cancers
Review

Near Infrared Photoimmunotherapy; A Review of Targets forCancer Therapy
Takuya Kato, Hiroaki Wakiyama, Aki Furusawa, Peter L. Choyke and Hisataka Kobayashi *Molecular Imaging Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health,Bethesda, MD 20892, USA; takuya.kato@nih.gov (T.K.); hiroaki.wakiyama@nih.gov (H.W.);
aki.furusawa@nih.gov (A.F.); pchoyke@mail.nih.gov (P.L.C.)* Correspondence: kobayash@mail.nih.gov; Tel.: +1-240-858-3069

Simple Summary: Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer
treatment that uses an antibody-photoabsorber (IRDye700DX) conjugate (APC) that is activatedby NIR light irradiation. A major benefit of NIR-PIT is that only APC-bound cancer cells that are
exposed to NIR light are killed by NIR-PIT; thus, minimal damage occurs in adjacent normal cells.
NIR-PIT has now been applied to many cancers expressing various cell-surface target proteins using
monoclonal antibodies designed to bind to them. Moreover, NIR-PIT is not limited to tumor antigens
but can also be used to kill specific host cells that create immune-permissive environments in whichtumors grow. Moreover, multiple targets can be treated simultaneously with NIR-PIT using a cocktail
of APCs. NIR-PIT has great potential to treat a wide variety of cancers by targeting appropriate
tumor cells, immune cells, or both, and can be augmented by other immunotherapies.

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Citation: Kato, T.; Wakiyama, H.;
Furusawa, A.; Choyke, P.L.;
Kobayashi, H. Near Infrared
Photoimmunotherapy; A Review ofTargets for Cancer Therapy. Cancers
2021, 13, 2535. https://doi.org/
10.3390/cancers13112535
Received: 16 April 2021
Accepted: 18 May 2021
Published: 21 May 2021

Publisher’s Note: MDPI stays neutral

Abstract: Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatmentthat uses an antibody-photoabsorber (IRDye700DX) conjugate (APC) that is activated by NIR lightirradiation. In September 2020, the first APC and laser system were conditionally approved forclinical use in Japan. A major benefit of NIR-PIT is that only APC-bound cancer cells that are exposed
to NIR light are killed by NIR-PIT; thus, minimal damage occurs in adjacent normal cells. These earlytrials have demonstrated that in addition to direct cell killing, there is a significant therapeutic hostimmune response that greatly contributes to the success of the therapy. Although the first clinicaluse of NIR-PIT targeted epidermal growth factor receptor (EGFR), many other targets are suitablefor NIR-PIT. NIR-PIT has now been applied to many cancers expressing various cell-surface target
proteins using monoclonal antibodies designed to bind to them. Moreover, NIR-PIT is not limited
to tumor antigens but can also be used to kill specific host cells that create immune-permissive
environments in which tumors grow. Moreover, multiple targets can be treated simultaneously with
NIR-PIT using a cocktail of APCs. NIR-PIT can be used in combination with other therapies, such as
immune checkpoint inhibitors, to enhance the therapeutic effect. Thus, NIR-PIT has great potential
to treat a wide variety of cancers by targeting appropriate tumor cells, immune cells, or both, and canbe augmented by other immunotherapies.

with regard to jurisdictional claims inpublished maps and institutional affiliations.

Keywords: cancer; near-infrared photoimmunotherapy (NIR-PIT); target molecule; host immunity;
cancer therapy

Copyright: © 2021 by the authors.

1. Introduction

Licensee MDPI, Basel, Switzerland.

Near-infrared photoimmunotherapy (NIR-PIT) (IlliminoxTM ) is a newly developed
cancer treatment that induces selective immunogenic cell death in targeted cells [1]. NIRPIT utilizes an antibody–photoabsorber conjugate (APC) that is activated by NIR light,
typically administered as laser light. A large body of research has developed around NIRPIT, showing that it can kill many types of cancer cells by targeting unique transmembraneproteins overexpressed in cancer cells. Currently, a global phase III clinical trial of NIR-PIT

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2 of 20for inoperable head and neck cancer patients is currently underway using an anti-epidermal
growth factor receptor (EGFR)-antibody IRDye700DX (IR700) conjugate. In Japan, the first
APC (ASP-1929, AkaluxTM , Rakuten Medical Inc., San Diego, CA, USA) targeting EGFR
and utilizing a near infrared laser system (BioBladeTM , Rakten Medical Inc.) was approvedfor clinical use in September 2020. Although NIR-PIT has been primarily developed to use
antibodies against cancer membrane antigens, it can also kill normal host cells, especiallythose that promote cancer cell growth, by inhibiting the immune system locally [2].
Tumor-targeted NIR-PIT evokes a profound immune response. This can be augmented
with various checkpoint inhibitors. The first such agent, ipilimumab, was a monoclonal antibody against cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and was approved in
March 2011 to treat patients with late-stage melanoma. This class of immune checkpoint inhibitor (ICI) has continued to expand over the last decade, with many ICIs being approved.
Although these agents have been highly effective in some patients, immunotherapy-relatedside effects, termed immune-related adverse events (irAEs), have been widely reported invarious organs. irAEs occur because ICIs activate immunity throughout the body, not justin the tumor. These side effects can cause syndromes that mimic autoimmune disease innormal organs. Ideally, a comprehensive cancer therapy would selectively kill tumor cells
and activate local tumor immunity. None of the existing major therapies, surgery, radiation,or chemotherapy do this. Tumor- and immune cell-targeted NIR-PIT can kill tumor cells
selectively and deplete local immune suppressor cells in the tumor beds. This leads todramatically more active immune responses against tumors without inducing irAEs in
non-target tissues.
Numerous NIR-PIT for cancer and immune cells have been attempted over the past
decade, resulting in numerous research reports. This review summarizes these efforts
and serves to compile this work into one review to provide an update on NIR-PIT against
various effective molecular targets.
2. NIR-PIT
Historically, the term “photoimmunotherapy” meant targeted photodynamic therapy
using antibodies conjugated with conventional photosensitizers such as hematoporphyrin
based on cytotoxicity induced by reactive oxygen species (ROS) in the 1980s [3]. Various
combinations of conventional photosensitizers and antibodies were tested to improvetheir delivery and selectivity. Due to the unfavorable chemical design that conjugatedantibodies with hydrophobic chemicals, therapeutic effects were limited only on cells
in vitro or tumors with intra-tumoral or intra-spatial administration in vivo. Therefore,such conjugates were not successfully applied to cancer therapy via systemic administrationin vivo [4] because of insufficient delivery to the tumor due to rapid liver accumulation of
conjugates promoted by the hydrophobicity of conventional photosensitizers, resulting
in an insufficient accumulation of photosensitizers in the tumor [5]. Therefore, thesephotosensitizers have not been approved for clinical use. Furthermore, the cytotoxicity ofsuch conjugates relied on ROS that mainly induced apoptotic cell death, yet caused nonspecific damage on various parts of cells, including the cell membrane, in both targeted andadjacent non-targeted cells. In contrast, NIR-PIT (IlluminoxTM ) showed selective dedicatedcytotoxicity on target cells based on a photo-induced ligand release reaction rather than
ROS after the systemic intravenous injection of IR700-based APCs with various antibodies.
Additionally, NIR-PIT is a hybrid therapy of direct cancer cell killing induced by NIR light
exposure combined with rational immune activation. Therefore, NIR-PIT is totally different
from the old “photoimmunotherapy” and is now defined as IlluminoxTM .
Nanosized particles were used as carriers of photosensitizers due to the large loading
capacity [6]. Nanosized drugs have an advantage on delivery into tumor beds throughpermeable tumor vessels and are then retained in the tumor bed. This process is known as
the enhanced permeability and retention (EPR) effect [7]. However, the EPR effect offers
less than a twofold increase in nanodrug delivery compared to critical normal organs withinhomogeneous intratumor micro-distribution, resulting in failure to cure most cancers [8].

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Therefore, nanosized vehicles could be a good carrier for photothermal therapy (PTT),
but would not rationally be a good carrier of photosensitizers for targeted photodynamic
therapy (PDT). To overcome all these problems, NIR-PIT was developed as a novel cancer
treatment with high cell selectivity with minimal toxicity to adjacent cells.
NIR-PIT utilizes an antibody conjugated with the NIR photon-absorbing silicon phthalocyanine dye IR700 [1,9,10]. When injected intravenously, APCs bind to a specificcell membrane antigen. Once bound, NIR light (approximately 690 nm) exposure kills
only APC-bound target cells, sparing nearby cells which have no or minimal expressionof the target antigen [1,9,11,12]. Immediately after NIR light exposure to APC, axial ligands of the IR700 molecule dissociate from the molecule, converting the APC from veryhydrophilic to very hydrophobic. This change in the chemical characteristics of IR700 promotes conformational changes and aggregation of APCs that damage the cell membrane,
causing weakening and eventually rupture of the cell (Figure 1). NIR-PIT can clearly be
distinguished from conventional photodynamic therapy (PDT) or photothermal therapy(PTT), which rely on cytotoxic singlet oxygen and hyperthermia, respectively, and cause
non-selective damage to light-exposed cells [13,14]. In addition to direct cancer-cell killing,
NIR-PIT rapidly induces immunogenic cell death (ICD) [15,16], which initiates activationof the adaptive immune response, employing dead-cell-associated antigens, including calreticulin (CRT), adenosine triphosphate (ATP), high-mobility group box 1 (HMGB1), heatshock protein (Hsp) 70, and Hsp90 [17–20]. These danger signals activate local immaturedendritic cells (DCs) to stimulate the presentation of tumor antigens, which are released
from NIR-PIT-treated cancer cells. The DCs present these antigens to T cells, resulting
in priming and educating naive T cells to become cancer-specific CD8+ T cells [15,21].
Therefore, NIR-PIT has the potential to reset anti-tumor host immunity due to ICD. These
factors have contributed to the initial success of NIR-PIT.

Figure 1. Scheme for cellular cytotoxicity induced by NIR-PIT. Antibody-photoabsorber conjugatesbind to a specific cell membrane antigen. Immediately after NIR light exposure to APC, the wateroutside of the cells is flown into the target cell, leading to cell death. Adapted from Ref [10].

3. NIR-PIT Targeting Cancer Cells
Initially, NIR-PIT was developed to target cells expressing EGFR, human epidermal
growth factor receptor-2 (HER2), and prostate-specific membrane antigen (PSMA) [1]. Since
those early days, NIR-PIT has expanded to target a variety of transmembrane proteinsusing monoclonal antibodies designed to bind to these antigens. Here, we review many of
the targets that have been investigated for potential use in NIR-PIT using PubMed, with“photoimmunotherapy,” “near infrared,” and “IR700” as keywords (Table 1).

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Table 1. Correspondence of molecular targets for each tumor in NIR-PIT targeting cancer cells.
Malignant Neoplasma
Glioblastoma multiforme
Head and neck Ca.
Lung Ca.
MPM
Breast Ca.
Gastrointestinal Ca.
Esophageal Ca.
Gastric Ca.
Colorectal Ca.
Hepatic cell Ca.
cholangiocarcinoma
Pancreatic Ca.
GIST
Bladder Ca.
Prostate Ca.
Cervical Ca.
Ovarian Ca.
Malignant melanoma
LymphomaTarget Moleclue
EGFR

HER2

CD44

+
++
++

+
±

++
+

+

++

+

+

+

++
+
+

+
±

±
+
++

+

+

+
++
+
+
+
+

++

+
±
+
+

+
+
+
+

CEA

PDPN
+
+
+
+

MSLN

GPA33

TROP2

CDH-17++

PD-L1

Cancer Specific Target

+
++
++

DLL3 (SCLC)

+
+

+
+

+

+
+

+
+

+

+
+

+
+

+

+
+
+
+
+
+

GPC-3

c-KIT

++
+

±
+

+
+

+

+
+
+
+
++
++

PSMA

CD146
CD20, CD25, CLA

Ca., cancer; EGFR, epidernal growth factor receptor; HER2, human epidermal growth factor receptor-2; CEA, carcinoembryonic antigen; PDPN, podoplanin; MSLN, mesothelin; GPA33, glycoprotein A33 antigen;
TROP-2, tumor-associated calcium signal transducer 2; CDH-17, cadherin-17; PD-L1, programmed death-ligand 1; DLL3, delta-like protein 3; GPC-3, glypican-3; PSMA, prostate-specific membrane antigen; CLA,
cutaneous lymphocyte antigen; MPM, malignant plueral mesothelioma; GIST, Gastrointestinal stromal tumors; ++, highly expressing; +, moderately expressing; ±, weakly expressing.

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3.1. Epidermal Growth Factor Receptor
EGFR, belonging to the erythroblastosis oncogene B (ErbB) family, is a transmembranetyrosine kinase receptor [22]. Physiologically, EGFR regulates epithelial tissue development
and homeostasis. EGFR mutation and/or overexpression is observed in several humancancers (Figure 2A) and EGFR-targeted therapy has become a routine part of the treatment
of several cancers [23]. In several tumor types (e.g., head and neck, cervical, bladder, andesophageal cancer) EGFR overexpression has been associated with poor prognosis [24].
Hence, EGFR was considered a good target for early clinical trials of NIR-PIT. Multiple
studies have confirmed the therapeutic efficiency of EGFR-targeted NIR-PIT both forin vitro testing and in mouse xenograft models [1,25–29]. Cetuximab, which is a chimeric
IgG1 monoclonal antibody and a competitive inhibitor of EGFR ligand binding, wasapproved for head and neck squamous cell carcinoma (HNSCC), colorectal cancer, and nonsmall cell lung cancer (NSCLC) patients by the FDA. EGFR overexpression is very common
in HNSCC patients (up to 90%) [30]. Based on the relative ease of applying light to HNSCC
tumors and the poor outcomes for patients with recurrent disease with current therapies,
this setting was the logical starting point for clinical translation of NIR-PIT. The first phase
1/2 clinical trial of NIR-PIT took place in patients with recurrent HNSCC using cetuximabIR700 conjugates and successfully concluded in late 2017 [31]. The results suggested that
EGFR-targeted NIR-PIT was more effective than currently recommended second- and thirdline therapies for HNSCC. Cetuximab-IR700 was conditionally approved and registered forclinical use as the first EGFR-targeted NIR-PIT drug in Japan in 2020. Investigator-initiatedclinical trials against EGFR-expressing esophageal and stomach cancers using cetuximabIR700 started in 2019 for extending applications of the drug. Theoretically, EGFR-targeted
NIR-PIT could be applied to other types of cancers with EGFR overexpression [32–55].

Figure 2. The positivity proportion of (A) EGFR and (B) HER2 expressions in various cancers(ca.; carcinoma, ESCC; esophageal squamous cell carcinoma, EAC; esophageal adenocarcinoma).
((A) The numbers in the brackets show reference number. (B) Quoted from references [50,51]).

3.2. Human Epidermal Growth Factor Receptor 2
HER2, also known as HER2/neu, c-erbB2, or ERBB2, is a membrane tyrosine kinase
and another member of the ErbB family [22]. HER2 was found to be amplified in a humanbreast cancer cell line 35 years ago [56]. HER2 does not bind EGF-like ligands, relying
instead on heterodimerization with other ErbB family receptors for activation, which differs from other members of the ErbB family such as EGFR [57]. HER2 overexpression
creates spontaneous receptor homodimers or heterodimers with other ErbB family receptors, resulting in activated oncogenic downstream signaling (e.g., PI3K/Akt/mTOR
and MAPK) promoting cellular proliferation, survival, and angiogenesis [58]. In breast
cancer patients, HER2 positivity rates have been reported in the range of 15% to 20% [59].
HER2 is an established therapeutic target of breast cancer and several drugs, including
trastuzumab, pertuzumab, lapatinib, neratinib, and trastuzumab emtansine (T-DM1), havebeen approved for the treatment of HER2-positive breast cancer by the FDA. HER2 has
also been detected in approximately 20% of gastric cancers [60,61]. Trastuzumab andCancers 2021, 13, 2535

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fam-trastuzumab deruxtecan-nxki for HER2 positive gastric cancer patients have been
approved by the FDA. Moreover, HER2 amplifications or mutations and HER2 overexpression have been reported in various solid tumor types (Figure 2B) [60]. For example, theoverexpression rate of HER2 has been reported in 0% to 64% of esophageal cancer [62,63].
NIR-PIT utilizing trastuzumab-IR700 has been effective in xenograft models with a humanbreast cancer cell line, in pleural dissemination models by HER2 expressing NSCLC cells,
in esophageal carcinoma cell lines in vitro, and in subcutaneous tumor models and disseminated peritoneal models with several ovarian cancer cell lines [1,64–66]. Combination
therapy of HER2-targeted NIR-PIT and conventional chemotherapy such as 5-Fluorouracil(5-FU) rapidly induced significant tumor inhibition in a gastric cancer xenograft model [67].
Moreover, NIR-PIT with trastuzumab-IR700 and pertuzumab-IR700 conjugates showedstronger antitumor effects than with either mono conjugate in a gastric cancer xenograftmodel [68]. Therefore, HER2-targeted NIR-PIT is a promising therapy for various HER2
overexpressed tumors.
3.3. Cancer Stem Cell Markers
CD44, a non-kinase transmembrane glycoprotein, is a well-known marker of cancer
stem cells (CSCs) and mediates intercellular adhesion, regulating epithelial to mesenchymaltransition and cancer progression [69]. Various types of cancers, including most head andneck, gastric, lung, colorectal, and breast cancers, and hepatocellular carcinoma express
CD44, making it a valuable diagnostic and prognostic marker [70]. Thus, CD44 is an
important target for antibody-based therapies [71]. CD44-targeted NIR-PIT significantly
suppressed tumor progression and prolonged survival in CD44-expressing syngeneic
mouse models of oral squamous cell carcinoma [72]. Moreover, combined CD44-targeted
NIR-PIT with programmed cell death protein 1 (PD-1) or CTLA4 checkpoint blockade
was more effective in combination than as single therapies in syngeneic mouse models,
including a minimally immunogenic tumor [21,73,74]. Combined CD44-targeted NIR-PIT
with PD-1 blockade eradiated more than 70% of established tumors [21]. AC133, whichis also a stem cell-specific glycosylation-dependent epitope of CD133, is a CSC marker
found in many tumors [75]. AC133-targeted NIR-PIT suppressed tumor progression
and prolonged survival for both subcutaneous and orthotopic tumor models of AC133
positive glioblastoma stem cells [76]. Thus, NIR-PIT targeting CSCs can induce significant
therapeutic responses.
3.4. Prostate-Specific Membrane Antigen
PSMA is a well-established cell membrane marker of prostate cancer, and is expressedhighest in poorly differentiated, metastatic, and hormone-refractory prostate cancer [77].
The expression level of PSMA is associated with the stage and grade of the prostate cancer,
but the expression is low in normal tissues [78]. PSMA-targeted NIR-PIT significantly
suppressed tumor growth and prolonged survival in mouse prostate tumor models [77].
Furthermore, PSMA-targeted small and bivalent antibody fragments, including diabodies(Db) and minibodies (Mb), were effective therapies using NIR-PIT. Localized prostatecancer would be amenable to focal therapy with a PSMA-directed NIR-PIT using lightfibers inserted in the prostate. Because of its selectivity, it would be unlikely to damage
nerves or sphincter muscles, yet could result in effective tumor control by direct killing andinduction of anti-tumor host immunity. Therefore, NIR-PIT targeting PSMA is a reasonableapproach for prostate cancer.
3.5. Carcinoembryonic Antigen
Carcinoembryonic antigen (CEA), a glycoprotein involved in cell adhesion, is alreadyused as a tumor marker in various cancers, and the expression levels of CEA are related
to prognosis in colorectal cancer patients [79–81]. More than 50 years after its initial
discovery, CEA is still an excellent marker for differentiating colorectal cancer from normal
tissues (98.8% of cases) and detecting positive lymph nodes [82]. Therefore, CEA is a

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promising target for cancer therapy. NIR-PIT targeting CEA suppressed tumor progression
significantly not only in a xenograft model of gastric cancer but also in the orthotopic
pancreatic tumor model without adverse effects [83,84]. For pancreatic cancer, complete
tumor resection can improve overall survival. NIR-PIT could be used as an adjuvant
to surgery, treating any residual pancreatic cancer. This was tested in a patient-derived
orthotopic xenograft model using CEA-targeted NIR-PIT [85]. Moreover, surgery with
CEA-targeted NIR-PIT reduced recurrence by targeting residual cancer cells [86]. Thus,
CEA-targeted NIR-PIT could be an adjuvant to conventional cancer therapies.
3.6. Podoplanin
Malignant pleural mesothelioma (MPM) is a malignant tumor that originates frommesothelial cells and has an extremely poor prognosis [87,88]. Podoplanin (PDPN) is a
type I transmembrane glycoprotein that is expressed in lymphatic endothelial cells, type I
alveolar epithelial cells, and podocytes of the glomeruli. The antibody (D2-40) for PDPN
has been used for a long time as a specific pathological diagnostic marker to distinguish
lymphatic vessels from blood vessels and to verify the presence of MPM [89–91]. PDPN isalso upregulated in various tumors, including MPM, angiosarcomas, chondrosarcomas,
osteosarcomas, germ-cell tumors, gliomas, glioblastomas, dysgerminomas of the ovary, andsquamous cell carcinomas of the skin, esophagus, lung, cervix, and head and neck [92,93].
For NIR-PIT, the lung and thoracic cavity can effectively transmit NIR light over long distances, because it consists mainly of air. PDPN-targeted NIR-PIT using the NZ-1 antibody
demonstrated tumor suppression in both xenograft and orthotopic MPM models [87].
3.7. Mesothelin
Mesothelin (MSLN) is a cell-surface glycoprotein and tumor differentiation marker
expressed in several tumors, including lung cancer, pancreatic cancer, ovarian cancer, andMPM [94]. MSLN can be used as a systemic diagnostic biomarker and is a target for
antibody-based therapies [94,95]. MSLN-targeted NIR-PIT has been shown to be effective
in mouse xenograft models [96]. NIR-PIT targeting MSLM is a promising candidate for thetreatment of mesothelin-expressing tumors.
3.8. Glycoprotein A33 Antigen
Glycoprotein A33 antigen (GPA33) could be another target of NIR-PIT in colorectal
cancer because it is highly expressed in over 95% of human colorectal cancers, more than
60% of gastric cancers, and 50% of pancreatic cancers [97]. Additionally, GPA33 exhibits
limited expression in the normal intestinal epithelium [98]. GPA33-targeted NIR-PIT
demonstrated tumor growth suppression in a xenograft model [99].
3.9. Tumor-Associated Calcium Signal Transducer 2
Tumor-associated calcium signal transducer 2 (TROP2) is a 46kD glycoprotein thatplays a multifunctional cellular role, including transducing of cytoplasmic calcium. TROP2
is overexpressed in many epithelial cancers, including gastrointestinal tumors, cholangiocarcinoma, and prostate and pancreatic cancer [100]. Furthermore, it corelates with a poor
prognosis in various cancers [101]. TROP2-targeted NIR-PIT inhibited tumor growth incholangiocarcinoma and pancreatic cancer models [102]. Thus, TROP2-targeted NIR-PIT isan attractive candidate for TROP2 expressing tumors.
3.10. Cadherin-17
Cadherin-17 (CDH-17), a cell surface biomarker specific for gastrointestinal cancers,
is a cell−cell adherent junctional molecule and plays an important role in cancer celladhesion, progression, and metastasis [103]. Moreover, CDH-17 is highly expressed on colorectal, gastric, and pancreatic adenocarcinoma [104]. CDH17-targeted NIR-PIT inhibitedtumor growth in a pancreatic and gastric xenograft model [105]. Thus, CDH-17 is anotherpromising agent for NIR-PIT in gastrointestinal cancers.

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3.11. Delta-Like Protein 3
Delta-like protein 3 (DLL3) is a potential therapeutic target molecule for small cell lungcancer (SCLC) and other neuroendocrine tumors that is minimally expressed in normal
tissues [106]. However, rovalpituzumab tesirine, which is the first antibody targeting
DLL3, was terminated on August 2019 because of the failure of the TAHOE (NCT03061812)
and MERU (NCT03033511) clinical trials [107]. Notwithstanding this result, NIR-PIT
targeting DLL3 in SCLC xenograft model showed marked antitumor effects [108]. NIR-PIT
targeting DLL3 using rovalpituzumab could easily be translatable into the clinic sincerovalpituzumab has already been used in a human clinical trial [109].
3.12. Glypican-3
Glypican-3 (GPC3), a 65-kDa membrane-bound heparin sulfate proteoglycan thatis highly expressed in hepatic cell carcinoma (HCC) but not in normal tissue, is being
evaluated as a potential therapeutic target for HCC [110,111]. GPC3-targeted NIR-PIT
inhibited tumor growth compared to an untreated control in a xenograft model [112].
Moreover, the combination of GPC3-targeted NIR-PIT and the nanoparticle albumin-boundpaclitaxel enhanced the therapeutic effect [113].
3.13. c-KIT
Gastrointestinal stromal tumors (GISTs) are the most common submucosal tumors(SMTs) of the alimentary tract [114]. SMTs are derived from mesenchymal cells and are
generally covered with normal mucosa, so it is difficult to distinguish GIST from other typesof SMTs by conventional endoscopy [115,116]. However, c-KIT is expressed in over 90% of
GISTs on the cell membrane; therefore, it can be a molecular target for NIR-PIT [115,116].
Moreover, c-KIT-targeted NIR-PIT using anti-CD117 antibody induced acute necrotic cell
death and was very effective in a GIST tumor model [115]. Thus, NIR-PIT targeting c-KIT
could be a novel effective technology for GISTs.
3.14. CD20
Lymphoma is a diverse group of B-cell, T-cell, and natural-killer cell tumors, but the
majority of lymphomas (approximately 90%) are of B-cell origin [117]. Malignant cells
that originate from the B-cell lineage often express B-cell differentiation antigens such as
CD19 and CD20, which can be targeted by monoclonal antibodies [118]. Utilizing antiCD20 monoclonal antibody rituximab, CD20-targeted NIR-PIT against B-cell lymphoma
significantly inhibited tumor growth [119], and the therapeutic effect of NIR-PIT was
better than that of radioimmunotherapy against a xenograft model of aggressive B-cell
lymphoma [120].
3.15. Cutaneous Lymphocyte Antigen and CD25
Mycosis fungoides is a rare cancer, however, it is the most common subtype of cutaneous T-cell lymphoma [121]. It is reported that mycosis fungoides cells express cutaneous
lymphocyte antigen (CLA) [122]. CLA-targeted NIR-PIT specifically killed a mycosis fungoides cell line in vitro [123]. Thus, NIR-PIT could treat such locoregional lymphomas.
Additionally, CD25, which is an α-subunit of interleukin-2 receptor and overexpressedin most cases of adult T-cell leukemia/lymphoma (ATLL), is thought to arise from theregulatory T cell (Treg) [124]. Therefore, CD25 is potentially a good target molecule forcutaneous lesions of ATLL and for Treg killing.
3.16. CD146
CD146 has been identified as a melanoma cell adhesion molecule. The expression of
CD146 in melanoma is high and is found in 70% of primary melanomas and 90% of theirlymph node metastases [125]. Wei et al. reported that CD146-targeted NIR-PIT inhibitedtumor growth in a CD146-positive melanoma xenograft model [126].

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3.17. H-Type Lectin/β-D-Galactose Receptors
Non-antibody mediated NIR-PIT has proven to be more difficult than antibody mediated NIR-PIT. However, there are some advantages of using non-antibody targeting agents
such as galactosyl serum albumin (GSA), because GSA as a target is ubiquitous among
ovarian cancers, whereas it is difficult to find a single antibody that will bind to all types ofovarian cancers. GSA is comprised of galactose molecules conjugated via carboxyl groups
to an albumin molecule, and it binds to β-D-galactose receptors, also called H-type lectins,
which are overexpressed on the surface of many ovarian tumors [127]. Although a large
amount of NIR light exposure was needed for effective NIR-PIT therapy because GSA
ligands were quickly internalized after binding to β-D-galactose receptors, GSA-targeted
NIR-PIT specifically killed ovarian cancer cells (SHIN3) in vitro and suppressed tumor
growth in a peritoneal disseminated model [128]. This may be especially useful when
specific antibodies are not available for ovarian cancers. The development of similar nonantibody-based forms of NIR-PIT could therefore be an effective way of extending theutility of this novel therapy to other tumors.
3.18. Programmed Death-Ligand 1
PD-1 (CD279) is a T-cell immune checkpoint that suppresses autoimmunity, leading
to immune tolerance of cells expressing programmed death-ligand 1 (PD-L1, CD274) [129].
Upregulated PD-L1 on these cells contributes to the development of T-cell exhaustion [130].
PD-L1 is expressed in various types of normal cells (including placenta, vascular endothelium, pancreatic islet cells, muscle, hepatocytes, epithelium, and mesenchymal stem cells,
as well as on B cells, T cells, DCs, macrophages, and mast cells), but it is overexpressed inmany cancer cells, including melanoma, renal cell carcinoma, NSCLC, thymoma, and ovarian and colorectal cancer [129,131]. Furthermore, PD-L1 overexpression is associated with apoor prognosis [132]. Several antibodies against PD-1/PD-L1 have recently been developed
for clinical application by the FDA. PD-L1-targeted NIR-PIT using avelumab, which isa fully humanized IgG1 anti-PD-L1 monoclonal antibody (mAb), significantly inhibitedtumor growth and prolonged survival in a xenograft model [133]. Thus, PD-L1-targeted
NIR-PIT is another potential cancer therapy.
4. NIR-PIT Targeting Non-Cancer Cells
Cancer immunotherapy is aimed at activating or boosting the activation of the immune
system to attack cancer cells through endogenous immunity mechanisms [134]. The
pioneering checkpoint inhibitor ipilimumab, which is a mAb that targets CTLA4, wasapproved for advanced melanoma in 2011 [135]. Over the past several years, other novel
immunotherapies, including checkpoint inhibitor mAbs that target PD-1 or PD-L1 andchimeric antigen receptor T-cell therapies, have been developed and approved for clinicaluse [134,136–138]. Despite these major advances, the clinical use of immunotherapies faces
several challenges related to both efficacy and safety. Although the immune upregulation
provoked by these therapies results in an enhanced antitumoral response, it can also
cause numerous inflammatory and autoimmune manifestations that can be severe, andon occasion, fatal [139]. For that reason, an alternative approach is to inhibit or kill
immunosuppressive cells only within the tumor bed but not systemically. Hence, a new
type of NIR-PIT targeting non-cancer immune-related cells is being developed.
4.1. CD25
Within the tumor tissue, T cells and natural killer (NK) cells that can recognize cancer
cells are often present in large numbers, but their cytotoxic function is suppressed by
nearby immunosuppressor cells such as Tregs [140,141]. Controlling tumor-infiltrating
CD4+ CD25+ Foxp3+ Tregs has been considered an essential step for enhancing anticancer
immune reactions [142,143]. However, when targeting CD25, activated effector cells that
also express CD25 are depleted in addition to Treg cells, and this could interfere withactivation of effector cells [144]. Furthermore, systemic blockade of immunosuppressiveCancers 2021, 13, 2535

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regulatory mechanisms runs the risk of inducing autoimmune adverse events. Thus, CD25targeted NIR-PIT was developed to selectively deplete CD4+ CD25+ Foxp3+ Tregs in tumor
beds to induce the activation of antitumor effector cells [2]. CD25-targeted NIR-PIT caused
necrotic cell death against CD25-expressing HT-2-A5E cells in vitro, but in other cancer cells,
no therapeutic effect was detected. In an in vivo syngeneic model, CD25-targeted NIR-PIT
induced regression of treated tumors with rapid activation of tumor-infiltrating CD8+ T andNK cells and activation of antigen-presenting cells. Furthermore, the therapeutic effects of
CD25-targeted NIR-PIT extended to distant non-treated tumors in a tumor-specific manner,because activated CD8+ T and NK cells were increased in the non-treated tumor after
this therapy.
CD25 is also known as the IL-2 receptor α-chain. The IL-2 receptor is expressed not
only on Tregs but also on activated lymphocytes such as CD8+ T cells and NK cells [145].
Remaining circulating anti-CD25-mAb-IR700 could block these receptors, hindering T-cell
expansion [146]. Okada et al. reported that anti-CD25-F(ab’)2 NIR-PIT was more effective
than anti-CD25-IgG NIR-PIT in suppressing tumor growth and inducing abscopal effects
in vivo. The faster clearance of anti-CD25-F(ab’)2 -IR700 and the absence of antibodydependent cellular cytotoxicity (ADCC) or complement-dependent cellular cytotoxicity(CDC) contributed to the therapeutic efficacy of CD25-targeted NIR-PIT by minimizing thecirculating APCs with their ability to block the IL-2 receptor. Thus, local CD25-targeted
NIR-PIT produces tumor type-specific, systemic antitumor effects, which alter the growthof distant tumors of the same tumor type. Since expression and regulation of CD25 has
been well investigated and known, local CD25-targeted NIR-PIT is a promising method for
reducing immunosuppression caused by Tregs and thus augmenting effector cell-mediatedcancer cell killing.
4.2. CTLA4
A monoclonal antibody against CTLA4 was approved as an immune checkpoint
inhibitor for clinical use in 2011. CTLA4 and CD28 are expressed on T cells and share
the binding ligands CD80 and CD86 [147,148]. CD28 is constitutively expressed on T
cells, but CTLA4 is expressed only on activated T cells and binds to CD80/86 on antigenpresenting cells with higher affinity than CD28 [148–150]. Thus, blockade with an antiCTLA4 antibody of the binding between CTLA4 and CD80/86 promotes priming inlymph nodes and enhances antitumor immunity [151]. CTLA4 antibody has demonstrated
dramatic responses in some patients, but it has also been reported that irAEs were observed
in up to 90% of patients treated with a CTLA4 antibody [132,152]. To address this problem,
CTLA4-targeted NIR-PIT was developed to deplete CTLA4-expressing cells only in thetumor microenvironment. CTLA4-targeted NIR-PIT caused a T-cell mediated antitumor
effect in four syngeneic tumor models of cancer and eradiated more than 50% of establishedtumors. The balance of effector T cells and Tregs after CTLA4-targeted NIR-PIT was
improved, favoring effector intratumoral CD8+ T cells and resulting in T-cell activation inregional lymph nodes. Furthermore, CTLA4-targeted NIR-PIT induced an abscopal effectin over half of the tumor-bearing mice. It is believed that residual anti-CTLA4-IR700 mightenhance antitumor immunity by promoting T-cell priming. Thus, CTLA4-targeted NIR-PIT
is a promising antitumor therapy. However, CTLA4 exists in the cytoplasm in both normal
and cancer cells to a much larger extent than CTLA4 expressed on the cellular membrane.
Therefore, it is still unclear how the effects of CTLA4-targeted NIR-PIT affect these cellswith the large reserve of intracellular CTLA4.
4.3. CD206
Tumor-associated macrophages (TAMs) play essential roles in tumor invasion andmetastasis, and contribute to drug resistance [153]. TAM levels are correlated with poor
prognosis in various human cancers and are a major factor in the development of resistanceto chemo- or radiotherapy, as well as tumor recurrence [154]. TAM-targeted NIR-PIT using
an anti-CD206 antibody inhibited the growth of subcutaneous tumors and prevented theirCancers 2021, 13, 2535

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metastasis to the lungs in a tumor model in which M2 macrophages were induced withintumors by sorafenib treatment [153].
4.4. Fibroblast Activation Protein
Cancer-associated fibroblasts (CAFs) are the most abundant cell population in thetumor microenvironment (TME), and are thought to play an essential role in tumor progression, angiogenesis, metastasis, and immunosuppression [155–158]. Fibroblast activation
protein (FAP), a specific surface marker of CAFs, is recognized as the leading target
molecule for CAFs. Several reports have already demonstrated the efficacy of systemicallydepleting or targeting FAP+ cells in reducing tumor and stroma growth; however, serious
side effects have been observed in these approaches, such as cachexia and anemia [159].
Since rapid cell death was induced specifically and locally for CAFs, FAP-targeted NIR-PIT
suppressed tumor progression and overcame chemoresistance in an esophageal tumor
model without adverse effects [160]. Thus, the combined use of FAP-targeted NIR-PIT withconventional anticancer treatments can be expected to provide a more effective treatment
strategy [157,160].
4.5. CD47
CD47, a cell-surface protein that prevents cancer cell phagocytosis via the “don’t eatme” signaling, is highly expressed on several human cancers, including acute myeloid
leukemia, non-Hodgkin’s lymphoma, and bladder cancer [161]. CD47-targeted NIR-PIT increased direct cancer cell death and phagocytosis, resulting in inhibited tumor growth andimproved survival in a murine xenograft model of human bladder cancer [162]. Similarly,
calreticulin is a cell surface protein that confers an “eat me” signal known to counterbalance
the “don’t eat me” signal conferred by CD47 [161]. For this reason, increased prophagocytic calreticulin expression through CD47-targeted NIR-PIT could provide additional
tumor cytotoxicity.
4.6. Vascular Endothelial Growth Factor Receptor
Angiogenesis is regulated principally by interactions between vascular endothelial
growth factors (VEGFs) and vascular endothelial growth factor receptors (VEGFRs), andplays a key role in cancer growth and metastasis in various malignant tumors [163,164].
The overall survival in patients with metastatic or unresectable gastric cancer was significantly improved by treatment with an anti-VEGFR-2 antibody, ramucirumab, which
was approved by the FDA [165,166]. Applying the theory of NIR-PIT, VEGFR2-targeted
NIR-PIT led to reduced tumor growth and vascularity without phototoxic effects [167,168].
The application of NIR-PIT utilizing ramucirumab is anticipated to lead to an enhanced
antitumor effect.
5. NIR-PIT Targeting Forced Expression of New Specific Transmembrane Antigens
When no good target antigen exists on a cancer cell, gene transfer technology could
be used to make cells express an artificial target antigen, which could then be easily
targeted with an APC. For example, HER2-targeted NIR-PIT has promising antitumor
effects in HER2-positive cancers [1]. However, HER2 positivity rates in breast cancer
patients have been reported in the range of 15% to 20% [59]. The HER2-positive proportionin gastric cancers was estimated at only 12.2 to 22.1% [169], and gastric cancer itself
exhibits intertumoral HER2 heterogeneity [170]. To overcome the lack of molecular target
antigens, a replication-deficient adenoviral vector containing a gene that encodes the HER2
extracellular domain (HER2-ECD) was developed, and resulted in expression with HER2ECD on the surface of HER2-negative breast and gastric cancer cells [171]. The expressed
HER2-ECD did not trigger HER2 signaling pathways because of the lack of the HER2
intracellular kinase domain. Although exogenous HER2-ECD expression had no obvious
effect on the signaling pathway, ADCC activity by trastuzumab was strongly enhanced.
These results indicate that artificial exogenous domains expressed on the cell surface can beCancers 2021, 13, 2535

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targets for NIR-PIT cancer treatment. Using this theory, HER2-targeted NIR-PIT induced
direct cell membrane destruction of HER2-ECD-transduced HER2-negative breast cancer
cells [172]. Furthermore, in a mouse model of peritoneal dissemination of HER2-negative
gastric cancer, the combination of HER2-ECD administration and HER2-targeted NIR-PIT
inhibited peritoneal metastasis and prolonged survival without severe adverse effects [173].
However, an obvious flaw with this strategy is that it might be difficult to transfer a geneto all cancer cells. Therefore, none of the athymic mice were cured with either HER2-ECDtargeted ADCC or NIR-PIT. However, under normal immunity, as a consequence of themassive ICD induced by NIR-PIT, patients might acquire immunity against neo-antigens onunresponsive cancers without expressing recognizable transmembrane antigens other than
HER2, and this could result in successful treatment of the cancers. Thus, the combinationof gene transfer technology in which a target antigen is introduced into the cell followedby targeted NIR-PIT would expand and potentiate molecular-targeted therapy even fortarget-negative or attenuated cancer cells.
6. Combination Therapy of NIR-PIT
NIR-PIT can simultaneously target two or more kinds of cells by co-injecting two or
more different APCs, followed by exposure to NIR light. The combination approach for
NIR-PIT is greatly advantageous, as it allows for targeting a broader spectrum of tumors
more effectively, irrespective of enrichment of any one specific target. The combination
NIR-PIT using panitumumab-IR700 and trastuzumab-IR700 was more efficacious thaneither monotherapy alone in a bladder cancer xenograft model [28]. Furthermore, even if
the target is similar, when two different epitopes of HER2 were targeted on human gastriccancer cells using trastuzumab-IR700 (against domain IV of HER2) and pertuzumab-IR700(against domain II of HER2), the combination NIR-PIT demonstrated synergy in a HER2expressing xenograft model [68]. Moreover, this theory of NIR-PIT can be applied to target
not only other tumor markers but also other types of cells in combination. Combined
CD44- and CD25-targeted NIR-PIT simultaneously eliminates cancaer cells and Tregs
in tumors. The combined NIR-PIT induced significant tumor growth inhibition andresulted in prolonged survival compared to either type of NIR-PIT alone in several allograftmodels [174]. Thus, this combined regimen of NIR-PIT is another effective approach,especially for tumors with high target antigen expression and with abundant Tregs, toinduce long-term antitumor immunity.
7. Conclusions
We reviewed various antibodies that have been used for NIR-PIT, emphasizing thebroad spectrum of tumor antigens that can be targeted and introducing targeting of cells in
the tumor microenvironment (Figure 3). NIR-PIT has the advantage of adapting to cancers
in any organs where NIR light reaches. In the near future, NIR-PIT has great potential totreat a wide variety of cancers by targeting appropriate cancer cells, immune cells, or both.

Cancers 2021, 13, 2535

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Figure 3. Target molecule of NIR-PIT developed for cancer therapy. NIR-PIT can target not only
various surface molecules expressing cancer cells but also immunosuppressive cells that compose
the tumor microenvironment, such as regulatory T cells, tumor-associated macrophages, cancerassociated fibroblasts, and neovascularity.
Author Contributions: Conceptualization, T.K. and H.K.; investigation, T.K. and H.W.; resources,
H.K.; writing—original draft preparation, T.K. and H.W.; writing—review and editing, A.F., P.L.C.
and H.K.; visualization, T.K.; supervision, P.L.C. and H.K.; project administration, H.K.; funding
acquisition, H.K. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Intramural Research Program of the National Institutes of
Health, National Cancer Institute, and Center for Cancer Research (ZIA BC 011513).
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Conflicts of Interest: The authors have no conflict of interest to disclose.

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