HA22 (R490A) is a recombinant immunotoxin with increased antitumor activity without an increase in animal toxicity
HA22 (R490A) is a recombinant immunotoxin with increased antitumor activity without an increase in animal toxicity
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DOI：
10.1158/1078-0432.ccr-04-1939
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发表时间：
2005-02-15
影响因子：
11.5
通讯作者：
Pastan, I
中科院分区：
文献类型：
--
作者：
Bang, S;Nagata, S;Pastan, I
目的：RFB 4（dsFv）-PE 38（BL 22）是一种含有抗CD 22（Fv）和截短的假单胞菌外毒素A融合的重组免疫毒素，其在嘌呤类似物耐药的毛细胞白血病患者中诱导高完全缓解率。HA 22是BL 22的突变体，其在重链CDR 3中具有突变，导致细胞毒性活性增加。实验设计：将免疫毒素HA 22催化结构域（111）的精氨酸突变为丙氨酸。生产纯化的免疫毒素，并在细胞培养物中检测细胞毒活性，在小鼠中检测抗肿瘤活性和非特异性毒性。结果：HA 22（R490 A）对几种CD 22阳性细胞系的细胞毒作用与HA 22相似，比HA 22高2倍。当静脉注射时，HA 22（R490 A）对小鼠CA 46肿瘤具有比HA 22更强的抗肿瘤活性。HA 22和HA 22（R490 A）具有相似的LD 50（相似于1.3 mg/kg）和相似的血浆半衰期。R490 A突变还提高了抗间皮素重组免疫毒素SS 1（dsFv）-PE 38（SS 1 P）的细胞毒性。体外ADP-核糖基化测定显示HA 22 R490 A具有增加的活性。增加的细胞毒活性可能与ADP-核糖基化活性的增加有关。结论：蛋白质工程可用于增加重组免疫毒素的效力。由于HA 22（R490 A）具有增加的抗肿瘤活性而不增加动物毒性，因此具有该突变的免疫毒素是临床开发的候选物。
Purpose: RFB4 (dsFv)-PE38 (BL22) is a recombinant immunotoxin containing an anti-CD22 (Fv) fused to truncated Pseudomonas exotoxin A, which induces a high complete remission rate in patients with purine analogue-resistant hairy cell leukemia. HA22 is a mutant of BL22 with mutations in heavy-chain CDR3 resulting in increased cytotoxic activity. Our goal was to improve the activity of HA22.Experimental Design: Arg, which is located in the catalytic domain (111) of the immunotoxin HA22, was mutated to alanine. Purified immunotoxins were produced and tested for cytotoxic activity in cell culture and for antitumor activity and nonspecific toxicity in mice. ADP-ribosylation activity was also measured.Results: HA22 (R490A) is similar to2-fold more cytotoxic than HA22 on several CD22-positive cell lines. When injected i.v., HA22 (R490A) has more potent antitumor activity than HA22 against CA46 tumors in mice. HA22 and HA22 (R490A) have similar LD50S (similar to1.3 mg/kg) and similar plasma half-lives. The R490A mutation also improved the cytotoxicity of the antimesothelin recombinant immunotoxin SS1 (dsFv)-PE38 (SS1P). In vitro ADP-ribosylation assays show that HA22 R490A has increased activity. Increased cytotoxic activity is probably related to this increase in ADP-ribosylation activity.Conclusion: Protein engineering can be used to increase the efficacy of recombinant immunotoxins. Because HA22 (R490A) has increased antitumor activity without increased animal toxicity, immunotoxins with this mutation are candidates for clinical development.