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Engineering an all human component Antibody Enzyme Prodrug Therapy (ADEPT) to overcome its current clinical limitations.

机译：设计一种全人类成分的抗体酶前药疗法（ADEPT），以克服其当前的临床局限性。

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摘要

Immunogenicity caused by the use of non-human components in Antibody Directed Enzyme Prodrug Therapy (ADEPT) has limited its clinical application. To overcome this problem, we introduced rationally designed mutations in the substrate binding pocket of human purine nucleoside phosphorylase (hPNP) to change its substrate specificity. Our goal was to introduce minimal changes in the amino acid and structure of hPNP to develop a mutant enzyme, that unlike the wild type enzyme, accepts (deoxy)adenosine-based prodrugs as substrate. Amongst the different mutants of human PNP tested, a double mutant with amino acid substitutions Glu201Gln:Asn243Asp (hDM) is most efficient in cleaving the adenosine-based prodrugs, such as 2-fluoro-2'-deoxyadenosine (F-dAdo) to their corresponding cytotoxic drugs.;To target hDM to the tumor site, the enzyme was fused to either a human Anti-HER2/neu Peptide mimetic (AHNP) or to a human anti-HER2/ neu single chain Fv (C6MH3B1). Incubation of both fusion proteins, followed by wash to remove the unbound fraction resulted in a dose-dependent cytotoxicity with HER2/neu expressing cells in the presence of F-dAdo. Consistent with the greater affinity of C6MH3B1 for ECD HER2, cytotoxicity was seen at lower concentrations of hDM-C6MH3B1 than hDM-AHNP, making C6MH3B1 a better candidate for delivering hDM to tumor cells. The toxic drug is cytotoxic to tumor cells irrespective of their expression of tumor associated antigen or growth rate. This cytotoxicity should be restricted to the tumor microenvironment, since the endogenously expressed wild type enzyme does not use adenosine-based prodrugs as substrates.;We have successfully developed an ADEPT that is comprised of all human components (hDM-C6MH3B1). hDM, unlike the wild type enzyme is capable of cleaving the relatively non-toxic F-dAdo to its cytotoxic metabolite F-Ade, causing tumor cell apoptosis in vitro. The similarity of the structure of hDM with wild type hPNP, as well as our evaluation of possible MHCII binding peptides that result from the two introduced mutations or the fusion of the two proteins suggest that the likelihood that hDM-C6MH3B1 will elicit an immune response in human is small. We anticipate that targeting hDM to tumors followed by administration of F-dAdo will be cytotoxic to tumors, with minimal systemic toxicity and immunogenicity.
机译：在抗体定向酶前药治疗（ADEPT）中使用非人类成分引起的免疫原性限制了其临床应用。为了克服这个问题，我们在人嘌呤核苷磷酸化酶（hPNP）的底物结合口袋中引入了合理设计的突变，以改变其底物特异性。我们的目标是在hPNP的氨基酸和结构中引入最低限度的变化，以开发出一种突变酶，该酶不同于野生型酶，接受基于（脱氧）腺苷的前药作为底物。在测试的人类PNP的不同突变体中，具有氨基酸取代Glu201Gln：Asn243Asp（hDM）的双突变体最有效地裂解基于腺苷的前药，例如2-fluoro-2'-deoxyadenosine（F-dAdo）。为了将hDM靶向肿瘤部位，将酶与人抗HER2 / neu肽模拟物（AHNP）或人抗HER2 / neu单链Fv（C6MH3B1）融合。两种融合蛋白的孵育，然后洗涤以去除未结合的部分，导致在F-dAdo存在下，HER2 / neu表达细胞的剂量依赖性细胞毒性。与C6MH3B1对ECD HER2的更大亲和力一致，在hDM-C6MH3B1浓度低于hDM-AHNP的情况下，观察到了细胞毒性，这使C6MH3B1成为将hDM递送至肿瘤细胞的更好候选者。不管肿瘤相关抗原的表达或生长速率如何，该毒性药物对肿瘤细胞都具有细胞毒性。这种细胞毒性作用应仅限于肿瘤微环境，因为内源性表达的野生型酶不使用基于腺苷的前药作为底物。我们已经成功开发了由所有人类成分（hDM-C6MH3B1）组成的ADEPT。与野生型酶不同，hDM能够将相对无毒的F-dAdo裂解为其细胞毒性代谢物F-Ade，从而在体外引起肿瘤细胞凋亡。 hDM与野生型hPNP的结构相似性，以及我们对由两个引入的突变或两种蛋白融合产生的可能的MHCII结合肽的评估表明，hDM-C6MH3B1可能会在小鼠体内引发免疫反应。人是小。我们预计将hDM靶向肿瘤，然后给予F-dAdo将对肿瘤具有细胞毒性，同时具有最小的全身毒性和免疫原性。

著录项

  • 作者

    Afshar, Sepideh.;

  • 作者单位

    University of California, Los Angeles.;

  • 授予单位 University of California, Los Angeles.;
  • 学科 Chemistry Biochemistry.;Chemistry Pharmaceutical.;Health Sciences Immunology.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 126 p.
  • 总页数 126
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;药物化学;预防医学、卫生学;
  • 关键词

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