesis reduced neutralizing antitoxin antibodies by similar to 80% with no apparent loss in in vitro or in vivo activity. Because 2219KDEL7mut immunogenicity was significantly reduced and the drug was highly effective in vivo, we can now give multiple drug treatments with targeted toxins in future clinical trials. Mol Cancer Ther; 9(6); 1872-83. (C)2010 AACR.