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Near-Infrared Photoimmunotherapy of Cancer
Hisataka Kobayashi* and Peter L. Choyke
Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10,
Room B3B69, MSC1088, Bethesda, Maryland 20892-1088, United States

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CONSPECTUS: This Account is the ﬁrst comprehensive
review article on the newly developed, photochemistry-based
cancer therapy near-infrared (NIR) photoimmunotherapy(PIT). NIR-PIT is a molecularly targeted phototherapy forcancer that is based on injecting a conjugate of a near-infrared,
water-soluble, silicon-phthalocyanine derivative, IRdye700DX(IR700), and a monoclonal antibody (mAb) that targets an
expressed antigen on the cancer cell surface. Subsequent local
exposure to NIR light turns on this photochemical “death”
switch, resulting in the rapid and highly selective immunogenic cell death (ICD) of targeted cancer cells. ICD occurs as
early as 1 min after exposure to NIR light and results inirreversible morphologic changes only in target-expressing
cells based on the newly discovered photoinduced ligand release reaction that induces physical changes on conjugatedantibody/antigen complex resulting in functional damage on cell membrane. Meanwhile, immediately adjacent receptornegative cells are totally unharmed. Because of its highly targeted nature, NIR-PIT carries few side eﬀects and healing is rapid.
Evaluation of the tumor microenvironment reveals that ICD induced by NIR-PIT results in rapid maturation of immaturedendritic cells adjacent to dying cancer cells initiating a host anticancer immune response, resulting in repriming of polyclonal
CD8+T cells against various released cancer antigens, which ampliﬁes the therapeutic eﬀect of NIR-PIT. NIR-PIT can target
and treat virtually any cell surface antigens including cancer stem cell markers, that is, CD44 and CD133. A ﬁrst-in-human phase
1/2 clinical trial of NIR-PIT using cetuximab-IR700 (RM1929) targeting EGFR in inoperable recurrent head and neck cancer
patients successfully concluded in 2017 and led to “fast tracking” by the FDA and a phase 3 trial (https://clinicaltrials.gov/ct2/
show/NCT03769506) that is currently underway in 3 countries in Asia, US/Canada, and 4 countries in EU. The next step for
NIR-PIT is to further exploit the immune response. Preclinical research in animals with intact immune systems has shown that
NIT-PIT targeting of immunosuppressor cells within the tumor, such as regulatory T-cells, can further enhance tumor-cellselective systemic host-immunity leading to signiﬁcant responses in distant metastatic tumors, which are not treated with light.
By combining cancer-targeting NIR-PIT and immune-activating NIR-PIT or other cancer immunotherapies, NIR-PIT of a local
tumor, could lead to responses in distant metastases and may also inhibit recurrences due to activation of systemic anticancer
immunity and long-term immune memory without the systemic autoimmune adverse eﬀects often associated with immune
checkpoint inhibitors. Furthermore, NIR-PIT also enhances nanodrug delivery into tumors up to 24-fold superior to untreatedtumors with conventional EPR eﬀects by intensively damaging cancer cells behind tumor vessels. We conclude by describing
future advances in this novel photochemical cancer therapy that are likely to further enhance the eﬃcacy of NIR-PIT.

1. BACKGROUND
Three major cancer therapies; surgery, radiation, and chemotherapy, have been the traditional mainstays of oncology
treatment for over a half century. Each method aims to reducecancer burden while minimizing side eﬀects. However, eachtreatment is well-known to cause substantial damage to normal
cells, including immune cells, which becomes counterproductive to recovery and ultimately contributes to theoverall debilitation of the patient. A new approach, cancer
immunotherapy, seeks to use T-cell activating cytokines,
immune-checkpoint inhibitors, depletion of regulatory T-cells(Tregs), and cell-based therapies to selectively control tumor
growth. These methods have proven eﬀective in some patients
despite substantial side eﬀects. However, the current cancer
This article not subject to U.S. Copyright.
Published XXXX by the American Chemical
Societyimmunotherapies do not directly instigate cancer cell deathbut, rather, kill cancer cells by activating cytotoxic immune
cells.1,2 Large cancer burdens may overwhelm the hostimmune system’s ability to ﬁght the cancer. Meanwhile,
nonspeciﬁc oﬀ-target activation of the immune system cancause autoimmune-like damage to normal tissue. Theoretically,a therapy that selectively kills cancer cells while activating the
local host immune response would be ideal.
One such approach is near-infrared photoimmunotherapy(NIR-PIT).3 NIR-PIT diﬀers from conventional cancer
therapies in its selectivity for killing cancer cells while
Received: May 22, 2019

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ArticleFigure 1. Scheme for chemistry basis of NIR-PIT (top), physical changes conjugated proteins (middle), and single antibody molecule imaging
before and after NIR-PIT (bottom).

activating the host antitumor immune response. A ﬁrst-inhuman phase 1/2 clinical trial of NIR-PIT using cetuximabIR700 (RM1929) targeting EGFR in patients with inoperable
head and neck squamous cell cancer successfully concluded inlate 2017. A “fast-tracked” global phase 3 clinical trial began in2019 (https://clinicaltrials.gov/ct2/show/NCT03769506).
Early results suggest NIR-PIT is superior to existing second
and third line therapies for recurrent head and neck cancers.
Thus, NIR-PIT appears to be a promising new form of cancer
therapy.
NIR-PIT is based on the injection of a conjugate of an
antibody, which binds a cell surface marker on the cancer, anda photoactivating chemical (APC). A major feature of NIRPIT compared to other approaches is its speciﬁcity for cancer.
The speciﬁcity derives from targeting by the monoclonal
antibody (mAb). Cell killing is initiated by excitation of theantibody-bound photoactivating chemical, IRDye700DX(IR700), with near-infrared light at 690 nm. NIR light isnonionizing, causes no damage to DNA, is harmless to normal
cells and penetrates a few centimeters into the tissue. Since the
APC binds predominantly to cancer cells that overexpress thetargeted cancer-associated antigens, light activation results inselective cancer cell killing while not harming adjacent normal
cells including tumor inﬁltrating immune cells. Furthermore,by itself, IR700 is a water-soluble photo dye with nophototoxic or biotoxic properties of its own; therefore,
unbound IR700 that dissociates from the APC is safe and isreadily excreted in urine. The combination of the targetspeciﬁc APC and the limited exposure of light to the tumor
results in a highly targeted cancer therapy with minimal to nodamage to normal tissues. This theory has been borne out inearly phase 1/2 clinical trial results.
Importantly, unlike other traditional therapies, the highly
speciﬁc cancer cell death induced by NIR-PIT does notcompromise host immunity against cancer but even activates
multiclonal tumor-speciﬁc immune response. In fact, the rapid
nature of the cell death associated with NIR-PIT makes ithighly immunogenic. NIR-PIT rapidly releases cancer-speciﬁc
antigens and membrane damage danger signals which induce
activation of local dendritic cells, which prime and educate
̈ T cells leading to proliferation and cellcancer-speciﬁc naive
mediated cancer cell killing. This process is known as
immunogenic cell death (ICD) and NIR-PIT is perhaps thebest example of this mechanism of inducing host immunity.
Therefore, NIR-PIT could overcome problems of conventional
antibody-based therapy, including inhomogeneous or insuﬃcient delivery of antibodies or ADC and tumor heterogeneity
because NIR-PIT induced multiclonal immune response could
eliminate surviving cancer cells after NIR-PIT, even if
insuﬃcient APCs bind to cancer cells because of inhomogeneous expression of target antigens or uneven delivery or
insuﬃcient dosage. Additionally, since NIR-PIT does not have
limitation of repeated treatments, multiple NIR-PITs could
also help overcome these problems.
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ArticleFigure 2. Scheme and serial microscopic images for cellular cytotoxicity induced by NIR-PIT (see Video).

Figure 3. Biology of immunogenic cell death induced by NIR-PIT that leads to enhance antitumor host immunity against treated cancer cells.

2. MECHANISM OF CYTOTOXICITY
The molecular mechanisms of cell death caused by NIR-PIT
have recently been elucidated.4 Upon exposure to NIR,
photoinduced chemical changes to the IR700 molecule itself
and on the APC were identiﬁed. Under hypoxic or electron
donor-rich conditions, which are common in NIR-PIT-treatedtumor beds, IR700 undergoes photochemical ligand reactions
that release the hydrophilic side chains of IR700 and cause theremaining molecule to become very hydrophobic (Figure 1).
This chemical change leads to the formation of a Z-stackmultimer of silicon-phthalocyanine IR700 rings or waterinsoluble aggregates of APCs or APC-antigen complexes
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ArticleFigure 4. Selective depletion of regulatory T-cell (Treg) by NIR-PIT induced systemic antitumor host immunity.

leading to quenching of IR700 ﬂuorescence. The photochemical ligand release reaction leads to physicochemicalchanges within the APC-antigen complex, which reduces cell
membrane integrity because of damage to transmembranetarget proteins.
The mechanism of cellular cytotoxicity underlying NIR-PIT
was further investigated with three-dimensional dynamic live
cell microscopy, radioactive and ﬂuorescent probes, andbiological markers.5 For instance, three-dimensional dynamic
low coherence quantitative phase microscopy (3D-QPM) wasused to depict changes on the cellular membrane while dualplane inverted selective plane illumination microscopy(diSPIM) was used for depicting the release of cellular
contents immediately following NIR-PIT. The 3D-QPM
imaging showed that cells initially swell by approximately 3fold as water ﬂows into the cell following damage to the cell
membrane.(Figure 2).
Rapid swelling causes large tears in the membrane allowing
the release of intracytoplasmic contents into the extracellularspace. Observations made using diSPIM in cells expressing
cytoplasmic green ﬂuorescent protein (GFP) revealed that the
GFP was conﬁned within the cell during swelling but wasquickly dispersed once the cell membrane ruptured at whichtime the cell volume abruptly decreased. Cell bursting was notprevented by considerable amounts of NaN3, a singlet oxygen
quencher, or when temperatures were set to 4 °C. However,when cells were placed in a hyperosmotic buﬀer with 50 mM
dextran cell swelling was inhibited. Although organic andmacromolecular ﬂuorescent dyes were excluded from theduring cell swelling, H215O readily entered cells immediately
after NIR-PIT. The chemical and physical damage, which

induced rapid swelling of the cell and disruption of the cell
membrane are characteristic of ICD. NIR-PIT caused rapid
activation of stress markers including heat shock proteins 70
and 90, dying signals such as calreticulin, ATP and HMGB1,
which promote maturation of immature dendritic cells,followed by initiation of a host immune response against
released antigens from dying cancer cells (Figure 3).
To investigate whether oxidative changes of lipid molecules
caused by reactive oxygen species might account for the
weakening of the cellular membrane lipid bilayer, mass
spectroscopy was employed. Phosphatidylcholine was analyzed
in cells before and after NIR-PIT. The results showed that 16−
1 phosphatidyl choline, a major component of the lipid
membrane, showed minimal oxidation to a hyper-oxide lipid.
The amount was so minuteless than 1 ppmeven after
exposure to NIR light in 100-fold excess, that it was unlikely tobe responsible for the membrane disruption.4

3. MECHANISM OF IMMUNE ACTIVATIONNIR-PIT results in ICD that promotes maturation of immaturedendritic cells in the immediate microenvironment of thecancer cell.5 After cancer cell-targeted NIR-PIT, newly primed
CD8+T cells reacted to a larger repertoire of cancer antigens
compared with CD8+T cells before NIR-PIT, and proliferated
in treated tumor beds.6 Therefore, anticancer host immunity
was strengthened after cancer-cell targeted NIR-PIT largely
because of the re-education and subsequent proliferation of
CD8+T cells. While cancer targeted NIR-PIT itself may not
immediately kill all the cancer cells in a tumor, the hostimmune response appears to kill a high percentage of theremaining cells, at least in some instances. Thus, NIR-PIT can
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ArticleFigure 5. Combination of cancer-target NIR-PIT and immune-target NIR-PIT activates systemic antitumor host immunity for treating distantmetastasis and induces immune memory for avoiding recurrence.

Although this strategy appears eﬀective in animal models, it
could result in unwanted side eﬀects caused by the CPI.6 Usinga combination of NIR-PIT and a CPI, tumors started shrinking
immediately and disappeared several days after treatment.
Once tumors were eliminated with the combination therapy,
the animal’s immune system rejected any attempts toreinoculate the tumor in the same mouse suggesting thatthese mice had gained immunity against the initial tumor(Figure 5).

lead to complete eradication of the tumor after only one or twotreatments. The original testing of NIR-PIT was in immune
deﬁcient animals so the full extent of the immune response was
not realized until ﬁrst-in-human trials resulted in better than
expected results and was subsequently conﬁrmed in immune
competent animal models.
Conventional cancer immunotherapy includes the use of Tcell activating type 1 cytokines, such as IL-2 and IL-15,
immune-checkpoint inhibitors, such as anti-CTLA4 or antiPD1/PDL1 antibodies, and depletion of immune-suppressor
cells such as the negative regulatory T-cell (Treg) or the
myeloid derived suppressor cell (MDSC). These therapies
operate on the principle of activating pre-existing CD8+T cells
not only in tumor beds but also other parts of the body.
Therefore, they suﬀer from oﬀ-target eﬀects sometimes
mimicking autoimmune diseases. Unlike these therapies,
NIR-PIT locally enhances host immunity without systemicside eﬀects. Moreover, by selectively eliminating immunesuppressor cells in local tumor beds using immune-suppressor
cell-targeting antibodies against CD25 or CCR4 for Treg cells
and CXCR2 for MDSC, one could further enhance hostimmunity.
Local Treg cell depletion with Treg-targeted NIR-PIT
against CD25 is highly eﬀective in syngeneic mouse models.7
CD8+T and NK cells in treated tumor beds were fully
activated within a few hours after depletion of Tregs with NIRPIT. Interestingly, this Treg targeted NIR-PIT also had aneﬀect on nontreated tumors even though the treatment was
directed at only one targeted lesion, an example of the“abscopal” eﬀect. (Figure 4)
Direct antitumor NIR-PIT could be combined withconventional systemic cancer immunotherapy, including
immune-checkpoint inhibitors (CPI), to increase its eﬀectiveness by further activating CD8+T cells after NIR-PIT.

4. APPLICATIONS OF NIR-PIT
NIR-PIT can be applied to any cancer with overexpressedtarget membrane proteins for which there is a suitablemonoclonal antibody. NIR-PIT has been successfully performed with APCs targeting EGFR, HER2, PSMA,8 CEA,9
GPC3,10 mesothelin,11 CD25,12 CD20,13 PD-L1,14 CD44,15
CD133, Laminine33, and MUC1 in vivo and in vitro. Special
note is made of NIR-PIT directed at CD4416 and CD133,17
which are considered markers of cancer stem cells in breast
cancer and glioblastoma, respectively, Tumor regrowth was
greatly suppressed after CD44 or CD133-targeted NIR-PIT.
Additionally, mouse models of tumors located in the xenograft
ﬂank, peritoneally,18 pleurally,19 and solitary20 or miliary21 lungmetastasis, orthotopic cancers in athymic and immunocompetent mice,22,23 and spontaneous lung cancer in transgenic
mice24 were also successfully treated with NIR-PIT. Since 690
nm light can penetrate and treat cancers around 1 cm from thesurface or the light source, deeply seated tumors were also
treated with interstitial NIR light exposure using ﬁbro-optical
diﬀusers inserted through catheter needles25 or endoscopes,26
techniques that could be readily adapted to clinical practice.
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Article5. IMAGING EVALUATION OF NIR-PIT THERAPEUTIC
EFFECTS
There is no immediate change in tumor size after NIR-PIT.
When possible, direct observation demonstrates that the tumor
turns a whitish color, but it is not always possible to directly
observe a treated tumor. Therefore, the therapeutic eﬀects of
NIR-PIT can be monitored with several diﬀerent imaging
modalities. Light released after activating IR700 can bedetected with ﬂuorescence cameras and ﬂuorescence disappears after NIR-PIT because of the formation of dimers or
oligomers of phthalocyanine cores or precipitation of
conjugated proteins after both ligands detach following thephotochemical ligand release reaction. Therefore, decreased
IR700 emission on ﬂuorescence imaging after NIR PIT could
be an indicator that the photoinduced ligand release reaction
has occurred indicating adequate delivery of light at that site.4
This, however, may not indicate treatment success. Because of
the near-immediate cell death, imaging methods such as 18Fﬂuorodeoxyglucose positron emission tomography (18F-FDGPET) could be a rapid response marker of treatment success.
(Figure 6)27

due to vascular leakiness, following NIR-PIT one can observe
marked increases in permeability and leakage from vessels,
especially for macromolecules. This has been termed thesuperenhanced permeability and retention (SUPR) eﬀect todraw a distinction with EPR.29 Whereas EPR only allows for amodest delivery of nanosized therapeutic agents and similar
compounds, SUPR following NIR-PIT results in dramaticallyenhanced leakage by a factor of up to 24-fold. By inducing
immediate necrosis in the perivascular cancer cells, a space
forms between the vessels and the remaining tumor, allowing
the vessel to enlarge, while increasing blood volume anddecreasing blood velocity. (Figure 7)

Figure 7. Mechanism of superenhanced permeability and retention(SUPR) eﬀects induced by NIR-PIT

Consequently, there is improved delivery of nanosized
therapeutic agents into the treated tissue where they canremain and be eﬀective for several days. Therefore, acombination of NIR-PIT and nanosized anticancer agents
could be more eﬀective than either of the therapies alone andthis could be another mechanism by which any residual tumor
following NIR-PIT treatments could be eliminated. In a study
employing FDA approved liposome-encapsulated daunorubucin (DaunoXome)29 and nanoparticle albumin-bound paclitaxel (nab-palitaxel; Abraxane)30 in mouse xenograft models ofcancer, NIR-PIT in combination with either drug hadsigniﬁcantly better therapeutic eﬀects than with either therapy
alone. SUPR eﬀects also allow for enhanced delivery of otherantibodies and APCs with increased leakage into tumor beds
after initial NIR-PIT treatments.31−33 Mouse xenograft cancer
models showed that multiple applications of light following
single or multiple doses of APC slowed regrowth and increased
progression free survival. Antibody-drug conjugates (ADCs),such as photoactivatable drug release systems, could also be
incorporated in the series of treatments by (1) performing
NIR-PIT and inducing SUPR eﬀects and, then, (2) delivering
ADCs through the SUPR eﬀect and exposing the tumor site toa second dose of NIR light.34 Low molecular weight anticancer
agents that bind to proteins also behave similarly to nanosized
agents, making them applicable to increased delivery throughthe SUPR eﬀect.

Figure 6. Immediate decrease of glucose metabolism after NIR-PIT is
depicted by 18F-FDG PET.

This could be seen much earlier than actual physical changes
in the tumor can be measured. Additionally, there are twoadvanced imaging technologies; ﬂuorescence lifetime imaging
and bioluminescence imaging, which can evaluate acute NIRPIT treatment but these are limited to preclinical studies. By
detecting a shortened ﬂuorescence lifetime of IR700, acutenecrotic/immunogenic cell death can be inferred.28 By
depicting release and hydrolysis of ATP from dying cellswith necrotic/immunogenic cell death, bioluminescenceimaging also works as a good experimental tool for monitoring
acute NIR PIT eﬀects in luciferase expressing tumors in mousemodels.22

6. SUPERENHANCED UPTAKE AND RETENTION(SUPR) FOLLOWING NIR-PIT
Another unique feature of NIR-PIT is its immediate eﬀect onblood drug delivery. While some degree of enhanced
permeability and retention (EPR) is present in most tumors
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Article7. NIR-PIT TREATMENT OF CIRCULATING TUMORCELLS (CTCs)
Circulating tumor cells (CTCs) are thought to be one
mechanism by which tumors can metastasize. CTCs circulate
in the vasculature until they successfully graft in sites that
permit the cell to recruit other normal stromal cells crucial tothe development of the tumor microenvironment, as well asother CTCs. While CTCs are circulating, they are known toharbor characteristic cell surface markers that could readily betargeted with speciﬁc APCs. Continuous NIR illumination of
surface vessels, such as at the wrist or neck, performed withlight sources from bracelets or necklaces could be used toperiodically reduce CTCs. Reduced CTC levels by themselves
are associated with prolonged survivals and reduced risk ofmetastases. Thus, NIR-PIT directed at CTCs could be a meansof prolonging progression free survival.

Health in Bethesda, Maryland. Dr. Kobayashi was awarded his M.D.
and Ph.D. (Immunology/Medicine) by the Kyoto University in Japan.
He joined as a postdoctoral fellow the Nuclear Medicine Department
at the Clinical Center of the National Institutes of Health in 1995 andmoved to his current position in the Molecular Imaging Program at
NCI in 2004. His interest is in developing novel molecular imaging
agents and technologies especially for targeting cancers.
Peter L. Choyke is the Director of the Molecular Imaging Program at
the Center for Cancer Research of the National Cancer Institute in
Bethesda, Maryland. Dr. Choyke is a graduate of Jeﬀerson Medical
School and received training in Diagnostic Radiology at Yale
University and the University of Pennsylvania. He joined the
Diagnostic Radiology Department at the Clinical Center of the
National Institutes of Health in 1988 and formed the Molecular
Imaging Program at NCI in 2004. His interest is in accelerating thetreatment of cancer by using novel molecular imaging agents whichtarget speciﬁc features of cancers.

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8. NIR-PIT IN TISSUE ENGINEERINGThe new ﬁeld of tissue engineering allows stem cells to beplaced on speciﬁc scaﬀolds to grow new organs or heal
wounds. Unfortunately, during the growth of these cells,
teratomas may develop rendering the graft useless. Such
teratomas have characteristic cell surface markers that are
amenable to the development of APCs. NIR-PIT could be
used to eliminate teratomas without damaging the remainderof the 2D or 3D-graft and thus save the graft from being
discarded.35,36 This could improve throughput and lower costs
associated with tissue regeneration.

ACKNOWLEDGMENTS
This research was supported by the Intramural Research
Program of the National Institutes of Health, National Cancer
Institute, Center for Cancer Research (ZIA BC011513). We
also thank to Ms. Yu A. Nakamura for her assistance on
preparing this manuscript.

■

9. SUMMARY
Cancer-targeted NIR-PIT has great potential to become awidely applicable cancer therapy. NIR-PIT decreases thenumber of cancer cells and enhances host immune response ina highly selective manner reducing side eﬀects. When
combined with immune-activation therapies, NIR-PIT, notonly treats the local tumor, but also reduces or eliminates
systemic metastasis and prevents recurrence in some animalmodels. Since the eﬀects of NIR-PIT improve when hostimmunity is intact, NIR-PIT may eventually become a ﬁrst line
cancer therapy while other existing therapies, such as radiationand chemotherapy, that damage the immune system may berelegated to secondary and tertiary lines of therapy.

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ASSOCIATED CONTENTW Web-Enhanced Feature*

Video showing serial microscopic images for cellularcytotoxicity.

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AUTHOR INFORMATION

Corresponding Author*Phone: 240-858-3069. Fax: 240-402-3191. E-mail:Kobayash@mail.nih.gov.
ORCID

Hisataka Kobayashi: 0000-0003-1019-4112
Notes

The authors declare no competing ﬁnancial interest.
Biographies
Hisataka Kobayashi is the Chief scientist in the Molecular Imaging
Program at the National Cancer Institute of the National Institutes of
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DOI: 10.1021/acs.accounts.9b00273
Acc. Chem. Res. XXXX, XXX, XXX−XXX