Applying near-infrared photoimmunotherapy to B-cell lymphoma: comparative evaluation with radioimmunotherapy in tumor xenografts
Applying near-infrared photoimmunotherapy to B-cell lymphoma: comparative evaluation with radioimmunotherapy in tumor xenografts
Yusri-Dwi Heryanto 0 1
Hirofumi Hanaoka 0 1
Aiko Yamaguchi 0 1
Yoshito Tsushima 0 1
Takahito Nakajima 0 1
0 Department of Bioimaging Information Analysis, Gunma University Graduate School of Medicine , 39-22 Showamachi 3-chome, Maebashi 371-8511 , Japan
1 Department of Diagnostic Radiology and Nuclear Medicine, Gunma University Graduate School of Medicine , 39-22 Showa-machi 3-chome, Maebashi 371-8511 , Japan
Objective Radioimmunotherapy (RIT) has proven effective for patients with relapsed and refractory lymphoma. However, new types of therapy are strongly desired as B-cell lymphoma remains incurable for many patients. Photoimmunotherapy (PIT) is an emerging targeted cancer therapy that uses photosensitizer (IR700)-conjugated monoclonal antibodies (mAbs) to specifically kill cancer cells. To evaluate the usefulness and potential role of PIT for treating B-cell lymphoma in a comparison with RIT, we performed in vivo PIT and RIT studies with an IR700 or 90Y-conjugated anti-CD20 mAb, NuB2. Methods IR700 or 90Y were conjugated to NuB2. Since cell aggressiveness greatly affects the therapeutic effect, we selected both an indolent (RPMI 1788) and an aggressive (Ramos) type of B-cell lymphoma cell line. The in vitro therapeutic effect of PIT and the biodistribution profiles of IR700-NuB2 were evaluated. In vivo PIT and RIT studies were performed with 100 or 500 lg of IR700NuB2 and 150 lCi/20 lg of 90Y-NuB2, respectively, in two types of B-cell lymphoma-bearing mice. Results The in vitro studies revealed that Ramos was more sensitive than RPMI 1788 to PIT. The therapeutic effect of PIT with 500 lg IR700-NuB2 was superior to any other therapies against aggressive Ramos tumors, whereas RIT showed the highest therapeutic effect in indolent RPMI 1788 tumors. Since the uptake levels and intratumoral distribution of IR700-NuB2 were comparable in both tumors, a possible cause of this difference is the tumor growth rate. The PIT with 500 lg (IR700-NuB2) group showed a significantly greater therapeutic effect than the PIT with 100 lg group due to the higher and more homogeneous tumor distribution of IR700-NuB2. Conclusions PIT was effective for both indolent and aggressive B-cell lymphoma, and the higher dose provided a better therapeutic effect. In aggressive tumors, PIT was more effective than RIT. Thus, PIT would be a promising strategy for the locoregional treatment or control of B-cell lymphoma. Since PIT and RIT have distinctive advantages over each other, they could play complementary rather than competitive roles in B-cell lymphoma treatment.
B-cell lymphoma; Anti-CD20 antibody; Radioimmunotherapy; Photoimmunotherapy
Lymphoma is the most common hematological
malignancy with more than 450,000 new cases per year and
225,000 cancer deaths per year worldwide [
Lymphoma is a diverse group of B-cell tumors, T-cell
tumors, and natural-killer cell tumors, and the majority
of lymphomas (approx. 90%) are of B-cell origin [
The cornerstone of B-cell lymphoma treatment is
chemotherapy as a single agent or in combination with
immunotherapy and radiation therapy, which provide a
great effect in many cases. However, the relapse rate of
B-cell lymphoma is usually high, and sometimes
recurring tumors obtain resistance to these conventional
treatments. Thus, novel targeted therapies have been
developed in preclinical and clinical practice.
Radioimmunotherapy (RIT) with tumor-specific
monoclonal antibodies (mAbs) has proven effective for
patients with relapsed and refractory lymphoma [
RIT, mAbs are coupled to radioisotopes for delivering
cytotoxic radiation exposure specifically to lymphoma
cells. Two commercially available radiolabeled
anti-CD20 mAbs, 90Y-ibritumomab tiuxetan (Zevalin ) and
131Itositumomab (Bexxar ), demonstrated superior
therapeutic response compared to mAb alone [
RIT showed a potential for the treatment of aggressive
], it is currently approved only for
‘indolent’ (i.e., slow-growing) lymphoma. Tumor recurrence
is observed in approximately half of responders within
1 year after treatment even among indolent lymphomas,
although the response rate to RIT is as high as 70% [
Thus, B-cell lymphoma remains incurable for many
patients, and new and more effective types of therapy
are strongly desired.
Photoimmunotherapy (PIT) is an emerging targeted
cancer therapy that uses mAbs as a vehicle to deliver a
cytotoxic agent specifically to the tumor, as in RIT [
PIT uses mAbs conjugated with a photosensitizing
nearinfrared (NIR) phthalocyanine dye, IRDye700DX
(IR700). By irradiating NIR light to a tumor, the
photosensitizer-conjugated antibody-bound target cells are
specifically killed. PIT is relatively safe compared to
external radiotherapy and RIT because NIR light itself is
harmless and the photosensitizer-conjugated antibody
causes no toxicity without activation by NIR light. In
animal studies, PIT showed a very promising therapeutic
effect on many types of tumors including Burkitt’s
]. An early-phase clinical trial of PIT is
being conducted in patients with head and neck cancers.
However, further research is needed to assess the
usefulness of PIT for B-cell lymphoma, since PIT for
indolent lymphoma has not been evaluated and no
comparative study with existing methods has been
Both RIT and PIT use anti-tumor mAbs for delivering a
cytotoxic agent (a radionuclide or photosensitizer)
specifically to the tumor, but no study has compared and
contrasted the effectiveness of RIT and PIT. In this study, we
performed in vivo RIT and PIT experiments with 90Y- or
IR700-conjugated anti-CD20 mAbs. Since cell
aggressiveness plays a role in therapeutic effects, we selected
both an indolent-type and an aggressive-type B-cell
lymphoma cell line for tumor xenografts. Based on our results,
we discuss the usefulness and potential role of PIT for
B-cell lymphoma in comparison with RIT.
Materials and methods
IRDye700DX (IR700) NHS ester was obtained from
LICOR Biosciences (Lincoln, NE, USA). A murine
antiCD20 monoclonal antibody, NuB2, was kindly supplied by
Immuno-Biological Laboratories Co. (Takasaki, Japan).
All other chemicals were of reagent grade.
Synthesis of IR700–NuB2
The conjugation of IR700 NHS ester to NuB2 was
performed as described [
]. In brief, NuB2 (1 mg/500 ll,
6.7 nmol) was incubated with IR700 NHS ester (65 lg,
33.3 nmol) in 0.1 M Na2HPO4 (pH 8.5) at room
temperature for 2 h. The reaction mixture was purified with a
Sephadex G25 column (PD-10; GE Healthcare,
Piscataway, NJ, USA). The mAb concentration was determined
with a NanoDrop 1000 Spectrophotometer (Thermo
Scientific, Wilmington, DE, USA) by measuring the
absorption at 280 nm. The concentration of IR700 was measured
by absorption at 689 nm to confirm the number of
fluorophore molecules per mAb. The synthesis was controlled
to attach approximately 3–4 IR700 molecules to a single
Synthesis of radiolabeled NuB2
acid (SCN-Bn-DTPA; Macrocyclics, Dallas, TX, USA)
was used for labeling NuB2 with 90Y. SCN-Bn-DTPA in
dimethylformamide was added to NuB2 at 5 mg/ml in
50 mM borate-buffered saline (pH 8.5) at the molar ratio of
5:1. After incubation at 37 C for 24 h, DTPA–NuB2 was
purified using a Bio-Spin column (Bio-Rad Laboratories,
Hercules, CA, USA). For radiolabeling, 25–50 ll of a
solution of 90YCl3 (37 MBq, Nuclitec, Braunschweig,
Germany) was incubated with 50–100 ll of 0.25 M acetate
buffer (pH 5.5) for 5 min at room temperature, followed by
incubation with 100 lg of DTPA–NuB2 for 1 h at 40 C.
The 90Y-labeled antibody was purified using a Bio-Spin
column or PD-10 column. The radiochemical purity of
90Y-NuB2 was confirmed as [95% by Tec-Control
Chromatography Strips (Biodex Medical Systems, Shirley, NY,
USA) developed with saline. Iodine-125-labeled antibodies
were prepared according to standard protocols for the
Briefly, 740 kBq/2 ll of Na125I (PerkinElmer, Waltham,
MA, USA) and 1 lg of chloramine-T in 1 ll of 0.3 M
phosphate buffer were added to 40 lg of mAb in 100 ll of
0.3 M phosphate buffer. The 125I-labeled mAb was purified
using a Bio-Spin column.
We obtained the aggressive B-cell lymphoma cell line
Ramos (Burkitt’s lymphoma) and the indolent B-cell
lymphoma cell line RPMI 1788 (B lymphoblast) from the
American Type Culture Collection (ATCC, Manassas, VA,
USA). Cells were grown in RPMI 1640 medium (Wako
Pure Chemical Industries, Osaka, Japan) supplemented
with 10% heat-inactivated fetal bovine serum (Japan
Bioserum, Hiroshima, Japan) and 1% penicillin/streptomycin
(Wako Pure Chemical Industries) in tissue culture flasks.
Cell binding assay
A solution of 125I-NuB2 (containing 0.01 lg of antibody) was
added to 100 ll of cell suspension of Ramos or RPMI 1788
(2 9 106) cells with various amounts of nonlabeled antibody.
After incubation for 1 h at room temperature, the cell
suspension was centrifuged at 3000 rpm for 5 min. After the
supernatant was removed, the radioactivity of the cell fraction
was measured with a well-type c-counter (ARC7001; Hitachi
Aloka Medical, Tokyo, Japan) and compared with the initial
radioactivity. Bmax (antigen expression level) and Kd values
were determined by a Scatchard plot analysis. The
immunoreactivity of DTPA–NuB2 or IR700–NuB2 was
evaluated by the same methods using 125I-DTPA–NuB2 and
In vitro PIT
One hundred thousand cells were seeded into 12-well
plates with phenol red-free RPMI 1640 medium (Wako
Pure Chemical Industries) and incubated overnight. IR700–
NuB2 was then added to the culture at the final
concentration of 10 lg/ml. The cultures were incubated for 1 h.
After being washed with phosphate-buffered saline (PBS),
the cells were suspended again in the phenol red-free
medium. We divided the cell suspensions into one control
group and four treatment groups. The treatment groups
were irradiated with an NIR light-emitting diode
(Marubeni America, Santa Clara, CA, USA), which emits light
at the range 680–700 nm wavelength with different energy
density values: 0 (IR700–NuB2 only), 2, 5, and 10 J/cm2
(irradiation time was 40, 100 and 200 s, respectively). The
energy density was measured by optical power meter (PM
100; Thorlabs, Newton, NJ, USA). For the determination of
the cytotoxic effects of PIT, at 1 h after the light exposure,
propidium iodide (PI; Dojindo, Kumamoto, Japan) was
added to the cell suspension at the final concentration of
2 lg/ml and incubated at room temperature for 15 min.
Flow cytometry was performed with an Attune Acoustic
Focusing Cytometer (Applied Biosystems, Foster City, CA,
USA). The percentage of dead cells in the treatment group
was obtained by subtracting the mean percentage of dead
cells in the corresponding control group from the measured
Animal and tumor models
The animal experiments were conducted in accordance
with our institutional guidelines and were approved by the
institutional Animal Care Committee. Six- to
eight-weekold female KSN nude mice (Japan SLC, Shizuoka, Japan)
were inoculated with 3 9 106 of Ramos cells or 1 9 107 of
RPMI 1788 cells. Mice were monitored every 2 days for
their general health and tumor volumes.
For the determination of the tumor volume, the length
and the width were measured with an external caliper.
Tumor volumes were determined by the following formula:
tumor volume = length 9 width2 9 0.5. In vivo studies
were performed after the tumor volume reached
approximately [200 mm3. The average tumor volume of the
Ramos and RPMI 1788 tumors for the therapeutic studies
were 335.8 ± 3.8 and 312.3 ± 3.7 mm3, respectively.
In vivo PIT and RIT
Mice were randomly assigned to five groups of at least five
animals per group. These five groups were: (1) the control
group (no treatment), (2) the Ab-only group (IR700–NuB2
100-lg injection without NIR light exposure), (3) the
PIT100 group (PIT with IR700–NuB2 100-lg injection),
(4) the PIT500 group (PIT with IR700–NuB2 500-lg
injection), and (5) the RIT group (90Y-NuB2 150
lCi/20lg injection). NIR light was administered on day 1 and day
2 after the injection of IR700–NuB2 with the energy
density values 50 J/cm2 (irradiation time 17 min) and 100 J/
cm2 (irradiation time 34 min), respectively. Mice were
anesthetized with isoflurane during the procedure. Serial
fluorescence images, as well as white light images, were
obtained before and after each NIR light exposure (day 1
and day 2) using a Maestro In vivo Imaging System (CRi,
Woburn, MA) with an excitation filter at 671–705 nm and
the emission filter at 700 nm longpass. The images were
analyzed using ImageJ ver. 1.50i software [
]. The mice
were then monitored every 2 days for their general health
and tumor volumes. After 30 days or when a tumor volume
reached 2000 mm3, the mice were euthanized. The relative
tumor size was calculated by dividing the tumor volume by
each initial tumor volume. A complete response (CR) was
assigned when the relative tumor size became\0.1 and had
not increased within 30 days. A partial response (PR) was
assigned when the relative tumor size became \0.7. The
time to tumor progression was defined as the time when the
relative tumor size reached 2.5 [
Biodistribution studies were performed when the tumor
size became similar to that in the therapeutic studies
described above. A mixture of 125I-IR700–NuB2 and cold
IR700–NuB2 (10 kBq, protein dose: 100 or 500 lg) was
injected via the tail vein into tumor-bearing mice, and the
biodistribution studies were performed at 24 h after the
injection. Organs of interest were excised and weighed, and
the radioactivity was measured with a well-type c-counter.
The uptake data were calculated as the percentage-injected
dose per gram of tissue (%ID/g).
Fluorescence microscopy studies
Tumor xenografts were excised from nude mice 24 h after
the injection of IR700–NuB2 (100 or 500 lg) and
embedded in the OCT compound. Frozen sections (80 lm
thick) were prepared and fluorescence was assessed by
fluorescence microscopy (BZ9000; Keyence, Osaka,
Japan). Hematoxylin and eosin (HE) staining was then
Data are expressed as the mean ± standard error of the
mean (SEM). Statistical analyses were carried out using
IBM SPSS Statistics 22 software (IBM, Armonk, NY,
USA) and R Programming Language ver. 3.3.2 software.
We used unpaired t tests to compare differences between
pairs of groups. Differences in the relative tumor size at
day 8 and day 14 among the groups were evaluated by
oneway ANOVA followed by Tukey’s multiple comparison to
compare the effectiveness of the different treatments. We
used Spearman’s rank correlation coefficient to analyze the
correlation between the dead cell ratio and the power
intensity of NIR light. The Wilcoxon signed rank test was
used to test the differences in fluorescence intensity
between the PIT100 and PIT500 groups. Values of
p \ 0.05 were considered significant.
and 1.28 9 105 molecules per cell, respectively.
125IDTPA–NuB2 and 125I-IR700–NuB2 also showed binding
to cells and was inhibited by each of the NuB2 conjugates.
The calculated Kd values of NuB2, DTPA–NuB2 and
IR700–NuB2 toward Ramos cells were approximately
2.69 9 10-9, 5.54 9 10-9, and 5.33 9 10-9 M,
respectively, indicating that DTPA or IR700 conjugation has little
effect on the immunoreactivity of NuB2.
The percentage of cell death was significantly correlated
with the NIR light dose for both the Ramos (r = 0.88,
p \ 0.05) and RPMI 1788 cells (r = 0.90, p \ 0.05). We
found no significant difference between the control group
and the group treated with IR700–NuB2 only. The
percentage of cell death of Ramos cells was significantly
higher than that of RPMI 1788 cells at the same light
intensity (Fig. 1). For example, when exposed to 10 J of
NIR light, the percentage of dead Ramos cells was
approximately 26 ± 3.2%, whereas that in the RPMI 1788
cells was only 13 ± 0.3%.
In vivo PIT and RIT in Ramos tumor-bearing mice
One day after the injection of IR700–NuB2, the tumor
showed high fluorescence intensity (Fig. 2). After the first
exposure to NIR light, the fluorescence signal was
decreased due to the washout of IR700–NuB2 from dead
cells and partial photobleaching (Fig. 2a). IR700–NuB2
reaccumulated in the tumors by the time point before the
second NIR light exposure, though the intensity was lower
than that before the first NIR light exposure. The
fluorescence intensity of the PIT500 group was significantly
higher than that of the PIT100 group (p = 0.012),
The binding of 125I-NuB2 to Ramos and RPMI 1788 cells
was inhibited by NuB2 in a concentration-dependent
manner. The calculated CD20 expression levels of the
Ramos and RPMI 1788 were approximately 1.02 9 105
Fig. 1 The percentage of dead cells after treatment with PIT in vitro.
The data are mean ± SEM. There are significant differences between
dead cells in Ramos and RPMI 1788 cells at the same light intensity
(*p \ 0.05)
suggesting that as the dose of IR700–NuB2 increased, the
tumor accumulation of it increased.
Tumor growth was significantly inhibited in all four
treatment groups compared to the control group (Fig. 3).
Treatment with PIT (PIT100 and PIT500) and RIT resulted
in significantly better results compared to IR700–NuB2
alone (Ab only) on day 14 (p \ 0.05). The time to tumor
progression of the PIT500 group was significantly longer
than that of the RIT or PIT100 groups (25 ± 0.81,
than that of the PIT100 group on day 1 before PIT (*p = 0.012).
b Typical fluorescence image on day 1 before and after PIT in the
PIT100 group and PIT500 group
16 ± 0.80 and 14 ± 0.40 days, respectively, p \ 0.05).
Importantly, five of the six mice in the PIT500 group and
three of the eight mice in the PIT100 group showed PRs
after treatment, whereas no mice in the RIT or Ab-only
group showed tumor size reduction.
In vivo PIT and RIT in RPMI 1788 tumor-bearing mice
Similarly to the Ramos tumors, IR700–NuB2 accumulated
in the RPMI 1788 tumors at 1 day after the injection, and
the fluorescence signal decreased by PIT treatments
(Fig. 4). The accumulation of IR700–NuB2 in the PIT500
group was significantly higher than that in the PIT100
group (p = 0.001).
On average, the time to tumor progression of the RPMI
1788 control group was much slower than that of the
Ramos control group (10.7 ± 0.66 and 4.5 ± 0.22 days,
respectively, p \ 0.01). Tumor growth was significantly
inhibited in all four treatment groups compared to the
control groups (Fig. 5). The response rates to each
treatment are summarized in Table 1. RIT treatment was the
most effective compared to the other groups, and all mice
in the RIT groups achieved a CR after treatment. The
effectiveness of treatment in the PIT500 group was better
than that of the PIT100 group. The effectiveness of PIT and
RIT was more prominent in the RPMI 1788 tumors
compared to the Ramos tumors.
Biodistribution and fluorescence microscopy results
At 24 h after the injection, 125I-IR700–NuB2 showed
accumulation in both the Ramos and RPMI 1788 tumors
(Fig. 6). In both Ramos tumor-bearing mice and RPMI
1788 tumor-bearing mice, the tumor accumulation level of
125I-IR700–NuB2 (% dose/g) was not significantly
different between the use of 100 and 500 lg of cold IR700–
higher than that of the PIT100 group on day 1 before PIT
(*p = 0.001). b Typical fluorescence image on day 1 before and
after PIT in the PIT100 and PIT500 groups
NuB2. These results indicated that the amount of IR700–
NuB2 accumulated in the tumor was larger in the 500
lginjected group than in the 100 lg-injected group, since the
total protein amount could be estimated by multiplying the
protein dose by the % dose. The accumulation level of
125IIR700–NuB2 in the RPMI 1788 tumors was significantly
higher than that in the Ramos tumors with the 500-lg dose
of cold IR700–NuB2 injection (p \ 0.05), but not
significantly higher with the 100-lg dose.
Ex vivo fluorescence imaging demonstrated high
accumulations of IR700–NuB2 in the tumors at 24 h after
injection (Fig. 7). The intratumoral distributions of IR700–
NuB2 in the 500 lg-injected group were more
homogeneous compared to the 100 lg-injected group, throughout
Fig. 6 Biodistribution of 125I-IR700–NuB2 with 100 or 500 lg of
cold IR700–NuB2 in Ramos or RPMI 1788 tumor-bearing mice 24 h
after injection. Data were calculated as the percentage of injected
dose per gram of tissue and are represented as the mean ± SEM
(n = 4). The tumor accumulation level in the RPMI 1788 tumors was
significantly higher than that in the Ramos tumors treated with an
injection of 500 lg of cold IR700–NuB2 (*p \ 0.05)
In this study, we selected the anti-CD20 antibody NuB2 and
prepared an antibody–radionuclide or
antibody–photosensitizer conjugate. Our experiments revealed that (1) PIT is
effective for both types of B-cell lymphoma and (2) the
therapeutic effect of PIT was better than that of RIT in
aggressive lymphoma. In addition, PIT has a potential to treat
larger tumors since a high-dose injection improves PIT’s
therapeutic effect without inducing significant toxicity to
normal organs. We observed herein that the therapeutic effect
of PIT was better than that of RIT in aggressive tumors but not
in indolent tumors.
row injected with 100 lg of IR700–NuB2. Lower row injected with
500 lg of IR700–NuB2
In our in vitro and in vivo PIT studies, PIT showed a
distinctive effect on Ramos and RPMI 1788 cell lines and
mouse xenograft models. In the aggressive Ramos tumors,
the therapeutic effect of PIT with 500-lg IR700–NuB2 was
superior to any other therapeutic interventions including
RIT, whereas in the indolent RPMI 1788 tumors, RIT
showed the highest therapeutic effect. The effectiveness of
PIT and RIT was more prominent in the RPMI 1788
tumors compared to the Ramos tumors. Although
125IIR700–NuB2 showed significantly higher accumulations in
the RPMI 1788 tumors compared to the Ramos tumors
with 500 lg IR700–NuB2, the amount of IR700–NuB2
accumulated in the Ramos tumor with 500-lg injection
would be larger than in the RPMI 1788 tumor with 100-lg
injection. Nevertheless, the PIT100 group of RPMI 1788
tumors showed better therapeutic effects than the PIT500
group of Ramos tumors. In addition, IR700–NuB2 showed
comparable intratumoral distributions in both tumor types.
Thus, a possible cause of this difference of therapeutic
effect is the differing properties of the two cell lines used,
such as tumor growth rate and sensitivity, rather than the
tumor accumulation levels of antibody.
None of the treatments was able to eliminate the Ramos
tumors, in part because the tumors’ growth rate exceeded
the cell death rate. Since both Ramos cells and RPMI 1788
cells have comparable expression levels of CD20 and since
IR700–NuB2 has a high affinity to CD20, a similar amount
of IR700–NuB2 would bind to both cell lines. However,
PIT had a greater therapeutic effect in vitro on Ramos cells
than RPMI 1788 cells. Thus, Ramos cells are more
sensitive to PIT than RPMI 1788 cells. Therefore, although
Ramos cells are more sensitive to PIT, the Ramos tumors
started to regrow earlier compared to the RPMI 1788
tumors, which is likely to be simply because of the Ramos
tumors’ faster growth rate.
To induce phototoxicity by PIT, a certain amount of
IR700–mAb should bind to the target [
]. The cytotoxic
effect of PIT can be enhanced by increasing the level of
bound IR700–mAb [
]. However, due to the
heterogeneity in the expression levels of CD20 and the physical
inaccessibility to IR700–NuB2, it would be difficult to
deliver an adequate amount of IR700–NuB2 to every cell.
Thus, because of the heterogeneous distribution of IR700–
NuB2, a minimal number of cells that escaped immediate
cell death after NIR light exposure would have survived
and started to regrow. Once an adequate number of IR700–
NuB2 is delivered to all cells, PIT would be the more
effective therapy not only for indolent but also for
aggressive B-cell lymphoma.
We employed a 59 higher dose of IR700–NuB2
(500 lg) in addition to the conventional dose (100 lg)
because we used tumors that were [49 larger ([200 mm2)
compared to the tumors described in previous reports
(\50 mm2) [
7, 12, 16
]. Consistent with an earlier study [
in our experiment the frozen sections in the PIT500 group
revealed higher and more homogeneous distributions of
IR700–NuB2 in the tumors compared to the PIT100 group.
As a result, the PIT500 group showed a significantly
greater therapeutic effect compared to the PIT100 group.
Unlike the use of a radiolabeled mAb where normal organ
toxicity restricts the higher-dose injection, high-dose
injection is possible for PIT because IR700–mAb itself
causes no phototoxicity without NIR light exposure. Our
results thus indicate that PIT with the higher dose has the
potential to treat tumors larger than those previously
reported. Even if IR700–mAb is unable to reach the deeper
part of a tumor at first, repeated light exposure would be
beneficial to treat the whole tumor. Since PIT can increase
the permeability of tumor vessels (as the superenhanced
permeability and retention (SUPR) effect [
]), a high
amount of IR700–mAb remaining in the circulation will be
delivered into the deep part of the tumor after NIR light
The tumor distribution of 90Y-NuB2 would be
heterogeneous since the protein dose was low (20 lg). However,
since RIT has a crossfire effect, 90Y-NuB2 showed a
therapeutic effect in both lymphoma models. Based on
specific activity of 90Y-NuB2, protein dose of 150 lCi
90Y-NuB2 was adjusted to 20 lg. Although the distribution
pattern of the antibody in the tumor would be improved by
increasing the protein dose, the excess amount of unlabeled
protein would decrease the tumor accumulation level of the
radiolabeled protein [
], which leads to the decrease in the
therapeutic effect. Higher dose of radioactivity would
improve the therapeutic effect of RIT; however, it is known
that the maximum tolerated dose for tumor-bearing mice is
less than 200 lCi. Indeed, in our previous RIT studies,
some mice have died after 200 lCi injection due to the
]. Therefore, we decided to use 150 lCi in
this study. Consistent with the accumulated clinical
evidence, RIT greatly suppressed the tumor growth rate of
indolent RPMI 1788 tumors compared with the aggressive
Ramos tumors. Since the therapeutic effect of RIT depends
on the total radiation dose, the Ramos cells could grow
before a lethal dose of radiation was delivered, which led to
the relatively early regrowth of the Ramos cells. On the
other hand, since the growth rate of the RPMI 1788 cells
was slow, there was enough time to deliver a lethal dose of
radiation to all cells. For indolent lymphomas, RIT should
be selected prior to PIT, because of RIT’s excellent
therapeutic effect and convenience (it requires only a single
injection). However, PIT might be an option for large
tumors for which the outcome of RIT is inadequate [
The limited tissue penetration of NIR light
(approximately 2 mm) is the main disadvantage of PIT. In many
lymphoma cases, PIT may not be a feasible approach
because these tumors are usually located in deep tissues.
However, a light fiber can be extended to a lymphoma
located in deep tissues with the use of a device such as an
endoscope, laparoscope, or image-guided percutaneous
needle. Although limited penetration may also be a
significant challenge for large tumors, a light fiber could
potentially treat a lesion as large as a cylinder-shaped area
with a diameter of 4 cm and a height of the fiber’s length.
Thus, with the use of multiple light fibers, we can expose
the entire region of large tumors. Since PIT requires NIR
light exposure to each lesion, it might be perceived as a
local therapy rather than systemic therapy. However, PIT
causes cell rupture and release of many molecules from the
cytosol and nucleus which could induce maturation of the
dendritic cells and activating a patient’s innate immune
], and it thus may be able to kill entire cancer
cells distributed throughout the body. Another possible
treatment for disseminating tumors is combination therapy
with other systemic therapy like chemoradiation therapy,
because PIT can cause a rapid and strong therapeutic effect
against locoregional tumors without inducing significant
toxicity to normal organs.
To our knowledge, this is the first report that directly
compared the therapeutic effects of PIT and RIT. Our
findings indicate that PIT and RIT are complementary
rather than competitive. PIT, RIT or both should be
selected on a case-by-case basis for patients considering
the characteristics of each tumor such as its type, size,
number, site, and more. Although the precise differences in
the therapeutic effects of PIT and RIT for other tumor types
remain to be established, the present study demonstrated
that PIT and RIT can be complementary treatments.
Photoimmunotherapy was effective for both indolent and
aggressive B-cell lymphomas, and PIT with a high dose of
IR700–NuB2 showed a better therapeutic effect and
potential to treat large tumors. The therapeutic effect of
PIT was better than that of RIT against aggressive tumors,
but not indolent tumors. PIT could thus be a promising
strategy for the locoregional treatment of lymphomas.
Since PIT and RIT each has unique advantages, they could
play complementary rather than competitive roles in B-cell
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1. Ferlay J , Soerjomataram I , Dikshit R , Eser S , Mathers C , Rebelo M , et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012 . Int J Cancer . 2015 ; 136 : E359 - 86 .
2. Sabattini E , Bacci F , Sagramoso C , Pileri SA . WHO classification of tumours of haematopoietic and lymphoid tissues in 2008: an overview . Pathologica . 2010 ; 102 : 83 - 7 .
3. Hernandez MC , Knox SJ . Radiobiology of radioimmunotherapy: targeting CD20 B-cell antigen in non-Hodgkin's lymphoma . Int J Radiat Oncol . 2004 ; 59 : 1274 - 87 .
4. Read ED , Eu P , Little PJ , Piva TJ. The status of radioimmunotherapy in CD20? non-Hodgkin's lymphoma . Target Oncol . 2015 ; 10 : 15 - 26 .
5. Hohloch K , Lankeit HK , Zinzani PL , Scholz CW , Lorsbach M , Windemuth-Kieselbach C , et al. Radioimmunotherapy for firstline and relapse treatment of aggressive B-cell non-Hodgkin lymphoma: an analysis of 215 patients registered in the international RIT-Network . Eur J Nucl Med Mol Imaging . 2014 ; 41 : 1585 - 92 .
6. Dillman RO . Radiolabeled anti-CD20 monoclonal antibodies for the treatment of B-cell lymphoma . J Clin Oncol . 2002 ; 20 : 3545 - 57 .
7. Mitsunaga M , Ogawa M , Kosaka N , Rosenblum LT , Choyke PL , Kobayashi H . Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules . Nat Med . 2011 ; 17 : 1685 - 91 .
8. Nagaya T , Sato K , Harada T , Nakamura Y , Choyke PL , Kobayashi H . Near infrared photoimmunotherapy targeting EGFR positive triple negative breast cancer: optimizing the conjugate-light regimen . PLoS One . 2015 ; 10 : e0136829 .
9. Watanabe R , Hanaoka H , Sato K , Nagaya T , Harada T , Mitsunaga M , et al. Photoimmunotherapy targeting prostate-specific membrane antigen: are antibody fragments as effective as antibodies ? J Nucl Med . 2015 ; 56 : 140 - 4 .
10. Sato K , Nagaya T , Choyke PL , Kobayashi H . Near infrared photoimmunotherapy in the treatment of pleural disseminated NSCLC: preclinical experience . Theranostics . 2015 ; 5 : 698 - 709 .
11. Sato K , Choyke PL , Kobayashi H . Photoimmunotherapy of gastric cancer peritoneal carcinomatosis in a mouse model . PLoS One . 2014 ; 9 : e113276 .
12. Nagaya T , Nakamura Y , Sato K , Harada T , Choyke PL , Kobayashi H . Near infrared photoimmunotherapy of B-cell lymphoma . Mol Oncol . 2016 ; 10 : 1404 - 14 .
13. Schneider CA , Rasband WS , Eliceiri KW . NIH Image to ImageJ: 25 years of image analysis . Nat Methods . 2012 ; 9 : 671 - 5 .
14. Arpino G , Gutierrez C , Weiss H , Rimawi M , Massarweh S , Bharwani L , et al. Treatment of human epidermal growth factor receptor 2-overexpressing breast cancer xenografts with multiagent HER-targeted therapy . J Natl Cancer Inst . 2007 ; 99 : 694 - 705 .
15. Nakajima T , Sato K , Hanaoka H , Watanabe R , Harada T , Choyke PL , et al. The effects of conjugate and light dose on photo-immunotherapy induced cytotoxicity . BMC Cancer . 2014 ; 14 : 389 .
16. Hanaoka H , Nagaya T , Sato K , Nakamura Y , Watanabe R , Harada T , et al. Glypican-3 targeted human heavy chain antibody as a drug carrier for hepatocellular carcinoma therapy . Mol Pharm . 2015 ; 12 : 2151 - 7 .
17. Sano K , Nakajima T , Choyke PL , Kobayashi H . Markedly enhanced permeability and retention effects induced by photoimmunotherapy of tumors . ACS Nano . 2013 ; 7 : 717 - 24 .
18. Kobayashi H , Choyke PL . Super enhanced permeability and retention (SUPR) effects in tumors following near infrared photoimmunotherapy . Nanoscale . 2016 ; 8 : 12504 - 9 .
19. Hoeben BA , Molkenboer-Kuenen JD , Oyen WJ , Peeters WJ , Kaanders JH , Bussink J , et al. Radiolabeled cetuximab: dose optimization for epidermal growth factor receptor imaging in a head-and-neck squamous cell carcinoma model . Int J Cancer . 2011 ; 129 : 870 - 8 .
20. Hanaoka H , Katagiri T , Fukukawa C , Yoshioka H , Yamamoto S , Iida Y , et al. Radioimmunotherapy of solid tumors targeting a cell-surface protein, FZD10: therapeutic efficacy largely depends on radiosensitivity . Ann Nucl Med . 2009 ; 23 : 479 - 85 .
21. Witzig TE , Molina A , Gordon LI , Emmanouilides C , Schilder RJ , Flinn IW , et al. Long-term responses in patients with recurring or refractory B-cell non-Hodgkin lymphoma treated with yttrium 90 ibritumomab tiuxetan . Cancer . 2007 ; 109 : 1804 - 10 .
22. Ogawa M , Tomita Y , Nakamura Y , Lee M-J , Lee S , Tomita S , et al. Immunogenic cancer cell death selectively induced by near infrared photoimmunotherapy initiates host tumor immunity . Oncotarget . 2017 ; 8 : 10425 - 36 .