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Photoimmunotechnology as a powerful biological tool for molecular-based elimination of target cells and microbes, including bacteria, fungi and viruses


Microbial pathogens, including bacteria, fungi and viruses, can develop resistance to clinically used drugs; therefore, finding new therapeutic agents is an ongoing challenge. Recently, we reported the photoimmuno-antimicrobial strategy (PIAS), a type of photoimmunotechnology, that enables molecularly targeted elimination of a wide range of microbes, including the viral pathogen severe acute respiratory syndrome coronavirus 2 and the multidrug-resistant bacterial pathogen methicillin-resistant Staphylococcus aureus (MRSA). PIAS works in the same way as photoimmunotherapy (PIT), which has been used to treat recurrent head and neck cancer in Japan since 2020. Both PIAS and PIT use a monoclonal antibody conjugated to a phthalocyanine derivative dye that undergoes a shape change when photoactivated. This shape change induces a structural change in the antibody–dye conjugate, resulting in physical stress within the binding sites of the conjugate and disrupting them. Therefore, targeting accuracy and flexibility can be determined based on the specificity of the antibody used. In this protocol, we describe how to design a treatment strategy, label monoclonal antibodies with the dye and characterize the products. We provide detailed examples of how to set up and perform PIAS and PIT applications in vitro and in vivo. These examples are PIAS against microbes using MRSA as a representative subject, PIAS against viruses using severe acute respiratory syndrome coronavirus 2 in VeroE6/TMPRSS2 cells, PIAS against MRSA-infected animals, and in vitro and in vivo PIT against cancer cells. The in vitro and in vivo protocols can be completed in ~3 h and 2 weeks, respectively.

Key points

  • Photoimmunotherapy, an effective treatment for head and neck cancer, is a technique that uses a monoclonal antibody labeled with a phthalocyanine derivative to disrupt the binding site after photoactivation.

  • This technique allows for both ‘target specificity’ and ‘flexibility in target selection’, leading to the development of a novel antimicrobial strategy that enables targeted elimination of microbes, regardless of the target species or drug resistance status of the target.

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Fig. 1: Proposed mechanism of photoimmunotechnology and the target cells and microbes, including bacteria, fungi and viruses.
Fig. 2: Sample preparation for in vitro photoimmunotechnology.
Fig. 3: Experimental workflow for in vitro photoimmunotechnology.
Fig. 4: Overview of in vitro PIAS on the viral pathogen SC.
Fig. 5: Overview of PIAS on MRSA-colonized rats.
Fig. 6: Binding of mAb conjugates to the target cells and the bactericidal effect of PIAS.
Fig. 7: Effects of PIT on cancer cells.

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Data availability

Data of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.


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We acknowledge the staff at the National Institute of Infectious Diseases for providing SC isolate (EPI_ISL_408667) and thank JCRB Cell Bank in Japan for VeroE6/TMPRSS2 cells. We also thank Y. Hatayama and T. Morita for viral experiments, Y. Dan and H. Yamada (Hitachi High-Technologies), H. Saito and T. Tachibana (Jikei University) for preparing and acquiring SEM images, H. Ishizaka, and colleagues in our laboratory for technical support, and H. Kanuka, Y. Kinjo, Y. Manome, H. Tajiri, M. Saruta and A. Chiba (Jikei University) for their support and comments. We also thank the University of Alabama at Birmingham Bacterial Pathogenesis & Physiology Journal Club for comments on PIAS study. This study was partly supported by AMED (grant number: JP22fk0108611h0802 to T.I.), JSPS KAKENHI (grant numbers: JP22K07076 to T.I.; 21H03811, 18H03523, 26710010 and JP26670487 to M.M.; and JP17K16233 to K.I.), Takeda Science Foundation to M.M., and Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research grant number: ZIA BC 011513 to H.K. and The Jikei University Research Fund to M.M. and T.I.

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Authors and Affiliations



T.I., H.K. and M.M. designed the study, performed experiments and analyses, and wrote the manuscript. K.I. and T.N. performed the experiments and analyses. A.R. and K.M. contributed to the viral study. All authors commented critically on the manuscript.

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Correspondence to Tadayuki Iwase or Makoto Mitsunaga.

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Nature Protocols thanks Tayyaba Hasan, Mingdong Huang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Mitsunaga, M. et al. Commun. Biol. 5, 647 (2022):

Mitsunaga, M. et al. Nat. Med. 17, 1685–1691 (2011):

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Supplementary Figs. 1 and 2.

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Source data for Fig. 7b.

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Iwase, T., Ito, K., Nishimura, T. et al. Photoimmunotechnology as a powerful biological tool for molecular-based elimination of target cells and microbes, including bacteria, fungi and viruses. Nat Protoc 18, 3390–3412 (2023).

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