Feature Article

F eature Article

堀場雅夫賞 受賞者論文

Elucidation of the Mechanism of Near-Infrared Light-Induced Cell Death and Method Establishment for
Measuring Therapeutic Effects

Masao Horiba Awards

堀場雅夫賞 受賞者論文

Elucidation of the Mechanism of Near-Infrared Light-Induced Cell
Death and Method Establishment for Measuring Therapeutic Effects
近赤外光応答性細胞死誘導プローブの作用機構解明と治療効果計測基盤の構築

SATO Kazuhide
佐藤 和秀

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment thatinvolves the application of a near-infrared light probe (IR700) to a specific antibody that binds to antigens on the surface of cancer cells. However, despite its
impressive efficacy and innovation, the detailed mechanism of cell death has
remained unclear. From the viewpoints of analytical chemistry, condensed matterchemistry, and optics, it has been clarified that NIR-PIT cell death is the world’s
first photochemical-induced cell death, and the cell death can be measured andquantified by IR700 near-infrared fluorescence spectroscopy at 700 nm, thus
establishing the basis for a therapeutic biomarker. Due to the novelty of this cell
death, this therapy was recognized as a new cancer modality and was implemented clinically in Japan ahead of the rest of the world.

がん細胞表面の抗原に結合する特異的な抗体に近赤外線に反応するプローブ（IR700）をつけ，局所的に近赤外線を当てる事で治療を行う近赤外光線免疫療法（NIR-PIT）
は新しいがん治療として注目を集めている。しかしながら，その素晴らしい効果や革新性とは裏腹に，詳しい細胞死のメカニズムは不明であった。
分析化学，物性化学，光学の観点から，NIR-PITの細胞死が世界初の光化学誘導細胞死である事が解明され，その細胞死がIR700の700 nmの近赤外蛍光分
光測定により計測・定量でき，治療バイオマーカーとしての基盤が確立された。
この細胞死の新規性により，新しいがんのモダリティとして本治療が認められ，世界に先駆けて日本で臨床実装された。

Introduction
Near-Infrared photoimmunotherapy (NIR-PIT) is a nextgeneration cancer-targeting therapy in which antibodies
specific to antigens on the surface of cancer cells areattached to probes that react to near-infrared light and arethen locally irradiated with near-infrared light (Figure 1) [1].
This photo-targeted therapy is a futuristic, and multidisciplinary treatment method in which probes (chemistry) andantibodies (pharmacology and biology) are conjugatedunder optimal conditions (conjugation chemistry), andnear-infrared light (optics and physics) is added (Figure 1).
Irradiation with near-infrared light at 690 nm, the peakabsorption of the probe, immediately expands the cancer
cells and induces cell death. It is currently undergoinginternational Phase III trials (LUZERA-301) [2,3] and wasapproved by PMDA in Japan in September 2020 for thetreatment of recurrent and previously treated stage IV
head and neck cancer [4]. It is now being recognized as

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No.55 October 2021

the fifth cancer treatment after surgery, chemotherapy,
radiotherapy, and cancer immunotherapy. So far, various application studies of NIR-PIT therapy have been
conducted all over the world, and further development of
this therapy is expected from here. Although this therapy
has attracted attention for its innovation, the detailed
mechanism of cell death in NIR-PIT had not been fully
elucidated at the time of the introduction of Phase III trials.
This was a barrier to its approval and implementation.
In this review, we will provide an overview of NIR-PIT,
which is attracting attention as a “fifth cancer therapy”,
its cell death mechanism, and the prediction of its efficacy
by near-infrared spectroscopic imaging derived from its
elucidation.

Technical Reports

Overview of NIR-PIT

Figure 1 Overview of NIR-PIT 
NIR-PIT is a new therapeutic method in which an antibodyIR700 complex, in which IR700DX, a light-absorbing substance,
is added to the antibody, binds to cancer-specific antigens thatare highly expressed on the surface of target cancer cells, andis then exposed to NIR light.

About NIR-PIT
There are four known treatment methods for cancer:(1) surgery (surgical and endoscopic resection), (2)chemotherapy (anti-cancer drugs), (3) radiotherapy, and (4)cancer immunotherapy. These existing cancer treatments
cause injury not only to cancer cells but also to surrounding
normal tissues and organs. In anticancer drug therapy,
chemical substances that cause damage to both normal
cells and cancer cells are administered, but since thedosage is based on an extremely narrow dosage design thatallows normal cells to recover earlier than cancer cells, thedosage is strictly determined, and if an overdose occurs,
not only the cancer but also the individual may die. In
addition, surgery requires the resection of normal tissues
surrounding the cancer as margins, and radiation therapy
inevitably causes injury to normal cells within and throughthe irradiation area. Therefore, it would be ideal if there
were a cancer-targeted therapy that could superspecificallydamage only cancer cells in the body without damaging
normal cells and tissues.
Based on the above idea, Dr. Hisataka Kobayashi andhis colleagues at the National Cancer Institute (NCI)
in USA announced the development of Near-Infrared
Photoimmunotherapy (NIR-PIT) in 2011[1]. NIR-PIT is aphoto-targeted therapy that combines near-infrared lightand antibody photo-probe adducts, in which IR700, a lightabsorbing probe, is covalently attached to antibodies thatbind to cancer-specific antigens. NIR-PIT is super-selective
because it is doubly targeted by NIR-light and antibodies(Figure 1).
NIR-light is used in TV remote controls, CD players, etc.,
and is a safe light that has almost no adverse effects whenirradiated to the human body. IR700 is a probe that absorbs

light in the near-infrared region of 690 nm and emits
fluorescence at 700 nm, and was originally developed by
LICOR as a dye for microscopic observation in biology.
Antibodies are biomaterials that have been actively usedin medicine, biology, and engineering in recent years,
and are among the existing biomolecules that can bind
specifically and stably to molecules on the surface of cell
membranes. Among the existing biomolecules, antibodies
can bind specifically and stably to cell surface molecules[4].
As described above, NIR-PIT is a multidisciplinary therapy
that combines biology, pharmacology, chemistry, optics,
engineering, and physics.

Mechanisms of cell death in NIR-PIT
It has been proposed that oxidative stress is the main
mechanism of cell death using photosensitive substances[5].
This pathway is often considered to be one in which theenergy difference between photoexcitation and photoemission of a photosensitive substance acts on mitochondria andother organs, resulting in cell injury by oxidative stressagents such as singlet oxygen and free radicals, leading
to cell apoptosis. The detailed mechanism of cell death in
NIR-PIT has been unknown for a long time, which has beena barrier to its clinical implementation. Since the cell death
reaction in NIR-PIT proceeded even after the cell function
was stopped at 4°C, and the inhibition of cell death was not
sufficiently effective even when oxidative stress inhibitors(free radical scavengers) were added, it was inferred thatthe oxidative stress was not the main mechanism for
NIR-PIT. Therefore, it is inferred that the mechanism of
NIR-PIT is not based on oxidative stress as in conventional
photodynamic therapy (PDT). [5]
NIR-PIT has a very different therapeutic mechanism
compared to conventional cancer cell death. When the
antibody-IR700 conjugate bound to the cell surface isirradiated with near-infrared light (690 nm), the cell surface
is rapidly injured, the cell morphology changes, and thenthe cell bursts, leaking the cytoplasmic contents and causing cell death within a few minutes. Thus, the mechanism
is necrosis (rapid destruction of cells) rather than apoptosis,
which is the conventional slow death of cancer cells. Sincenear-infrared light irradiation itself is harmless to cells, it
can only damage the target cells to which the antibodyIR700 conjugate binds. In fact, when cells with and without
antibody-IR700 conjugates were co-cultured and irradiatedwith NIR light at the same time, cell death was observed
only in the cells with antibody-IR700 conjugates, while
the neighboring cells without antibody-IR700 conjugateswere not injured at all. The antibody-IR700 conjugate canselectively destroy not only cells in vitro but also target cells
in vivo in space and time. [6]No.55 October 2021

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F eature Article

堀場雅夫賞 受賞者論文

Elucidation of the Mechanism of Near-Infrared Light-Induced Cell Death and Method Establishment for
Measuring Therapeutic Effects

Aggregation by photochemical reaction of IR700 and detection of released products by mass spectrometry
Figure 2 A
 ggregation by photochemical reactionof IR700 and detection of released products by mass spectrometry
			 a. The photochemical reaction of IR700
as the starting point of NIR-PIT action:
the silanol group attached to the siliconof the silicon phthalocyanine ring ofIR700 is released by near-infrared lightirradiation, and as a result, the hydrophilic nature of IR700 suddenly
becomes hydrophobic and aggregates.
As a result of aggregation, the fluorescence is lost.
			 b. The silanol groups released from thesupernatant after irradiation weredetected by mass spectrometry (IT-TOF
MS), proving the reaction.
			 c. IT-TOF-MS analysis showed a peak ofsilanol group only at 16 J/cm2 nearinfrared light irradiation. By productscan (MS3) analysis, the structure wasdetermined and proved to be silanolgroup.

Originally, IR700 dye (IR700 DX) was developed andmarketed with the merit that its fluorescence hardly fades
compared to other dyes (AlexaFluor680, etc.) during thenumber of scans in the fluorescence observation environment of microscope slide samples. However, the accumulation of in vitro and in vivo experiments with NIR-PIT
clearly showed that the fluorescence of IR700 faded, which
was a contradiction. As a hypothesis to resolve this discrepancy, we hypothesized a change in the chemical nature ofIR700 due to photochemical reactions. The hypothesis isthat the chemical nature (hydrophilic) of IR700 is rapidlychanged to hydrophobic by the release of the silanol side
chain by near-infrared light. The hydrophobized IR700
forms aggregates in the aqueous solution, and as a result of
the aggregation, the fluorescence is lost from the solution
in a series of photochemical reactions (Figure 2a)[7]. It
was thought that the aforementioned discrepancy could
be explained by the difference between the inclusion stateof the slide samples using non-water-soluble inclusionmaterial and the water-rich biological environment.
Based on these hypotheses, in order to prove thismechanism, we attempted to measure the silanol groups(molecular weight 500) above and below the silicon phthalocyanine ring of IR700, which are thought to be released
upon aggregation, by mass spectrometry. IT-TOF-MS wasable to detect the peak of the molecular weight only in thecase of light irradiation (Figure 2b). In addition, it has
been found that this liberation reaction is enhanced in the24

No.55 October 2021

absence of oxygen and in an environment rich in electron
donors, while the presence of electron absorbers inhibits
the reaction. This indicates that this reaction is independentof oxygen and free radicals, and has the advantage that thereaction proceeds more easily in tumors under hypoxic
conditions.
Next, in order to prove that the above reaction also
occurred in the antibody-IR700 adduct, cetuximab-IR700(cet-IR700) was irradiated in the near-infrared to provethat silanol could be measured and detected by mass
spectrometry. In addition, the solution of cet-IR700 was
confirmed by the product scan method to be of IR700
origin, not antibody origin, with the structure of the silanolgroup (Figure 2c).
SDS-PAGE analysis was performed to study the effect ofphotochemical reaction of IR700 on antibody IR700 adductand antibody IR700 adduct-antigen protein complex. First,
we found that NIR light irradiation (peak 690 nm NIR
light laser irradiation) caused the antibody (cetuximab)band to smear on SDS-PAGE, resulting in the loss of IR700
fluorescence. The photochemical reaction of IR700 resulted
in the protein denaturation, i.e., aggregation of the added
antibody proteins (Figure 3a). As shown in the figure, thedenaturation increased in a near-infrared light-dependent
manner, and the aggregation was almost complete at 64 J/
cm2. It is also clear that the near-infrared fluorescence islost due to aggregation. In order to investigate the effectTechnical Reports
Examination of protein aggregation and loss of fluorescence of cet-IR700 by near-infrared irradiation andchanges in the antibody-IR700 adduct-antigen protein complex using SDS-PAGE

Figure 3 E
 xamination of protein aggregation and loss of fluorescence of cet-IR700 by near-infrared irradiation andchanges in the antibody-IR700 adductantigen protein complex using SDS-PAGE			a. The band of cet-IR700 became a smear in a light-dependent manner and lost its fluorescence, indicating that it was aggregated.
			b. When cet-IR700 was bound to an antigen protein (EGFR) and irradiated with near-infrared light, the band of the antigen protein also became
smeared, indicating that not only cet-IR700 but also its binding partner antigen was aggregated. With aggregation, fluorescence was also lost.

of the photochemical change of cet-IR700 on the binding
partner antigen protein, we conducted a near-infraredirradiation experiment with cet-IR700 bound to EGFR
protein (antibody-IR700 adduct-antigen protein complex)(peak 690 nm near-infrared laser irradiation measured
at 16 J/cm2). SDS-PAGE showed that not only cetuximab
but also EGFR protein, the antigen, was aggregated(Figure 3b). In addition, the loss of NIR fluorescence ofIR700 was observed. From the above, it was confirmedthat the complex of antibody IR700 adduct and antigen
was aggregated by light irradiation. This indicates that thephotochemical aggregation of IR700 causes the antibody toaggregate and also causes the antigen protein, the binding

partner of the antibody, to aggregate.
Although we were able to prove the photochemical reaction
by mass spectrometry above, it is necessary to capture
the change visually. This mechanism can be clarified byimaging the changes in a single antibody molecule andquantitatively measuring the photochemical reaction.
Therefore, we attempted to perform nano-imaging in liquid
by atomic force microscopy (AFM). Panitumumab-IR700(pan-IR700) was adhered to a mica substrate and the topof the substrate was filled with 10 mM phosphate buffer
containing 50 mM MgCl2 for nano-imaging in liquid.
Macroscopic views of 1.0 μm and microscopic images ofObservation and Quantification of Changes in panitumumab-IR700 (pan-IR700) under Near-Infrared Light
Irradiation by Nano-Imaging in Liquid Using FM-AFM

Figure 4 O
 bservation and Quantification of Changes in panitumumab-IR700 (pan-IR700) under Near-Infrared Light Irradiation by Nano-Imaging in Liquid
Using FM-AFM The changes of pan-IR700 before and after near-infrared light irradiation were investigated by nano - imaging in liquid using FM-AFM.
			a. The pan-IR700 molecules depicted on the mica substrate aggregated and became larger after light irradiation, and adhered to the mica substrate.
			b. pan-IR700 molecules depicted on the mica substrate have a Y-shape, but they aggregate and completely change their structure after lightirradiation.
			c. Results of change quantification by liquid nano-imaging using FM-AFM; the height and area significantly increased only in the group where
Pan-IR700 was irradiated with NIR-light.

No.55 October 2021

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堀場雅夫賞 受賞者論文

Elucidation of the Mechanism of Near-Infrared Light-Induced Cell Death and Method Establishment for
Measuring Therapeutic Effects

Relationship of IR700 fluorescence and in vitro cell death, in vivo anti-tumor effects, respectively

Figure 5 Relationship of IR700 fluorescence and in vitro cell death, in vivo anti-tumor effects, respectively
a. IR700-fluorescence in vitro was detected before and after NIR-PIT with flow cytometry.
b. Cytotoxicity in vitro was detected before and after NIR-PIT with luciferase activities.
c. Positive correlations between decrease of IR700 fluorescence and cytotoxicity is detected (r = 0.9779).
d. in vivo NIR-PIT regimen is showed.
e. Mouse tumor inoculation outline and light dose.
f. Representative image of treated mice.
g. Positive correlations between decrease of IR700 fluorescence and anti-tumor effects is detected (r =0.6316).

41.7 nm were taken. In the macroscopic image, a single
molecule of pan-IR700 attached to the mica substrate
was agglomerated and expanded as an oligomer by nearinfrared light irradiation (measured value: 16 J/cm2). In
the micro-image, a single molecule of pan-IR700 in the
shape of a Y-shape underwent structural changes upon lightirradiation, and expanded, deformed, and aggregated as anoligomer (Figure 4a, b). As a control, pan-IR700 alone (nolight irradiation) and antibody (pan) plus light irradiation(16 J/cm 2) were tested in the same way for quantification.
As a result of quantification of the height and Area values
over time, only the combination of pan-IR700 and lightirradiation (16 J/cm 2) was found to be dominant, with theheight and Area values expanding over time (Figure 4c).
Therefore, nano-imaging confirmed that the antibodyIR700 agglomerates upon near-infrared light irradiation.
In order to analyze the relationship between photochemical reactions and cell death in vitro, we analyzed
the relationship between the loss of IR700 fluorescenceon the cell membrane before and after light irradiationand cell death. It is known that near-infrared fluorescenceis lost in a dose-dependent manner, and measuring thenear-infrared fluorescence of IR700 in IR700 antibody
adducts was thought to reflect the degree of protein
aggregation. Changes in near-infrared fluorescence were
quantified by flow cytometry (Figure 5a), and cell death
was quantified by luciferase activity (Figure 5b), and therespective changes were analyzed as a scatter plot. The
results showed a positive correlation between the decreasein IR700 fluorescence and the increase in cell death (r =0.9779) (Figure 5c). This result suggests that cell death can

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be measured from the amount of decrease in near-infrared
fluorescence in target cells bound with IR700 antibody
adducts, proving that IR700 photochemistry is strongly
associated with cell death in NIR-PIT.
To analyze the relationship between the photochemical
reaction and the in vivo anti-tumor effect, we analyzed
the relationship between the loss of IR700 fluorescence intumors before and after light irradiation and the anti-tumor
effect (Figure 5d). As shown in Figure 5e, three tumors
were created in the same individual and irradiated with 0,50, and 100 J/cm2 of near-infrared light, respectively, andthe amount of change was measured. The change in IR700
fluorescence was quantified by the 700 nm channel of the
PEARL imager, and the anti-tumor effect was quantified
by the luciferase activity, which was analyzed in relation to
the scatter plot (Figure 5f). The results showed a positive
correlation between the decrease in IR700 fluorescence andthe increase in antitumor effect (r = 0.6316) (Figure 5g).
This result suggests that the anti-tumor effect can bemeasured from the amount of decrease in IR700 fluorescence (700 nm) across the target tumor bound with IR700
antibody adducts, proving that the photochemical reactionof IR700 is strongly related to the anti-tumor effect of NIRPIT. Since this decrease in IR700 fluorescence may predict
therapeutic efficacy, optimization of real-time imaging ofIR700 fluorescence is underway.

Conclusion
The above results suggest that the initiation point of cell
death in NIR-PIT is the hydrophobization of IR700 by

Technical Reports
Mechanism of cell destruction by NIR-PIT

Figure 6 Mechanism of cell destruction by NIR-PIT 
The antibody-IR700 complex binds to cancer-specific antigens on the cell surface. When the cells are irradiated with near-infrared light, thesilanol group is released from IR700 and rapid hydrophobization occurs, resulting in the formation of aggregates including antibody and antigen
proteins. This causes physical damage to the cell membrane. The osmotic disparity causes water to flow into the cell from outside the cell, causing the cell to rupture. Thus, the cell contents are disseminated to the surrounding area as necrosis to produce active cell destruction.

near-infrared light irradiation and the subsequent aggregation of antibody and antigen proteins[7]. In this study, theaggregation of antibody-IR700 adducts by near-infrared
light irradiation was revealed by nano-imaging, and theresults were convincing enough to propose a new conceptof cell death in NIR-PIT. Integrating these analyticalchemical data, cell culture, and animal studies, the authors
propose the following mechanism of action of NIR-PIT(Figure 6). The antibody-IR700 complex, which binds
to cancer-specific antigens on cell membranes, suddenly
aggregates upon NIR irradiation and acts as a physical cell
membrane barrier by denaturing the cell membrane antigen
to which it binds. This mechanism was considered to bea novel photocell death that is completely different fromprevious concepts. The clarification of this mechanism
proved the superiority of NIR-PIT as a new anti-cancer
modality, and led to its limited approval in Japan as a “fifth
cancer treatment” ahead of other countries. In addition,
as a further application, it is thought that it is possible topredict the effect of treatment in real time by measuring
and evaluating near-infrared fluorescence before and after
treatment as described in this paper, and devices are being
developed that apply intraoperative fluorescence imaging
devices. We look forward to the development of futureapplications.

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28;4(11):1559-1569.

SATO Kazuhide
佐藤 和秀
Designated Assistant Professor
Institute for Advanced Research, Respiratory Medicine / S-YLC,
Nagoya University.

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