Directed Evolution of Stabilized Monomeric CD19 for Monovalent CAR Interaction Studies and Monitoring of CAR-T Cell Patients

被引：13

作者：

Laurent, Elisabeth ^{[1
,2
]}

Sieber, Anna ^{[1
]}

Salzer, Benjamin ^{[3
]}

Wachernig, Anna ^{[1
]}

Seigner, Jacqueline ^{[4
]}

Lehner, Manfred ^{[3
]}

Geyeregger, Rene ^{[3
]}

Kratzer, Bernhard ^{[5
]}

Jager, Ulrich ^{[6
]}

Kunert, Renate ^{[1
]}

Pickl, Winfried F. ^{[5
]}

Traxlmayr, Michael W. ^{[4
]}

机构：

[1] BOKU Univ Nat Resources & Life Sci, Dept Biotechnol, A-1190 Vienna, Austria

[2] BOKU Univ Nat Resources & Life Sci, BOKU Core Facil Biomol & Cellular Anal, A-1190 Vienna, Austria

[3] St Anna Childrens Canc Res Inst, A-1090 Vienna, Austria

[4] BOKU Univ Nat Resources & Life Sci, Dept Chem, A-1190 Vienna, Austria

[5] Med Univ Vienna, Inst Immunol, Ctr Pathophysiol Infectiol & Immunol, A-1090 Vienna, Austria

[6] Med Univ Vienna, Dept Internal Med, Div Hematol & Hemostaseol, A-1090 Vienna, Austria

来源：

ACS SYNTHETIC BIOLOGY | 2021年 / 10卷 / 05期

基金：

奥地利科学基金会;

关键词：

CD19; chimeric antigen receptor (CAR); yeast surface display; directed evolution; protein engineering; FMC63; THERMAL-STABILITY; SIGNAL PEPTIDE; B-LYMPHOCYTES; RAPID METHOD; PROTEINS; ANTIGEN; EXPRESSION; MOUSE; TISAGENLECLEUCEL; SELECTION;

D O I：

10.1021/acssynbio.1c00010

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

CD19 is among the most relevant targets in cancer immunotherapy. However, its extracellular domain (ECD) is prone to aggregation and misfolding, representing a major obstacle for the development and analysis of CD19-targeted therapeutics. Here, we engineered stabilized CD19-ECD (termed SuperFolder) variants, which also showed improved expression rates and, in contrast to the wild type protein, they could be efficiently purified in their monomeric forms. Despite being considerably more stable, these engineered mutants largely preserved the wild type sequence (>98.8%). We demonstrate that the variant SF05 enabled the determination of the monovalent affinity between CD19 and a clinically approved FMC63-based CAR, as well as monitoring and phenotypic characterization of CD19-directed CAR-T cells in the blood of lymphoma patients. We anticipate that the SuperFolder mutants generated in this study will be highly valuable tools for a range of applications in basic immunology and CD19-targeted cancer immunotherapy.

引用

页码：1184 / 1198

页数：15

共 62 条

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ZOLA, H ;

MACARDLE, PJ ;

BRADFORD, T ;

WEEDON, H ;

YASUI, H ;

KUROSAWA, Y .

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