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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

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Bioconjugate Chemistry

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Near-infrared photochemoimmunotherapy by photoactivatable bifunctional

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antibody-drug conjugates targeting HER2-positive cancer

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

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Kobayashi3, Hisao Tajiri1

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

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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

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

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

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

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

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

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

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

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ACS Paragon Plus Environment


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ABSTRACT

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

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

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

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

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

7

linked

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

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

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

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

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

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

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

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

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

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

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

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

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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

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TOC Graphic

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INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Trastuzumab emtansine

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

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

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


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

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

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

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

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

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

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

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

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effectiveness of NIR-PIT treatment.

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

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

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

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

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

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agent for NIR-photochemoimmunotherapy.

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RESULTS

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

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

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

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



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

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

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

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are

shown

in

Figure1.

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

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

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

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each. MW: molecular weight.

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HER2 expression in vitro

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


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

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

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

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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 ±

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

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

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

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(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

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

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

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

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

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

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

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

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

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

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

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signals (Figure S2). 7



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

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

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

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

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

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


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

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

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as means ± SEM (n = 3).

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Fluorescence microscopy

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

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

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

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

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

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

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

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

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

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T-DM1-IR700 (Figure 3C).

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Figure 3. Fluorescence microscopy.

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

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

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

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

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

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NIR light irradiation in vitro

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

9

t-test).

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

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

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

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

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

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

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

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

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

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

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


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

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

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cells (Figure 5B).

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

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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

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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,

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and a reduction in the number of cells.

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


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

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

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

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

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

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

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

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

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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.

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

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

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

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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).

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

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(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

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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

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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

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


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

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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).

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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,

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

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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).

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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

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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 ... - ACS Publications

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