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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Therapeutic enzyme deimmunization by combinatorial T-cell epitope removal using neutral drift
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Therapeutic enzyme deimmunization by combinatorial T-cell epitope removal using neutral drift

机译：使用中性漂移通过组合性T细胞表位去除治疗性酶脱免疫

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摘要

A number of heterologous enzymes have been investigated for cancer treatment and other therapeutic applications; however, immunogenicity issues have limited their clinical utility. Here, a new approach has been created for heterologous enzyme deimmunization whereby combinatorial saturation mutagenesis is coupled with a screening strategy that capitalizes on the evolutionary biology concept of neutral drift, and combined with iterative computational prediction of T-cell epitopes to achieve extensive reengineering of a protein sequence for reduced MHC-Ⅱ binding propensity without affecting catalytic and pharmacological properties. Escherichia coli L-asparaginase Ⅱ (EcAll), the only nonhuman enzyme approved for repeated administration, is critical in treatment of childhood acute lymphoblastic leukemia (ALL), but elicits adverse antibody responses in a significant fraction of patients. The neutral drift screening of combinatorial saturation mutagenesis libraries at a total of 12 positions was used to isolate an EcAII variant containing eight amino acid substitutions within computationally predicted T-cell epitopes-of which four were nonconser-vative-while still exhibiting k_(cat)/K_M= 10~6 M~(-1) s~(-1) for L-Asn hydrolysis. fuerther, immunization of HLA-transgenic mice expressing the ALL-associated DRB1~*0401 allele with the engineered variant resulted in significantly reduced T-cell responses and a 10-fold reduction in anti-EcAII IgG titers relative to the existing therapeutic. This significant reduction in the immunogenicity of EcAII may be clinically relevant for ALL treatment and illustrates the potential of employing neutral drift screens to achieve large jumps in sequence space as may be required for the deimmunization of heterologous proteins.
机译：已经研究了许多用于癌症治疗和其他治疗应用的异源酶。但是，免疫原性问题限制了其临床实用性。在这里，已经为异源酶脱免疫创造了一种新方法，其中组合饱和诱变与利用中性漂移的进化生物学概念的筛选策略相结合，并与T细胞表位的迭代计算预测相结合，以实现对A细胞抗原表位的广泛改造。蛋白质序列可降低MHC-Ⅱ的结合倾向，而不影响催化和药理特性。大肠杆菌L-天冬酰胺酶Ⅱ（EcAll）是唯一被批准重复给药的非人类酶，对治疗儿童急性淋巴细胞白血病（ALL）至关重要，但在相当一部分患者中引起不良抗体反应。使用组合饱和诱变文库的总共12个位置的中性漂移筛选来分离EcAII变体，该变体在计算预测的T细胞表位内包含八个氨基酸取代-其中四个是非保守的，同时仍表现出k_（cat）对于L-Asn水解，/ K_M = 10〜6 M〜（-1）s〜（-1）。此外，用工程改造的变体免疫表达ALL相关DRB1〜* 0401等位基因的HLA转基因小鼠，相对于现有治疗剂，可显着降低T细胞反应，并使抗EcAII IgG滴度降低10倍。 EcAII免疫原性的这种显着降低可能在临床上与ALL治疗有关，并说明了采用中性漂移筛选以实现异源蛋白质脱免疫所需的序列空间大跳跃的潜力。

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  • 作者

    Jason R. Cantor; Tae Hyeon Yoo; Aakanksha Dixit; Brent L. lverson; Thomas G. Forsthuber; George Georgiou;

  • 作者单位

    Department of Chemical Engineering, University of Texas, Austin, TX 78712;

    Department of Chemical Engineering, University of Texas, Austin, TX 78712;

    Department of Biology, University of Texas, San Antonio, TX 78249;

    Department of Chemistry and Biochemistry, University of Texas, Austin, TX 78712;

    Department of Biology, University of Texas, San Antonio, TX 78249;

    Department of Chemical Engineering, University of Texas, Austin, TX 78712,lnstitute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712,Section of Molecular Genetics and Microbiology, University of Texas, Austin, TX 78712,Department of Biomedical Engineering, University of Texas, Austin, TX 78712;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    directed evolution; protein engineering;

    机译：定向进化;蛋白质工程;

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