针对B细胞恶性肿瘤独特型的基于抗体的治疗药物的研发。

Generation of antibody-based therapeutics targeting the Idiotype of B-cell Malignancies.

作者信息

Weiss Emily, Sarnovsky Robert, Ho Mitchell, Arons Evgeny, Kreitman Robert, Angelus Evan, Antignani Antonella, FitzGerald David

机构信息

Biotherapy Section, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health; 9000 Rockville Pike, 37/5124 Bethesda, MD 20892.

出版信息

Antib Ther. 2019 Jan;2(1):1-10. doi: 10.1093/abt/tby012. Epub 2018 Dec 27.

DOI:10.1093/abt/tby012

PMID:30801054

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6383771/

Abstract

BACKGROUND

A feature of many B-cell tumors is a surface-expressed immunoglobulin (sIg). The complementarity determiningregions (CDRs)of the sIg, termed the 'idiotype', are unique to each tumor. We report on a phage selection strategy to generate anti-idiotype therapeutics that react with sIg CDR3 sequences: the MEC1 B-cell tumor line was used as proof of concept.

METHODS

To create a mimetic of the MEC1 idiotype, CDR3 sequences from heavy and light chains of the sIg were grafted into a scFv framework scaffold. Using the Tomlinson I phage library of human scFvs, we enriched for binders to MEC1 CDR3 sequences over unrelated CDR3 sequences.

RESULTS

By ELISA we identified 10 binder phage. Of these, five were sequenced, found to be unique and characterized further. By flow cytometry each of the five phage bound to MEC1 cells, albeit with different patterns of reactivity. To establish specificity of binding and utility, the scFv sequences from two of these binders (phage 1, and 7) were converted into antibody-toxin fusion proteins (immunotoxins) and also cloned into a human IgG1 expression vector. Binder-1 and -7 immunotoxins exhibited specific killing of MEC1 cells with little toxicity for non-target B-cell lines. The full-length antibody recreated from the binder-1 scFv so exhibited specific binding.

CONCLUSION

Our results establish the utility of using engrafted CDR3 sequences for selecting phage that recognize the idiotype of B-cell tumors.

摘要

背景

许多B细胞肿瘤的一个特征是表面表达免疫球蛋白（sIg）。sIg的互补决定区（CDR），即所谓的“独特型”，对每个肿瘤来说都是独特的。我们报告了一种噬菌体筛选策略，用于生成与sIg CDR3序列反应的抗独特型治疗剂：使用MEC1 B细胞肿瘤系作为概念验证。

方法

为了创建MEC1独特型的模拟物，将sIg重链和轻链的CDR3序列嫁接到单链抗体（scFv）框架支架中。利用人类scFv的Tomlinson I噬菌体文库，我们富集了与MEC1 CDR3序列结合的噬菌体，而非与无关CDR3序列结合的噬菌体。

结果

通过酶联免疫吸附测定（ELISA），我们鉴定出10种结合噬菌体。其中5种进行了测序，发现是独特的，并进一步进行了表征。通过流式细胞术，这5种噬菌体中的每一种都与MEC1细胞结合，尽管反应模式不同。为了确定结合的特异性和实用性，将其中两种结合噬菌体（噬菌体1和7）的scFv序列转化为抗体-毒素融合蛋白（免疫毒素），并克隆到人类IgG1表达载体中。结合噬菌体1和7的免疫毒素对MEC1细胞表现出特异性杀伤作用，对非靶B细胞系几乎没有毒性。从结合噬菌体1的scFv重建的全长抗体也表现出特异性结合。