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Construction of Genetically Modified Lactococcus lactis Producing Anti-human-CTLA-4 Single-Chain Fragment Variable

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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) single-chain 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.

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Acknowledgements

This work was supported by a Grant-in-Aid for JSPS Fellows, JP201922578. We thank the Research Center for Support of Advanced Science, Shinshu University, for use of their facilities.

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Authors and Affiliations

  1. Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, 8304 Minamiminowa, Kamiina, Nagano, 399-4598, Japan

    Fu Namai, Aito Murakami, Asami Ueda, Masami Tsukagoshi, Suguru Shigemori, Tasuku Ogita, Takashi Sato & Takeshi Shimosato

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  1. Fu Namai
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  2. Aito Murakami
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  3. Asami Ueda
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  4. Masami Tsukagoshi
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  5. Suguru Shigemori
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  6. Tasuku Ogita
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  7. Takashi Sato
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  8. Takeshi Shimosato
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Contributions

FN, AM, AU, MT, and SS performed the experiments and analyzed the data; TSa and TO contributed reagents, materials, and/or analytical tools; TSh designed the research project. FN and TSh wrote the paper.

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Correspondence to Takeshi Shimosato.

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Namai, F., Murakami, A., Ueda, A. et al. Construction of Genetically Modified Lactococcus lactis Producing Anti-human-CTLA-4 Single-Chain Fragment Variable. Mol Biotechnol 62, 572–579 (2020). https://doi.org/10.1007/s12033-020-00274-8

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