News Press Research Release
Title

Spatiotemporal depletion of tumor-associated immune checkpoint PD-L1 with near-infrared
photoimmunotherapy promotes antitumor immunity
Key Points

We successfully evaluated and developed near-infrared photoimmunotherapy targeting PD-L1, animmune checkpoint molecule, and found that PD-L1-targeted near-infrared photoimmunotherapy
synergistically exerts significant anti-tumor effects via cancer immunity, despite the limited
expression of PD-L1. We also found that metastatic tumors without light irradiation also exhibited
antitumor effects via cancer immunity. Translated with www.DeepL.com/Translator (free version)

We have shown that near-infrared photoimmunotherapy targeting PD-L1 synergistically activatesanti-tumor immunity by light-induced destruction of cancer cells, activation of tumor immunity byimmune checkpoint molecules, and modification of the cancer microenvironment.

This study proposes near-infrared photoimmunotherapy as an alternative treatment for patients who
are not eligible for near-infrared photoimmunotherapy based on high expression of cancer-specificantigens, and is expected to contribute as a basic knowledge for the application of near-infrared
photoimmunotherapy targeting PD-L1 to humans.

Summary 1
Graduate student Shunichi Taki (1st author) at the department of Respiratory Medicine, Nagoya
University Graduate School of Medicine (Dean: Kenji Kadomatsu),, Assistant Professor Kazuhide Sato(corresponding author), at Institute for Advanced Research, Nagoya University (Director: Yoshiyuki Suto),
and their collaborators succeeded in developing near-infrared photoimmunotherapy(NIR-PIT) targeting
PD-L1, an immune checkpoint molecule.
PD-L1, a ligand for PD-1, an immune checkpoint protein, has been detected in various solid tumors, andimmune checkpoint inhibitors targeting PD-1/PD-L1 have shown efficacy in cancers of various organs.
Even if the expression of PD-L1 in tumors is low (TPS (Tumor Proportion Score) of ≥1%), they can beused as first-line therapy and are known to have certain efficacy. However, the effect is not sufficient, andthere is a need to develop technology that can enhance the effect of immune checkpoint inhibitors.
NIR-PIT is a new cancer treatment reported in 2011 by Dr Hisataka Kobayashi and his colleagues at theNational Cancer Institute (NCI/NIH). A complex of antibodies that specifically recognize proteinsexpressed by cancer cells and a photosensitive substance IR700 is synthesized, and irradiated with nearinfrared light around 690nm while bound to the target proteins on the cell surface, the cancer cells aredestroyed. It is a novel therapeutic technology that is expected to become the fifth cancer treatment, andwas approved in Japan in September 2020 for the treatment of recurrent and previously treated head andneck cancer that highly expresses EGFR, ahead of the rest of the world. For the future expansion ofindications, there is a need to consider new targets, and especially the combination with cancer
immunotherapy is considered to be ideal.
In this study, we synthesized a complex of mouse anti-PD-L1-F(ab')2 antibody and IR700, anddemonstrated the effects of NIR-PIT on tumors by targeting PD-L1 in cell and animal experiments.
Although PD-L1 expression is low in mouse tumors as well as in humans, sufficient anti-tumor effects wereobtained by activating cancer immunity. It was also found that the treatment had a certain effect on tumors
in metastatic areas (photo-abscopal effect). This treatment is expected to be used in clinical practice in thefuture as an application of NIR-PIT to patients whose tumors do not have high expression of specific target
antigens.
This research was supported by the Program for Developing Next-generation Researchers (Japan Scienceand Technology Agency); JSPS KAKENHI (18K15923, 21K07217); CREST; FOREST-Souhatsu (JST);The Uehara Memorial Foundation; Takeda Science Foundation; and Princess Takamatsu Cancer ResearchFund, etc.
The paper was published in Journal for ImmunoTherapy of Cancer, a scientific journal jointly publishedby BMJ publishing group (electronic version dated November 1, 2021).

Summary 2
Research BackgroundPD-L1, a ligand for the immune checkpoint protein PD-1, is detected in a variety of solid tumors. PD-L1
is present in tumor cell membranes and works to weaken the immune response of CD8(+) T cells and evade
immune surveillance. PD-1/PD-L1-targeted immune checkpoint inhibitors have been shown to be effectivein the treatment of cancers of various organs, and have some efficacy as first-line therapy even when thetumor expression of PD-L1 is low (TPS (Tumor Proportion Score) ≥1%). However, the effect is not
sufficient, and there is a need to develop technology that can enhance the effect of immune checkpointinhibitors.
NIR-PIT is a new cancer treatment method developed by Dr. Hisataka Kobayashi and his colleagues at
the National Institutes of Health/National Cancer Institute (NCI/NIH) in 2011. A complex of antibodies
that specifically recognize proteins expressed by cancer cells and a photosensitive substance IR700 issynthesized, and irradiated with near-infrared light around 690nm while bound to the target proteins on thecell surface, the cells are destroyed. These cell death mechanisms have been clarified in 2018 by Sato et al,
the lead author of this release, as a new concept of cell death based on photochemical reactions (Sato K,et.al. ACS Cent Sci. 2018 Nov 28;4(11):1559-1569. doi: 10.1021/acscentsci.8b00565.). Since it can targetand destroy cancer cells in a different way, it is expected to be the "fifth cancer treatment" following surgery,radiation, chemotherapy, and cancer immunotherapy. NIR-PIT was first approved in Japan in September
2020 for the treatment of recurrent and previously untreated head and neck cancer that highly expresses
EGFR under the PMDA's prior application system.
However, conventional NIR-PIT, including this approval, requires cancer-targeting antigens that arehighly expressed on cancer cells, which limits the number of patients who can receive the therapy. It isnecessary to deliver this innovative light-targeted cancer therapy to a wide range of patients so that it canbe used as a general standard treatment. To solve this problem, we focused on PD-L1, an immune
checkpoint molecule that is not highly expressed but is widely expressed in low to moderate levels in cancer,
and planned to develop NIR-PIT targeting cancer immunity. In addition, we found that this treatment
method also enhances the effect of cancer immune checkpoint inhibitors (Figure 1).

Figure 1. Aim of this research

Research Results
We prepared PD-L1 F(ab')2-IR700 by synthesizing a complex of PD-L1 F(ab')2 and photosensitizer IR700.
PD-L1 F(ab')2-IR700 is thought to reduce systemic side effects by eliminating non-specific binding of PDL1 antibodies. It is also smaller in size than IgG and is expected to have higher tumor penetration. PD-L1

F(ab')2-IR700 was used to treat murine tumor cells (lung cancer, colorectal cancer, prostate cancer,melanoma). The expression of PD-L1 was low (about 1/100) compared to the expression of EGFR intumors, which is currently approved in humans. Since the effect of this therapy depends on the expression
level of cancer antigens on the surface of the cancer cell membrane, the effect of NIR-PIT targeting PD-L1in cell experiments was limited compared to that in tumors with high expression of EGFR, and more intenselight energy was required. The effect of NIR-PIT was also observed in cell experiments when sufficient
light energy was provided. In summary, the effects of NIR-PIT targeting PD-L1 in cell experiments werelimited and not suitable for therapeutic application.
However, when we examined the effects of single NIR-PIT using PD-L1 F(ab')2-IR700 in a mousesyngeneic tumor model, we found significant suppression of tumor growth and prolongation of survival,
which was not expected from the effects of cell experiments. Furthermore, in a mouse syngeneic metastasis
model, irradiation of only one tumor with near-infrared light resulted in suppression of tumor growth not
only in the irradiated tumor but also in the tumor not irradiated with near-infrared light, and significantly
prolonged survival.
We continued to analyze anti-tumor immunity to elucidate the discrepancy between the therapeutic effectsof cell and animal experiments described above. It was confirmed that CD8(+) T cells and NK cells, which
are key players of anti-tumor immunity, were activated inside the tumor. In addition, detailed analysis ofthe tumor microenvironment revealed that bone marrow-derived immunosuppressive cells (MDSCs) werereduced. We found that the partial tumor necrosis induced by NIR-PIT, the immune checkpoint effect of
PD-L1 F(ab')2, and the removal of MDSCs from the tumor microenvironment acted synergistically toactivate anti-tumor immunity and exert a remarkable anti-tumor effect. In addition, analysis of the blood ofmice suggested that the anti-tumor immunity was enhanced systemically. This systemic activation of antitumor immunity was thought to have had an effect on tumors that had not been treated with NIR-PIT andthat had not been exposed to light (Figure 2).

Figure 2. Creation of PD-L1 F(ab')2 and development of NIR-PIT application to tumors and its mechanism.

Research Summary and Future Perspective
NIR-PIT targeting PD-L1 showed sufficient anti-tumor effects even at low to moderate expression levels.
Furthermore, NIR-PIT targeting PD-L1 was shown to have anti-tumor effects not only at the site ofexposure to near-infrared light but also on metastatic tumors. The results of this study indicate that NIRPIT can be applied to patients without high expression of appropriate specific cancer antigens, as it isconsidered to be a next-generation type of cancer immunotherapy that differs in concept from conventional
NIR-PIT targeting highly expressed targets. Thus, it is considered to be possible to expand the indication
to a wide range of cancer patients, and since PD-L1 antibody is already clinically approved, this treatment
method is considered to be easy to apply clinically.
This treatment is expected to be used in the future in clinical practice as a proposal of NIR-PIT as analternative treatment for patients who are not eligible for NIR-PIT based on the application of highexpression of cancer-specific antigens. This study will contribute to the basic knowledge for theimplementation of NIR-PIT targeting PD-L1 to human malignant tumors.

Publication

Shunichi Taki1a, Kohei Matsuokab, Yuko Nishinagaa, Kazuomi Takahashia, Hirotoshi
Yasuia, Chiaki Koikec, Misae Shimizuc, Mitsuo Satob, Kazuhide Satoa,c,d,e,fa

Respiratory Medicine, Nagoya University Graduate School of MedicinebDepartment of Integrated Health Sciences, Nagoya University Graduate School ofMedicine, Nagoya, JapancNagoya University Institute for Advanced Research, Advanced Analytical andDiagnostic Imaging Center (AADIC) / Medical Engineering Unit (MEU), B3 UnitdNagoya University Institute of Nano-Life-Systems, Institutes of Innovation for Future

Societye

FOREST-Souhatsu, CREST, JSTfNagoya University Institute for Advanced Research, S-YLC

Spatiotemporal depletion of tumor-associated immune checkpoint PD-L1 with near-infrared
photoimmunotherapy promotes antitumor immunity
Journal for ImmunoTherapy of Cancer, November 1 , 2021
DOI:http://dx.doi.org/10.1136/jitc-2021-003036

Japanese ver.: https://www.med.nagoya-u.ac.jp/medical_J/research/pdf/Jou_Imm_Can_211101.pdf