Near-Infrared Photochemoimmunotherapy by Photoactivatable

Subscriber access provided by Duke University Libraries

Article

Near-infrared photochemoimmunotherapy by photoactivatable bifunctional antibody-drug conjugates targeting HER2-positive cancer Kimihiro Ito, Makoto Mitsunaga, Takashi Nishimura, Masayuki Saruta, Takeo Iwamoto, Hisataka Kobayashi, and Hisao Tajiri Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00144 • Publication Date (Web): 12 Apr 2017 Downloaded from http://pubs.acs.org on April 13, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

1

Near-infrared photochemoimmunotherapy by photoactivatable bifunctional

2

antibody-drug conjugates targeting HER2-positive cancer

3 4

Kimihiro Ito1, Makoto Mitsunaga*, Takashi Nishimura, Masayuki Saruta, Takeo Iwamoto2, Hisataka

5

Kobayashi3, Hisao Tajiri1

6 7

1

8

School of Medicine, Minato, Tokyo 105-8461, Japan

9

2

Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University

Division of Molecular Cell Biology, Core Research Facilities for Basic Science, The Jikei University

10

School of Medicine, Minato, Tokyo 105-8461, Japan

11

3

12

Room B3B69, MSC1088, Bethesda, MD 20892-1088, USA

Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, NIH, Building 10,

13 14

*Correspondence should be addressed to: Makoto Mitsunaga, M.D., Ph.D.

15

Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University

16

School of Medicine, 3-25-8 Nishishinbashi, Minato, Tokyo 105-8461, Japan

17

Phone: +81-3-3433-1111; Fax: +81-3-3435-0569; E-mail: [email protected]

18

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 39

1

ABSTRACT

2

Near-infrared photoimmunotherapy (NIR-PIT) is a new class of molecular targeted cancer therapy based on

3

antibody-photoabsorber conjugates and NIR light irradiation. Recent studies have shown effective tumor

4

control, including that of human epidermal growth factor receptor 2 (HER2)-positive cancer, by selective

5

molecular targeting with NIR-PIT. However, the depth of NIR light penetration limits its use. Trastuzumab

6

emtansine (T-DM1) is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab

7

linked

8

antibody-drug-photoabsorber conjugates, T-DM1-IR700, which can work as both NIR-PIT and

9

chemoimmunotherapy agents. We evaluated the feasibility of T-DM1-IR700-mediated NIR light irradiation

10

by comparing the in vitro and in vivo cytotoxic efficacy of trastuzumab-IR700 (T-IR700)-mediated NIR

11

light irradiation in HER2-expressing cells. T-IR700 and T-DM1-IR700 showed almost identical binding to

12

HER2 in vitro and in vivo. Owing to the presence of internalized DM1 in the target cells, NIR-PIT using

13

T-DM1-IR700 tended to induce greater cytotoxicity than that of NIR-PIT using T-IR700 in vitro. In vivo

14

NIR-PIT using T-DM1-IR700 did not show a superior antitumor effect to NIR-PIT using T-IR700 in

15

subcutaneous small tumor models which could receive sufficient NIR light. In contrast, NIR-PIT using

16

T-DM1-IR700 tended to reduce the tumor volume and showed significant prolonged survival compared to

17

NIR-PIT using T-IR700 in large tumor models which could not receive sufficient NIR light. We

18

successfully developed T-DM1-IR700 conjugate which have a similar immunoreactivity to the parental

19

antibody with increased cytotoxicity due to DM1 and a potential for new NIR-PIT agent for targeting tumors

20

that are large and inaccessible to sufficient NIR light irradiation to activate the photoabsorber IR700.

to

the

cytotoxic

agent

maytansinoid

DM1.

Here,

2


we

developed

bifunctional

Page 3 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1


TOC Graphic

2 3

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 39

1

INTRODUCTION

2

Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor

3

family, which regulates cell proliferation, differentiation, and apoptosis through signal transduction by

4

forming homodimers or heterodimers.1 HER2 is commonly expressed on the cell membrane of various types

5

of cancers, and its overexpression is associated with tumor malignancy.2 Trastuzumab—a humanized

6

monoclonal antibody that targets HER2—manifests its antitumor activity by inducing antibody-dependent

7

cellular cytotoxicity, inhibiting ligand-independent HER2 signaling, blocking the active formation of HER2,

8

and preventing the cleavage of the extracellular domain of HER2.3, 4 Although trastuzumab is widely used

9

for treating HER2-expressing cancers, its therapeutic effect is rarely curative when it is used as a single

10

agent; therefore, it is mainly used in combination with chemotherapy.5,

6

11

(trastuzumab-DM1; T-DM1) is a recently developed antibody-drug conjugate (ADC) composed of a highly

12

potent cytotoxic drug—DM1 derived from maytansine—connected to trastuzumab via a non-reducible

13

thioether linker. In addition to retaining all the mechanisms of action of native unconjugated trastuzumab,

14

T-DM1 also has HER2-targeted cytotoxicity, which depends on DM1.7-9 On binding to HER2, T-DM1

15

undergoes internalization and lysosomal degradation. This process induces the intracellular release of

16

DM1-containing catabolites, which bind to tubulin and prevent microtubule assembly, resulting in mitotic

17

arrest, cell growth inhibition, and cell death.10, 11

Trastuzumab emtansine

18

Near-infrared photoimmunotherapy (NIR-PIT) is a new class of molecular targeted cancer therapy

19

based on an antibody-photoabsorber conjugate (APC) and NIR light irradiation. A photoabsorbing

20

phthalocyanine dye, IR700, which is conjugated with antibody, induces selective cytotoxicity only to 4


Page 5 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

APC-bound cells only when excited by NIR light at a specific wavelength of 690 nm. The APC shows

2

similar immunoreactivity to that of the native unconjugated antibody, resulting in highly selective binding to

3

the target molecules on the cell membrane, rapidly inducing membrane rupture and cellular necrosis by the

4

photo-activated IR700 after NIR light exposure without cytotoxic effects towards non-expressing cells.12-14

5

NIR-PIT using trastuzumab-IR700 (T-IR700) conjugates has been shown to cause HER2-targeted

6

phototoxicity in various HER2-expressing cancer mouse models, leading to strong antitumor effects.15-20

7

However, some cancer cells were found to survive and tumor recurrences were eventually seen in mouse

8

models after a single NIR-PIT treatment. Thus, it is necessary to develop a new method for enhancing the

9

effectiveness of NIR-PIT treatment.

10

Here, we developed an antibody-drug-photoabsorber conjugate (ADPC), trastuzumab-DM1-IR700

11

(T-DM1-IR700), which has potential applications in both in NIR-PIT and chemoimmunotherapy. We

12

assumed that NIR-PIT using T-DM1-IR700 is more useful than NIR-PIT using T-IR700 by increased

13

cytotoxicity due to DM1. Therefore, we compared the in vitro and in vivo cytotoxic efficacy of NIR-PIT for

14

HER2-expressing cells using T-IR700 or T-DM1-IR700 and evaluated the utility of T-DM1-IR700 as a new

15

agent for NIR-photochemoimmunotherapy.

16 17

RESULTS

18

In vitro characterization of T-IR700 and T-DM1-IR700 conjugates

19

On the basis of the concentrations of trastuzumab and IR700 determined by spectrometry, the conjugates

20

T-IR700 and T-DM1-IR700 were synthesized by covalently conjugating approximately 3 IR700 molecules

21

each to trastuzumab and T-DM1, respectively. We also examined IR700 conjugation to trastuzumab and 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 39

1

T-DM1 by mass spectrometry. LC/ESI-MS analyses showed that the peak of the molecular weights of

2

trastuzumab, T-IR700, T-DM1, and T-DM1-IR700 were approximately 148000, 150000–156000, 148000–

3

152000, and 152000–159000, respectively (Figure S1). Schematic structures of T-IR700 and T-DM1-IR700

4

are

shown

in

Figure1.

5 6

Figure 1. Schematic structures of T-IR700 and T-DM1-IR700

7

T-DM1 contains an average of 3–3.5 DM1 molecules linked to trastuzumab via a non-reducible thioether

8

linker (MCC linker). An average of 3 IR700 molecules are covalently conjugated to trastuzumab and T-DM1

9

each. MW: molecular weight.

10

11

HER2 expression in vitro

12

After 3-h incubation with 10 µg/mL T-IR700 or 10 µg/mL T-DM1-IR700, 3T3/HER2 cells showed strong 6


Page 7 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

IR700 fluorescence, and these signals were almost completely blocked by the addition of excess

2

unconjugated trastuzumab, suggesting HER2-specific binding of T-IR700 and T-DM1-IR700 (Figure 2A).

3

The ratios of the mean fluorescence intensities (MFIs) compared to that of the isotype control were 248.9 ±

4

3.4 for T-IR700, 258.0 ± 3.8 for T-DM1-IR700, 5.1 ± 0.1 for T-IR700 with trastuzumab blocking, and 4.9 ±

5

0.2 for T-DM1-IR700 with trastuzumab blocking, respectively (means ± SEM, n = 3). Similar to 3T3/HER2

6

cells, HCC-1419 cells also showed strong IR700 fluorescence with T-IR700 or T-DM1-IR700, and these

7

signals were blocked by the addition of excess unconjugated trastuzumab (Figure 2B). MFI ratios

8

(treatment:isotype control) were 91.2 ± 3.5 for T-IR700, 90.9 ± 3.0 for T-DM1-IR700, 1.9 ± 0.4 for T-IR700

9

with trastuzumab blocking, and 4.2 ± 0.8 for T-DM1-IR700 with trastuzumab blocking (means ± SEM, n =

10

3). In contrast, there were no significant differences in signal intensity between T-IR700 or T-DM1-IR700

11

treatment and the isotype control in HER2-negative BALB/3T3 cells (Figure 2C).

12

13

Similar HER2-specific binding capabilities of T-IR700 and T-DM1-IR700 in vitro

14

When cells were exposed to T-IR700 or T-DM1-IR700 for 24 h, IR700 signals increased in a

15

dose-dependent manner but were almost saturated by treatment with 3 µg/mL of IR700 conjugates in both

16

3T3/HER2 and HCC-1419 cells (Figure 2D). Therefore, we considered 3 µg/mL of IR700 conjugates as the

17

treatment dose for the in vitro study. Despite being HER2-negative, BALB/3T3 cells showed weak IR700

18

signals after 24-h exposure to 10 µg/mL and 30 µg/mL of the IR700 conjugates. These signals were not

19

blocked by the addition of excess unconjugated trastuzumab; therefore, we considered them as nonspecific

20

signals (Figure S2). 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 39

1 2

Figure 2. Expression of human epidermal growth factor receptor 2 and the dose-response binding of

3

T-IR700 and T-DM1-IR700 in vitro.

4

(A, B, C) Flow cytometry analysis revealed strong human epidermal growth factor receptor 2

5

(HER2)-specific binding of T-IR700 and T-DM1-IR700 in 3T3/HER2 and HCC-1419 cells but not in

6

NIH/3T3 cells after 3-h incubation with 10 µg/mL T-IR700 or 10 µg/mL T-DM1-IR700. Specific binding

7

was demonstrated by excess unconjugated trastuzumab blocking. (D) Flow cytometry analysis was 8


Page 9 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

performed after 24-h incubation with several concentrations of T-IR700 or T-DM1-IR700. T-DM1-IR700

2

and T-IR700 bound to HER2-positive cells in a dose-dependent manner at equal levels. Data are presented

3

as means ± SEM (n = 3).

4 5

Fluorescence microscopy

6

To detect the time-lapse co-localization of T-IR700 or T-DM1-IR700, fluorescence microscopy was

7

performed. IR700 fluorescence signals were detected predominantly on the cell surface of 3T3/HER2 and

8

HCC-1419 cells after 3-h incubation with T-IR700 or T-DM1-IR700, whereas only partial subcellular

9

localization was observed. Trastuzumab-photoabsorber conjugate showed gradual internalization into the

10

cytoplasm of HER2-positive cells in earlier studies.12, 21 Consistent with those studies, when cells were

11

incubated with T-IR700 or T-DM1-IR700 for 24 h, subcellular localization pattern of IR700 was not

12

different between 3T3/HER2 and HCC-1419 cells, indicating similar levels of internalization of T-IR700

13

and T-DM1-IR700 (Figure 3A, 3B). In contrast, HER2-negative BALB/3T3 cells did not show any

14

detectable fluorescence for IR700 under the same camera conditions after treatment with T-IR700 or

15

T-DM1-IR700 (Figure 3C).

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 10


Page 10 of 39

Page 11 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Figure 3. Fluorescence microscopy.

2

Fluorescence microscopy showed HER2-specific binding of T-IR700 and T-DM1-IR700 in 3T3/HER2 and

3

HCC-1419 cells but not in BALB/3T3 cells. IR700 fluorescence signals were detected mainly on the cell

4

surface after 3-h incubation and were gradually internalized. (A) 3T3/HER2, (B) HCC-1419, (C) BALB/3T3

5

cells. DIC: differential interference contrast. Scale bar = 30 µm.

6

7

Higher cytotoxicity of T-DM1-IR700-mediated NIR light irradiation than that of T-IR700-mediated

8

NIR light irradiation in vitro

9

A LIVE/DEAD assay showed that the percentage of cell death by T-DM1-IR700 single treatment was

10

significantly higher than that in the untreated control and by T-IR700 single treatment in HER2-positive

11

3T3/HER2 and HCC-1419 cells, whereas cytotoxicity of T-IR700 single treatment and the untreated control

12

did not differ significantly (Figure 4A, 4B). There was no difference in cytotoxicity between untreated

13

control and 1 J/cm2 of NIR light irradiation (without mAbs) in each cell line (Figure S3A). When cells were

14

treated with either T-IR700 plus NIR light or T-DM1-IR700 plus NIR light, the percentage of cell death

15

increased in a NIR light dose-dependent manner. In addition, cytotoxicity differed significantly between

16

T-IR700 single treatment and T-IR700 plus NIR light treatment, as well as between T-DM1-IR700 single

17

treatment and T-DM1-IR700 plus NIR light treatment. Furthermore, the percentage of cell death by

18

T-DM1-IR700 plus NIR light treatment was significantly higher than that by T-IR700 plus NIR light

19

treatment especially in low NIR light doses (Figure 4A, 4B). In contrast, no cytotoxicity associated with

20

T-IR700 single treatment, T-DM1-IR700 single treatment, T-IR700 plus NIR light treatment, or 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Page 12 of 39

T-DM1-IR700 plus NIR light treatment was detected in HER2-negative BALB/3T3 cells (Figure 4C).

2

A LDH cytotoxicity assay showed that T-DM1-IR700 single treatment resulted in greater

3

cytotoxicity than that by T-IR700 or T-DM1-IR700 single treatment in 3T3/HER2 cells (Figure 4D). When

4

cells were treated with either T-IR700 plus NIR light or T-DM1-IR700 plus NIR light, cytotoxicity increased

5

in a NIR light dose-dependent manner. Furthermore, T-DM1-IR700 plus NIR light treatment resulted in

6

greater cytotoxicity than that by T-IR700 plus NIR light treatment (Figure 4D). Similar cytotoxicity was

7

detected in HCC-1419 cells treated with 1 J/cm2 of NIR light irradiation (Figure 4E). In contrast, no

8

cytotoxicity associated with T-IR700 or T-DM1-IR700 single treatment or plus NIR light irradiation was

9

detected in BALB/3T3 cells (Figure 4F)

12


Page 13 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 4. T-DM1-IR700-mediated NIR light irradiation induced greater cytotoxicity in vitro

3

(A, B, C) A LIVE/DEAD assay in HER2-positive 3T3/HER2 and HCC-1419 cells showed that the 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 39

1

percentage of cell death by T-DM1-IR700 plus NIR light treatment was significantly higher compared to

2

that by T-IR700 plus NIR light treatment, especially in low NIR light doses. No cytotoxicity associated with

3

T-IR700 or T-DM1-IR700 single treatment or plus NIR light irradiation was found in HER2-negative

4

BALB/3T3 cells. Data are presented as means ± SEM (n = 3, *P < 0.05, **P < 0.01, Student’s t-test). (D, E,

5

F) A LDH cytotoxicity assay showed that T-DM1-IR700 plus NIR light treatment induced greater

6

cytotoxicity than that by T-IR700 plus NIR light treatment in both 3T3/HER2 and HCC-1419 cells. No

7

cytotoxicity associated with T-IR700 or T-DM1-IR700 single treatment or plus NIR light irradiation was

8

found in BALB/3T3 cells. Data are presented as means ± SEM (n = 3, *P < 0.05, **P < 0.01, Student’s

9

t-test).

10

To compare the long-term cytotoxic effects of T-IR700 plus NIR light or T-DM1-IR700 plus NIR

11

light treatment, a trypan blue dye exclusion assay and microscopic observation were performed. As shown

12

in Figure 5A, T-DM1-IR700 single treatment resulted in significant growth inhibition compared to that in

13

the untreated control or that by T-IR700 single treatment in 3T3/HER2 cells, whereas there was no

14

significant difference in growth inhibition between T-IR700 single treatment and the untreated control.

15

There was no difference in long-term growth inhibition between untreated control and 1 J/cm2 of NIR light

16

irradiation in both cell lines (Figure S3B, S3C). T-DM1 and T-IR700 plus NIR light treatment could not

17

enhance cytotoxicity compared with T-DM1-IR700 plus NIR light treatment or T-IR700 plus NIR light

18

treatment, caused by competition between T-DM1 and T-IR700 (Figure S4). Long-term growth inhibition

19

was apparent after T-IR700 plus NIR light or T-DM1-IR700 plus NIR light treatment. Importantly, the

20

growth inhibition was more prominent by T-DM1-IR700 plus NIR light treatment than that by 14


Page 15 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

T-DM1-IR700 single treatment or T-IR700 plus NIR light treatment. In contrast, no cytotoxicity associated

2

with T-IR700 or T-DM1-IR700 single treatment or plus NIR light irradiation was observed in BALB/3T3

3

cells (Figure 5B).

4

Microscopic observation revealed that T-DM1-IR700 single treatment induced a tendency of giant

5

cell formation and a reduction in the number of 3T3/HER2 cells, whereas T-IR700 single treatment did not

6

cause any morphological changes or a reduction in cell number compared to the untreated control (Figure

7

5C). T-IR700 plus NIR light treatment induced cell collapse caused by membrane rupture and a reduction in

8

the number of cells, and T-DM1-IR700 plus NIR light treatment induced giant cell formation, cell collapse,

9

and a reduction in the number of cells.

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 39

1 2

Figure 5. Long-term growth inhibition assay and morphological changes in response to 16


Page 17 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

T-DM1-IR700-mediated NIR light irradiation in vitro

2

(A) A trypan blue dye exclusion assay showed significant growth inhibition by T-DM1-IR700 plus NIR

3

light treatment compared to that with T-DM1-IR700 single treatment or T-IR700 plus NIR light treatment in

4

3T3/HER2 cells. Data are presented as means ± SEM (n = 3, **P < 0.01, ***P < 0.001, Student’s t-test). (B)

5

No cytotoxicity associated with T-IR700 or T-DM1-IR700 single treatment or plus NIR light irradiation was

6

observed in BALB/3T3 cells. (C) Microscopic changes in response to T-DM1-IR700 plus NIR light

7

treatment in 3T3/HER2 cells. T-DM1-IR700 single treatment induced giant cell formation and a reduction in

8

the number of cells, while T-IR700 single treatment did not cause any visible morphological changes

9

compared to the untreated control. T-IR700 plus NIR light treatment induced cell collapse and a reduction in

10

the number of cells, and T-DM1-IR700 plus NIR light treatment induced giant cell formation, cell collapse,

11

and a reduction in the number of cells. Scale bar = 100 µm.

12

13

In vivo biodistribution of T-IR700 and T-DM1-IR700

14

To examine the biodistribution of T-IR700 and T-DM1-IR700 in a xenograft tumor model, serial

15

fluorescence images were obtained before and after the injection of T-IR700 or T-DM1-IR700. IR700

16

fluorescence intensities of 3T3/HER2 tumors were strong and similar for T-DM1-IR700 and T-IR700 1 day

17

after the treatment, gradually decreasing thereafter (Figure 6A, B).

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 39

1 2

Figure 6. In vivo biodistribution of T-IR700 and T-DM1-IR700.

3

(A) 3T3/HER2 tumor xenografts (right dorsum) visualized by IR700 fluorescence were similar after

4

intravenous injection of T-DM1-IR700 and T-IR700. (B) Quantitative analysis showed comparable levels of

5

IR700 fluorescence in 3T3/HER2 tumors between T-DM1-IR700 treatment and T-IR700 treatment. Data are

6

presented as means ± SEM (n = 3).

7 8

Comparison of in vivo antitumor effect between T-IR700-mediated NIR light irradiation and

9

T-DM1-IR700-mediated NIR light irradiation

10

In large tumor experiments, tumor xenografts reached 200 mm3 in volume approximately 12 days after

11

subcutaneous injection of two million 3T3/HER2 cells. Tumors were irradiated with 100 J/cm2 of NIR light

12

twice 1 day after intravenous injection of T-IR700 or T-DM1-IR700 (day 1 and 8 after initial intravenous

13

injection) under the guidance of IR700 fluorescence, because the highest IR700 accumulation was observed

14

at that time point. As compared with the non-NIR-PIT groups, significant antitumor effects were observed in

15

the groups of both T-IR700 plus NIR light irradiation and T-DM1-IR700 plus NIR light irradiation (Figure 18


Page 19 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

7A). Comparing the 2 NIR-PIT groups, T-DM1-IR700 plus NIR light irradiation treatment tended to reduce

2

the tumor volume compared to T-IR700 plus NIR light treatment. In addition, T-DM1-IR700 plus NIR light

3

treatment showed significant prolonged survival compared to T-IR700 plus NIR light treatment (Figure 7B).

4

Pathological analysis showed that massive granulation with inflammatory changes and giant cells formation

5

were observed in the tumor nodules treated with T-DM1-IR700 plus NIR light irradiation (Figure S5).

6

In small tumor experiments, tumor xenografts reached 30 mm3 in volume approximately 6 days

7

after subcutaneous injection of one million 3T3/HER2 cells. Consistent with large tumor experiment,

8

significant antitumor effects were observed in the groups of both T-IR700 plus NIR light irradiation and

9

T-DM1-IR700 plus NIR light irradiation compared with the non-NIR-PIT groups, however, there was no

10

difference in the tumor volume and there was no significant difference in survival between these 2 NIR-PIT

11

treatment groups (Figure 7C, 7D).

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 39

1 2

Figure

3

T-DM1-IR700-mediated NIR light irradiation.

4

(A) Large tumor experiments: Strong antitumor effects were observed in the groups of T-IR700 plus NIR

5

light irradiation and T-DM1-IR700 plus NIR light irradiation compared to the untreated control group.

6

T-DM1-IR700 plus NIR light irradiation treatment tended to reduce the tumor volume compared to T-IR700

7

plus NIR light treatment. Data are presented as means ± SEM (n = 10 in each group, 11 days after initial

8

treatment; ***P = 0.001: T-IR700 plus NIR light irradiation vs. untreated control, ****P < 0.0001:

7.

In

vivo

antitumor

effects

of

T-IR700-mediated

20


NIR

light

irradiation

and

Page 21 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

T-DM1-IR700 plus NIR light irradiation vs. untreated control; Mann–Whitney U test). (B) Large tumor

2

experiments: Survival was prolonged significantly when mice were treated with T-DM1-IR700 plus NIR

3

light irradiation compared to T-IR700 plus NIR light irradiation. (n = 10 in each group, **P = 0.0077:

4

T-DM1-IR700 plus NIR light irradiation vs. T-IR700 plus NIR light irradiation; log-rank test). (C) Small

5

tumor experiments: Strong antitumor effects were observed in the groups of T-IR700 plus NIR light

6

irradiation and T-DM1-IR700 plus NIR light irradiation compared to the untreated control group. There was

7

no difference in the tumor volume and there was no significant difference in survival between the 2 NIR-PIT

8

groups. Data are presented as means ± SEM (n = 10 in each group, 11 days after initial treatment; ****P

Near-Infrared Photochemoimmunotherapy by Photoactivatable

Recommend Documents