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Prokaryotic expression of MLAA-34 and generation of a novel human ScFv against MLAA-34 by phage display technology.

ABSTRACT: MLAA-34 is a newly identified monocytic leukemia-associated antigen that is overexpressed in acute monocytic leukemia specifically, thus providing a novel target for the therapy of acute monocytic leukemia. In this study, we first expressed MLAA-34 protein in Escherichia coli (E.coli) BL21 (DE3) cells and purified it by nickel ion affinity chromatography with high purity (>90%). Then, MLAA-34 was used as antigen for biopanning anti-MLAA-34 single chain antibody fragment (ScFv) from a fully human ScFv library, and a high affinity ScFv named MA1 was selected by phage-ELISA. Finally, after expression of MA1, we found that MA1 can specifically bind with U937 MLAA-34 positive cells, and the binding affinity of MA1 was at the nanomolar level. Furthermore, inhibition of U937 cell proliferation indicated that the novel antibody MA1 has the potential to be used as a therapeutic agent for acute monocytic leukemia.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC5503596 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Prokaryotic expression of MLAA-34 and generation of a novel human ScFv against MLAA-34 by phage display technology.

Zhang Yang Y Zhang Pengyu P He Aili A Yang Yun Y Wang Jianli J Zhang Hui H Zhang Wanggang W

Oncotarget 20170601 24

MLAA-34 is a newly identified monocytic leukemia-associated antigen that is overexpressed in acute monocytic leukemia specifically, thus providing a novel target for the therapy of acute monocytic leukemia. In this study, we first expressed MLAA-34 protein in Escherichia coli (E.coli) BL21 (DE3) cells and purified it by nickel ion affinity chromatography with high purity (>90%). Then, MLAA-34 was used as antigen for biopanning anti-MLAA-34 single chain antibody fragment (ScFv) from a fully human ...[more]

PMID: 28388565

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Project description:BACKGROUND:Lymphocyte-activation gene (LAG)3 is a 498 aa transmembrane type I protein acting as an immune inhibitory receptor. It is expressed on activated lymphocytes, natural killer cells and plasmacytoid dendritic cells. In activated lymphocytes, LAG3 expression is involved in negative control of cell activation/proliferation to ensure modulation and control of immune responses. In view of its deregulated expression in tumor-infiltrating lymphocytes, LAG3, together with the additional immune checkpoint inhibitors CTLA4 and PD1, is considered a major target in order to reverse the immunosuppression typically mounting in oncologic diseases. Since many patients still fail to respond to current immune checkpoints-based therapies, the identification of new effective immune inhibitors is a priority in the ongoing fight against cancer. RESULTS:We identified a novel human single-chain variable fragment (scFv) Ab against a conformational epitope of LAG3 by in vitro phage display technology using the recombinant antigen as a bait. This scFv (referred to as F7) was characterized in terms of binding specificity to both recombinant antigen and human LAG3-expressing cells. It was then rebuilt into an IgG format pre-optimized for clinical usage, and the resulting bivalent construct was shown to preserve its ability to bind LAG3 on human cells. Next, we analyzed the activity of the anti-LAG3 scFvF7 using two different antigen-specific CD8+ T lymphocyte clones as target cells. We proved that the reconstituted anti-LAG3 F7 Ab efficiently binds the cell membrane of both cell clones after peptide-activation. Still more significantly, we observed a striking increase in the peptide-dependent cell activation upon Ab treatment as measured in terms of IFN-γ release by both ELISA and ELISPOT assays. CONCLUSIONS:Overall, the biotechnological strategy described herein represents a guiding development model for the search of novel useful immune checkpoint inhibitors. In addition, our functional data propose a novel candidate reagent for consideration as a cancer treatment.

Dataset Information

Prokaryotic expression of MLAA-34 and generation of a novel human ScFv against MLAA-34 by phage display technology.

Publications

Prokaryotic expression of MLAA-34 and generation of a novel human ScFv against MLAA-34 by phage display technology.

Similar Datasets

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