Construction of Genetically Modified Lactococcus lactis Producing Anti-human-CTLA-4 Single-Chain Fragment Variable
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ORIGINAL PAPER
Construction of Genetically Modified Lactococcus lactis Producing Anti‑human‑CTLA‑4 Single‑Chain Fragment Variable Fu Namai1 · Aito Murakami1 · Asami Ueda1 · Masami Tsukagoshi1 · Suguru Shigemori1 · Tasuku Ogita1 · Takashi Sato1 · Takeshi Shimosato1 Accepted: 16 September 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Lactic acid bacteria are human commensal organisms that have immunomodulatory and metabolism-promoting effects. In addition, due to the increasing demand for biopharmaceuticals, genetically modified lactic acid bacteria (gmLAB) that produce recombinant proteins are expected to be used as microbial therapeutics and next-generation probiotics. In this study, we constructed a gmLAB strain that produces anti-human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) singlechain fragment variable (CTLA4scFv) for possible use in a cancer treatment strategy using gmLAB. CTLA-4, an immune checkpoint molecule, suppresses the anti-cancer immune response; thus, inhibition of CTLA-4 signaling is important in cancer therapy. In this study, we designed a CTLA4scFv composed of a heavy and light chain of the variable region from anti-human CTLA-4 antibody connected by a flexible peptide linker. CTLA4scFv was expressed using nisin controlled gene expression (NICE) system, a lactococcal inducible gene expression system, and the DNA sequence encoding CTLA4scFv was inserted downstream of the PnisA promoter of the gene expression vector pNZ8148#2. Furthermore, expression of recombinant CTLA4scFv was confirmed by Western blotting, and the immunoreactivity of recombinant CTLA4scFv against human CTLA-4 protein was examined using ELISA. We speculate that gmLAB producing bioactive CTLA4scFv will become an attractive approach for cancer treatment. Keywords gmLAB · Next-generation probiotics · Lactococcus lactis · Single-chain fragment variable · CTLA-4 · NICE system
* Takeshi Shimosato shimot@shinshu‑u.ac.jp Fu Namai 19hs503h@shinshu‑u.ac.jp Aito Murakami 20as117a@shinshu‑u.ac.jp Asami Ueda 19as102h@shinshu‑u.ac.jp Masami Tsukagoshi 20as113j@shinshu‑u.ac.jp Suguru Shigemori shigemoris@shinshu‑u.ac.jp Tasuku Ogita ogitat@shinshu‑u.ac.jp Takashi Sato satotak@shinshu‑u.ac.jp 1
Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, 8304 Minamiminowa, Kamiina, Nagano 399‑4598, Japan
Introduction Lactic acid bacteria (LAB) are human commensal organisms that reside primarily in the digestive tract. LAB are often used in food preparation, such as fermentation of livestock products, and have been certified as Generally Recognized As Safe by the US Food and Drug Administration (FDA) because of their long history of use [1]. In addition, the functionality of LAB as probiotics, such as their immunomodulatory and metabolism-promoting effects, has been widely studied for promoting good quality of life [2–4]. Furthermore, due to the increasing demand for biopharmaceuticals in recent years, LAB are expected to be used for disease prevention and
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