European Journal of Medicinal Chemistry 252 (2023) 115298

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European Journal of Medicinal Chemistry

journal homepage: www.elsevier.com/locate/ejmech

Research paper

Synthesis and biological evaluation of theranostic Trastuzumab–SN38

conjugate for Near-IR fluorescence imaging and targeted therapy of HER2+
breast cancer
Dmytro Kobzev, Chandrashekhar Prasad, Dipak Walunj, Hodaya Gotman, Olga Semenova,
Andrii Bazylevich, Leonid Patsenker, Gary Gellerman *
Department of Chemical Sciences, The Faculty of Natural Sciences, Ariel University, Ariel, 40700, Israel

A R T I C L E I N F O A B S T R A C T

Keywords: Here, we report on the design, synthesis, and biological evaluation of a new theranostic antibody drug conjugate
Targeted cancer therapy (ADC), Cy5-Ab-SS-SN38, that consists of the HER2-specific antibody trastuzumab (Ab) connected to the near
Theranostic ADC infrared (NIR) pentamethine cyanine dye Cy5 and SN38, which is a bioactive metabolite of the anticancer drug
Controlled drug release
irinotecan. SN38 is bound to an antibody through a glutathione-responsive self-immolative disulfide carbamate
Disulfide carbamate linker
Self-immolative linker
linker. For the first time, we explored this linker in ADC and found that it to reduce the drug release rate, which is
Trastuzumab important for safe drug delivery. The developed ADC exhibited specific accumulation and nanomolar anti-breast
cancer activity on HER2-positive (HER2+) cell lines but no effect on HER2-. Animals treated with this ADC
exhibited good tolerance. In vivo studies have shown that the ADC had good targeting ability for HER2+ tumors
with much higher anticancer potency than trastuzumab itself or a mixture of trastuzumab with SN38. Side-by-
side HER2+/HER2-xenograft at the 10 mg/kg dose exhibited specific accumulation and reduction of HER2+
tumor but not accumulation or growth inhibition of HER2-counterpart. The self-immolative disulfide linker
implemented in this study was proven to be successful, broadening its utilization with other antibodies for
targeted anticancer therapy in general. We believe that the theranostic ADCs comprising the glutathione-
responsive self-immolative disulfide carbamate linker are applicable for the treatment and fluorescent moni­
toring of malignancies and anticancer drug delivery.

1. Introduction has a poor prognosis with a median overall survival of 15 months [12].
In addition, conventional chemotherapy may cause severe side effects.
Antibody–drug conjugates (ADCs) consisting of an antigen-specific The combination of an antibody with a highly potent drug in an ADC
antibody linked to a potent drug are widely used in the treatment of affords more efficient target-specific treatment and significantly reduced
different types of cancer [1–4]. These conjugates can be additionally toxicity.
equipped with a fluorescent dye to yield theranostic ADCs, enabling Currently, there are two FDA-approved ADCs in clinics to target and
real-time monitoring of drug delivery (Fig. 1A) [5,6]. treat HER2+ breast cancer: trastuzumab-emtansine (T-DM1) and
In ADCs, antibodies are mainly used as receptor-specific carriers for trastuzumab-deruxtecan, and several others are in clinical development
drug delivery through the intracellular uptake of the entire ADC via [13]. Emtansine is a powerful antimitotic macrocyclic drug that is linked
endocytosis [7–9]. Among others, trastuzumab (Herceptin), a human­ to trastuzumab through a succinimidyl trans-4-(maleimidylmethyl)
ized monoclonal antibody (Ab) that is a HER2-positive (HER2+) in­ cyclohexane-1-carboxylate (SMCC) noncleavable linker that releases the
hibitor launched in the late 1990s, was the first immunotherapeutic drug only after full degradation of the linker and antibody in lysosomes
agent to improve survival in women with metastatic HER2+ breast [14–17]. The second ADC, trastuzumab-deruxtecan (deruxtecan is a
cancer (BC) [10,11]. On the other hand, cancer treatment with con­ potent, low nanomolar Topo I inhibitor), contains a tetrapeptide-based
ventional chemotherapeutic drugs for patients in the metastatic stage proteolytically cleavable linker that secures the intracellular release of

* Corresponding author.
E-mail addresses: [email protected] (D. Kobzev), [email protected] (C. Prasad), [email protected] (D. Walunj), [email protected] (H. Gotman),
[email protected] (O. Semenova), [email protected] (A. Bazylevich), [email protected] (L. Patsenker), [email protected] (G. Gellerman).

https://doi.org/10.1016/j.ejmech.2023.115298
Received 28 January 2023; Received in revised form 16 March 2023; Accepted 17 March 2023
Available online 22 March 2023
0223-5234/© 2023 Elsevier Masson SAS. All rights reserved.
D. Kobzev et al. European Journal of Medicinal Chemistry 252 (2023) 115298

carbonate linker and thereby improve safety of drug delivery. Cy5 dye
was chosen as one for the most bright long-wavelength emitting markers
suitable for in vitro and in vivo imaging; this dye exhibits no detectable
cytotoxicity, which is important for our experiments [37–39].

2. Results and discussion

2.1. Synthesis of Cy5-Ab–SS–SN38

To assemble SN38, Ab, and Cy5 into Cy5-Ab-SS-SN38, among

several other known disulfide-based self-immolative linkers [40], we
decided to choose 3-((2-aminoethyl)disulfaneyl)propanoic acid re­
ported in previous study [41]. We anticipate that this linker, at the
N-terminus, will form a more proteolytically stable carbamate group
with 20-OH of SN38 than the reported carbonate (Scheme 1). To the best
of our knowledge, this linker has not been previously reported for ADC
construction. First, the more reactive phenolic 10-OH was protected by
Fig. 1. General structure of fluorescently monitored theranostic ADC, Dye­
the t-butoxycarbonyl (Boc) group using Boc2O with pyridine in DCM,
–Ab–Linker–Drug (A) and Cy5-Ab-SS-SN38 (B) synthesized and evaluated in
followed by the reaction with 4-nitrophenyl chloroformate to afford the
this work. The investigated self-immolative linker is highlighted with magenta.
activated intermediate, 10-O-Boc-SN38–20-O-PNC, as described in
previous study [42]. Then, the Fmoc-SS-CO2H linker, synthesized ac­
the drug from Ab in cancer cells [18]. Another potent and
cording to previous publication [43], was loaded on 2-Cl-Trt chloride
low-nanomolar-range Topo I inhibitor, SN38, is synthetically more
resin, and after Fmoc deprotection (20% piperidine–DMF), it reacted
accessible than deruxtecan and successfully used as a drug in Sacitu­
with 10-O-Boc-SN38–20-O-PNC in the presence of DIPEA. The product
zumab govitecan (Trodelvy) ADC to treat triple-negative breast cancer
was cleaved from the solid support (30% TFA–DCM), affording
(BC), potentiating its utility as a drug in a trastuzumab-based ADC
SN38-SS-CO2H in satisfactory yield after HPLC purification.
[19–21].
In the next stage, Cy5-Ab-SS-SN38 was synthetized by two sequen­
Utilization of an appropriate linker is extremely important to provide
tial conjugations of Ab with NHS activated SN38-SS-CO2H followed
sufficient stability during systemic circulation and allows efficient
with NHS activated Cy5 by the straightforward, well-established, and
release of the drug inside the tumor cells [22–24]. The noncleavable
commonly used procedure [44] (Scheme 2).
linkers together with Ab decompose in the lysosomes through proteol­
ysis, which results in the release of the drug inside the cancer cells [25].
2.2. Characterization of Cy5-Ab-SS-SN38
Stimuli-responsive cleavable linkers based on disulfide (S–S), glucuro­
nide, short peptides, and pH-sensitive hydrazone break at the target sites
The absorption spectra of Cy5, SN38-SS-CO2H, and Cy5-Ab-SS-
due to activation by, e.g., enzymes, glutathione, or pH gradients that
SN38 measured in 0.1 M PBS buffer pH 7.4 are shown in Fig. 2A. The
characterize the environment of abnormal cells [23,26,27]. For
Cy5-Ab-SS-SN38 conjugate has three absorption bands related to Ab
instance, disulfide (S–S) linkers between the drug and Ab are cleaved at
(Herceptin, λmax = 280 nm), SN38-SS-CO2H (λmax = 379 nm), and Cy5
a physiologically relevant concentration of glutathione in cancer cells
(λmax = 650 nm). The spectrophotometrically determined dye-to-
[28], resulting in controlled drug release. S–S linkers are known to be
antibody ratio (Cy5/Ab) in this conjugate was 0.7, and the drug-to-
effectively applied in glutathione-responsive PEGylated graphene
antibody ratio (SN38/Ab) was 2.0, which was in good agreement with
quantum dot-based nanoparticles for the diagnosis and treatment of
the MALDI-TOF data mentioned above. The Cy5 dye exhibited almost no
breast cancer and in calicheamicin-bearing ADCs to treat leukemias [29,
aggregation when bound to Ab, which could be seen from a minor in­
30]. Camptothecin (CPT) bound to oleic acid through a disulfide car­
crease in the shoulder (at ~600 nm) on the short-wavelength slope of
bonate linker to yield a nanoaggregative system was described in pre­
the main absorption band [45].
vious studies [31]. However,the carbonate linker cleavage was
The Cy5-Ab-SS-SN38 conjugate was characterized by MALDI-TOF.
relatively fast (the cleavage rate half-life was approximately 8 h), and
Based on the MW for trastuzumab (148,225 Da, Fig. S1) and the con­
therefore, the drug might cause toxic side effects upon delivery.
jugate (150,018 Da, Fig. S2), the molar ratio of the components in Cy5/
ADCs that incorporate glutathione-responsive self-immolative disul­
Ab/SS-SN38 was calculated to be approximately 1:1:2.
fide linkers and target HER2+ breast cancer have yet to be reported. Self-
immolative linkers afford spontaneous drug release as a result of a
2.3. Self-immolative release mechanism of SN38
breakdown of the stimuli-sensitive bond [32,33]. Fluorescent dyes used in
ADCs for in vivo real-time monitoring of drug delivery should efficiently
The cleavage mechanism for various SS-based self-immolative
absorb and emit in the biologically transparent red or near-IR spectral
linkers has recently been discussed, e.g., in Ref. [40]. Here, we moni­
region. In general, pentamethine and heptamethine dyes such as Cy5 or
tored the release of SN38 from SN38-SS-CO2H using LC‒MS analysis.
Cy7 are employed in theranostic ADCs [34–36].
Similar to the reported method [31], we utilized dithiothreitol (DTT) as
In this work, we synthesized a theranostic trastuzumab–SN38 con­
a reductive stimulus agent to monitor the release rate of SN38. As shown
jugate labeled with the near-IR fluorescent dye Cy5 and evaluated this
in Fig. 3A and B, SN38-SS-CO2H rapidly degraded into the SN38-SH
Cy5-Ab-SS-SN38 ADC (Fig. 1B) on a mouse xenograft model for the real-
intermediate in the presence of 10 mM DTT in 10 mM PBS at pH 7.4 with
time monitored targeted therapy of HER2+ breast cancer. HER2-
τ1/2–0.55 h, which was then slowly hydrolyzed into active SN38
negative (HER2-) cells were used as a negative control. In this ADC,
(τ1/2–21 h, Fig. S3). Thus, the reduction of the disulfide bond in
SN38 is conjugated to trastuzumab via a self-immolative 3-((2-amino­
SN38-SS-CO2H into SN38-SH leads to spontaneous intramolecular
ethyl)disulfaneyl)propanoic acid (disulfide carbamate) linker. SN38 is
decomposition of the carbamate bond, resulting in the release of SN38,
used in our work as the only one accessible low-nanomolar Topo I
which is the rate-limiting step of the overall reaction (Fig. 3C). These
inhibitory drug against HER2+ breast cancer, which is suitable for
findings are in good agreement with a previously reported study
conjugation to antibody. Disulfide carbamate linker was taken to reduce
examining the DTT-stimulated release of camptothecin (CPT) from a
the drug release rate compared to the previously investigated disulfide
disulfanyl-ethyl carbonate (ETCSS) linker [31]. At the same time, the

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D. Kobzev et al. European Journal of Medicinal Chemistry 252 (2023) 115298

Scheme 1. Synthesis of SN38-SS-CO2H.

Scheme 2. Synthesis of Cy5-Ab-SS-SN38 conjugate.

disulfide carbamate linker provided slower drug release (~21 h) than fluorescence signal from Cy5 gradually increased after incubation of
the carbonate linker (~8 h [31]), which should help to improve the SKBR3 and BT474 (HER2+) cells with Cy5-Ab-SS-SN38, reaching a
safety of drug delivery. plateau after 3 h. At the same time, there was no fluorescence from
MDA-MB-231 cells (Fig. 4A; Fig. S4) due to the lack of HER2+ receptors.
The fluorescence intensity of Cy5 was 2.5-fold higher in BT474 cells
2.4. Accumulation of Cy5-Ab–SS–SN38 on cancer cells
than in SKBR3 cells at all the observed time points, which was more
likely due to the higher number of HER2+ on BT474 cells [46].
The accumulation of Cy5-Ab-SS-SN38 in cancer cells was studied in
vitro in the SKBR3, BT474 and MDA-MB-231 cell lines. Cells were
incubated with Cy5-Ab-SS-SN38 over 0, 1, 3 and 6 h followed by
fluorescence microscopy imaging (Fig. 4A). As shown in Fig. 4B, the

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Fig. 2. Absorption spectra of Cy5 (dashed line), SN38-SS-CO2H (dash-dot line), and Cy5-Ab-SS-SN38 (solid line) in 0.1 M PBS pH 7.4 (A) and MALDI-TOF spectra of
antibody (B) and Cy5-Ab-SS-SN38 (C).

Fig. 3. Time-dependent LC‒MS spectra for the products formed upon SN38-SS-CO2H cleavage initiated by DTT (10 mM) in 10 mM PBS pH 7.4 (A), kinetic curves for
the DTT-mediated release of SN38-SH and SN38 from SN38-SS-CO2H (B), and an anticipated mechanism of DTT-induced SN38 release from SN38-SS-CO2H (C).

Fig. 4. Time-dependent fluorescence microscope images (A) and average integrated fluorescence intensities (B) of SKBR3 and BT474 (HER2+) cells and MDA-MB-
231 (HER2-) cells stained with Cy5-Ab-SS-SN38.

2.5. In vitro cytotoxicity SS-SN38 (ADC) compared to SN38, trastuzumab (Ab) and an equimolar
mixture of Ab and SN38 (SN38þAb) in the 0.4–1000 nM concentration
By using the CCK-8 assay, we evaluated the cytotoxicity of Cy5-Ab- range against BT474, SKBR3, and MDA-MB-231 cells. The fluorescence

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D. Kobzev et al. European Journal of Medicinal Chemistry 252 (2023) 115298

signal from the cells did not change between 3 h and 6 h (Fig. 4B) and, incubated with MDA-MB-231 (HER2-) cells, SN38 and SN38+Ab
therefore, to avoid early cleavage of the S–S linker (τ1/2–21 h, Fig. 3B) showed the most pronounced cytotoxicity (IC50 ~ 950 nM), while Cy5-
that could result in the toxicity to HER2-cells, we exposed the cells to Ab-SS-SN38 and Ab did not affect these cells in the investigated con­
drugs during 6 h and 16 h. Cytotoxicity was expressed through the cell centration range. Thus, the obtained results demonstrate that the
viability after the treatment. As shown in Fig. 5, after 6 h of exposure of trastuzumab-driven theranostic ADC Cy5-Ab-SS-SN38 accumulated
SKBR3 cells to ADC, washing, and subsequent incubation for 72 h, the specifically in HER2+ cell lines, increasing the cytotoxic effect of SN38.
ADC exhibited insufficient cytotoxicity with IC50 ~ 150 nM, while all Notably, the exposure time for accumulation of 16 h was sufficient to
the other drugs showed a negligible effect on all the investigated cell reach effective ADC toxicity to the cells.
lines. None of the tested drugs had a noticeable effect on BT474 cells Noticeably, according to the CCK-8 assay results, BT474 cells exhibit
after 6 h of the exposure period with subsequent incubation for 72 h. reduced sensitivity to the ADC treatment compared to SKBR3 cells,
After 16 h of drugs exposure to SKBR3 and BT474 cells and subsequent while according to the fluorescence microscope imaging data (Section
incubation for 72 h, the ADC, SN38, Ab, and SN38þAb exhibited 2.4) they contain elevated number of HER2 receptors (the images and
elevated cytotoxicity. The cytotoxicity of the ADC against SKBR3 and brighter, Fig. 4). We assume that such a phenomenon can be attributed
BT474 increased ~8.5-fold (IC50 ~ 10 nM) and ~4.1-fold (IC50 ~ 150 to different response of these two cell lines to the Cy5-Ab-SS-SN38
nM), respectively, compared to SN38 and mixture SN38þAb (IC50 for conjugate.
both drugs ~85 nM in SKBR3 and ~610 nM for BT474). Ab demon­
strated the lowest cytotoxicity and did not reach IC50 in the investigated
concentration range. On the other hand, when these drugs were

Fig. 5. Viability of SKBR3, BT474 (HER2+), and MDA-MB-231 (HER2-) cancer cells after 6 h and 16 h of exposure to drugs following washing and an additional 72 h
incubation. Concentration range of Cy5-Ab-SS-SN38 vs. SN38, trastuzumab (Ab), and an equimolar mixture Ab þ SN38was 0.04–1000 nM. The result shown for
each concentration represents the mean ± standard error calculated from triplicates.

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2.6. Fluorescently monitored targeted cancer therapy in vivo with Cy5- autofluorescence in imaging experiments. After 11 days of tumor
Ab–SS–SN38 development, groups 2, 4 and 5 were intravenously (IV) administered
(tail) every 7 days (until the end of the experiment) the Cy5-Ab-SS-
The Cy5-Ab-SS-SN38 conjugate was investigated for real-time flu­ SN38 conjugate (200 μg in 200 μL PBS, 10 mg/kg).
orescently monitored targeted therapy of HER2+ breast cancer in the
mouse xenograft model (Fig. 6). SKBR3 cells were selected for the 2.6.1. Imaging
xenograft following the in vitro results that indicated SKBR3 as a more The distribution of the conjugates in the mouse whole body was
responsive cell line to ADC treatment. Twenty six-week-old athymic monitored by an in vivo imaging system, CRi Maestro II. The images were
BALB/c female nude mice (Harlan Labs, Nes Ziona, Israel) were sub­ captured in white light and NIR channels 24 h postinjection. Accumu­
cutaneously inoculated in the dorsal right side with the human breast lation events were verified for each injection of the conjugate. At 24 h,
cancer cell lines MDA-MB-231 (10 mice) and SKBR3 (10 mice), 1 × 106 an intense fluorescence signal originating from the Cy5-Ab-SS-SN38
cells in PBS into nu/nu mice, 100 μL per mouse. Similarly, 5 more mice conjugate was observed in the lungs of MDA-MB-231 tumor-bearing
were inoculated simultaneously in the dorsal right side with SKBR3 and mice (group 2). At the same time, there was complete accumulation of
in the dorsal left side with MDA-MB-231 (two tumors). Tumors were Cy5-Ab-SS-SN38 in SKBR3 tumor-bearing mice (groups 4 and 5, Fig. 7).
allowed to establish until the tumor sizes reached approximately 30 As expected, the fluorescence signal from MDA-MB-231 tumor cells was
mm3 (11 days). Then, the mice were randomly separated into five very low because these cells lack HER2+ receptors. The tumor sizes
groups (5 mice per group). Group 1 (MDA-MB-231) and group 3 were monitored until the 9th (groups 1, 2), 10th (group 5), 16th (group
(SKBR3) were used as the controls for tumor growth and background 3), and 29th (group 4) days by the hands-on digital caliper method [47]

Fig. 6. Mice experiment setup.

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Fig. 7. Representative in vivo whole-body images of tumor-bearing mice (groups 2, 4 and 5) captured at 24 h after IV injection of the Cy5-Ab-SS-SN38 (10 mg/kg)
conjugate. The 1st injection was carried out on day 0, the 2nd injection on the 7th day, the 3rd injection on the 14th day, the 4th injection on the 21st day, and the
5th injection on the 28th day; 1st row – white light, 2nd row – fluorescence channel, 3rd row – their merge.

(the final tumor volumes were 949 ± 28 mm3). Evidently, at the 9th day,
the tumor volumes of live mice decrease as follows: group 2 (949 ± 28
mm3) ≥ group 1 (910 ± 33 mm3) > group 5, MDA-MB-231 (764 ± 43
mm3) > group 3 (192 ± 16 mm3) > groups 5, SKBR3 (33 ± 3 mm3) ≈
group 4 (30 ± 3 mm3) (Fig. 8), pointing on the prevalence of ADC vs.
other substances. By the 10th day, the MDA-MB-231 tumors of
ADC-injected group 5 reached 1015 ± 51 mm3, which was similar to the
negative control (group 1). Thus, the Cy5-Ab-SS-SN38 conjugate did not
affect MDA-MB-231 (HER2-) tumor growth.
In contrast, the slower developing SKBR3 tumors in control group 3
reached 991 ± 58 mm3 only on the 16th day. Meanwhile, the SKBR3
tumors in IV-injected groups 4 and 5 significantly decreased over time.
The experiment of SKBR3 tumor growth in group 5 (side-by-side MDA-
MB-231- and SKBR3-bearing mice), which initially was 27 ± 2 mm3,
was discontinued on the 10th day according to ethical regulation: the
size of the MDA-MB-231 tumor became too large (1015 ± 51 mm3).
Nonetheless, the group 4 tumor volume decreased to 10 ± 1 mm3 on the
Fig. 8. SKBR3 and MDA-MB-231 (MDA) tumor growth curves. 29th day (Fig. 7). Thus, Cy5-Ab-SS-SN38 administered IV at 10 mg/kg
led to an ~3-fold SKBR3 tumor decrease.
(Fig. 8). Then, all the tumors were resected for further imaging and flow The resected tumors were captured in white light (Fig. 9A), and the
cytometry analysis. tumor volumes were determined by a caliper. The MDA-MB-231 tumors
of groups 1, 2 and 5 were 910 ± 42 mm3, 918 ± 18 mm3, and 1015 ± 63
2.6.2. Tumor development mm3, respectively. The SKBR3 tumors of groups 3, 4 and 5 were 1026 ±
As shown in Fig. 8, the MDA-MB-231 tumor in mice from groups 1 48 mm3, 14.5 ± 0.5 mm3 and 27 ± 1 mm3, respectively. The volumes of
(control), 2 and 5 (after IV injection of the conjugate) (Fig. 6) started the tumors were similar to those determined in vivo by the same caliper
rapidly developing on the 3rd day of observation. Meanwhile, the method (Fig. 6).
SKBR3 tumors in mice from group 3 (control) showed moderate growth We can conclude, therefore, that Cy5-Ab-SS-SN38 can efficiently
over 10 days followed by rapid growth. The accelerated development of prevent tumor growth selectively for HER2+ cancer cell lines (SKBR3),
MDA-MB-231 tumors compared to SKBR3 tumors was previously re­ causing a 3-fold decrease in the tumor during the observed period.
ported [18]. At the same time, the SKBR3 tumors on Cy5-Ab-SS-SN38
(ADC)-injected mice from groups 4 and 5 did not show a notable in­ 2.6.3. Flow cytometry
crease over 10 days but gradually decreased afterwards. By the 9th day, The resected tumors (Fig. 9A) were dissociated into single-cell sus­
the MDA-MB-231 tumors in control group 1 reached 910 ± 33 mm3, pensions, as described in the procedure [48], and the isolated cells were
demonstrating a 30-fold volume increase compared to their initial vol­ stained with Ann–FITC and propidium iodide (PI) to identify dead
ume (~30 mm3), which is similar to the tumors of ADC-injected group 2 apoptotic and necrotic cells, respectively, by flow cytometry. The cells
that remained unstained were considered alive. As anticipated, only live

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D. Kobzev et al. European Journal of Medicinal Chemistry 252 (2023) 115298

Fig. 9. Representative photographs of the tumors (A). Flow cytometry analysis of cancer cells taken from MDA-MB-231 (MDA) and SKBR3 mouse tumors. Q1 –
necrosis, Q2 – late apoptosis, Q3 – early apoptosis, Q4 – live cells. (B).

cells (98.4–99.4%) were detected for all the MDA-MB-231 tumor-­ development (10th day) and predominantly apoptosis at a late stage of
bearing groups (1, 2 and 5) and for the SKBR3 tumor-bearing control cancer development (29th day). As anticipated, the increasing number
group 3. of ADC injections increases the treatment efficacy; however, adminis­
Flow cytometry analysis of the SKBR3 cancer cells taken from group tering the five ADC doses instead of the two results in only a 1.6-fold
5 (SKBR3 tumor) resected on the 10th day (total, two IV injections) reduction in the number of live cells.
showed significant necrosis (52.3%) and insignificant early (2.1%) and Next, the tumors and mouse organs (heart, liver, spleen, and kidney)
late (6.1%) apoptosis (Fig. 9B). In contrast, analysis of the SKBR3 cancer were resected; the images were taken (Fig. 10) and compared to those
cells in group 4 resected on the 29th day (total of five IV injections) for the nonadministered control mice. All the organs except the brain
indicated almost no necrosis (2.5%) but significant early (36.7%) and late from control mice had very low autofluorescence (groups 1 and 3),
(36%) apoptosis. The number of live cells in group 5 (10th day) was which was taken as a threshold background. Upon using the Cy5-Ab-SS-
39.5%, which was 1.6-fold higher (24.9%) than that in group 4 (29th SN38 conjugate, the fluorescence intensity of the MDA-MB-231 tumors
day). Thus, the death mechanism for ADC-treated SKBR3 cells in the from groups 2 and 5 became more pronounced compared to other or­
tumor was predominantly necrosis at an early stage of cancer gans, and there was bright fluorescence of the SKBR3 tumors from

Fig. 10. Images of selected organs of tumor-bearing mice after resection. 1st row – white-light, 2nd row – fluorescence channel, 3rd row – their merging.

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groups 4 and 5, although SKBR3 tumors had much lower volumes procedure [49]. 1H NMR (400 MHz, CDCl3, ppm): δ 7.77 (d, J = 7.6 Hz,
compared to those of MDA-MB-231. Because the conjugate only negli­ 2 H), 7.60 (d, J = 7.5 Hz, 2 H), 7.37–7.45 (m, 2 H), 7.29–7.36 (m, 2 H),
gibly accumulated in the nontargeting healthy organs, it should not 5.19 (br. s., 1 H), 4.43 (d, J = 6.8 Hz, 2 H), 4.19–4.29 (m, 1 H), 3.53 (q, J
affect them. = 6.0 Hz, 2 H), 2.91–2.98 (m, 2 H), 2.72–2.86 ppm (m, 4 H). 13C NMR
(100 MHz, CDCl3, ppm): 175.69, 156.39, 143.82, 141.32, 127.71,
3. Conclusions 127.05, 125.02, 120.00, 66.81, 47.19, 39.68, 37.94, 33.72, 32.92.
HRMS (ESI) m/z calcd for C20H21NO4S2 [M+Na]+ = 426.0810, found:
In summary, we have developed a novel theranostic antibody‒drug 426.0815.
conjugate (ADC), Cy5-Ab-SS-SN38, based on the SN38 drug conjugated 4.2.2. Solid phase synthesis of (S)-3-((2-((((4,11-diethyl-9-hydroxy-
with the trastuzumab antibody via a self-immolative 3-((2-aminoethyl) 3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3′ ,4’:6,7] indolizino [1,2-
disulfaneyl)propanoic acid (disulfide carbamate) linker and additionally b]quinolin-4-yl)oxy)carbonyl) amino) ethyl)disulfanyl) propanoic acid
equipped with the fluorescent dye Cy5. Utilization of the disulfide (SN38-SS-CO2H).
carbamate linker compared to the carbonate linker reduced the drug
release rate compared to disulfide carbonate linkers (τ1/2–21 h vs. ~8 h). a) Loading of Fmoc-protected 3-((2-aminoethyl)disulfanyl)propanoic
This ADC provides a bright fluorescent signal enabling real-time moni­ acid (Fmoc-SS-CO2H). To a swelled 2-Cl-Trt chloride resin (200 mg,
toring of the ADC distribution in the body and accumulation in the 0.4–0.6 mmol/g loading) in a jacketed fritted peptide synthesis
tumor. The accumulation rate (time) for this ADC, determined through vessel, a solution of Fmoc-SS-CO2H (50 mg, 0.12 mmol, synthesized
the increase in the fluorescence signal in SKBR3 and BT474 cells in vitro in a 63% yield according to the procedure [43]) in dry DCM (4 mL)
to reach saturation, was found to be approximately 3 h. Self-immolative and DIPEA (0.125 μL, 0.72 mmol) was added, and the mixture was
release of SN38 from the ADC was defined. The IC50 on the SKBR3 cell shaken at room temperature for 1.5 h. The solid support was capped
line was determined to be ~8.5 times lower than that for SN38 (at cDrug with DCM–MeOH–DIPEA (5 mL, 17:2:1, v/v/v, 30 min) and washed
= 10 nM) and ~4.1 times lower than SN38 for the BT474 cell line (at with DCM (5 × 5 mL).
cDrug = 150 nM). b) Fmoc deprotection: The Fmoc protecting group was removed by
The study of the Cy5-Ab-SS-SN38 efficacy in the mouse xenograft treatment with 20% piperidine in DMF (2 × 5 mL, 15 min each) with
model to suppress SKBR3 and MDA-MB-231 breast cancer tumor growth subsequent washing with DCM (2 × 5 mL, 3 min) and DMF (2 × 5
allows us to conclude that this ADC efficiently prevents tumor growth mL, 3 min each).
selectively for SKBR3 (HER2+) cancer cell lines, causing a 3-fold c) Attaching of the drug to amine tether via carbamate linker: 10-O-
decrease in the tumor during the 29 days. At the same time, this ADC Boc-SN38–20-O-PNC (163 mg, 0.25 mmol) was dissolved in DMF (2
had no detectable effect on MDA-MB-231 (HER2-) tumors, confirming mL) containing DIPEA (125 μL, 0.72 mmol). Then, the obtained so­
its selectivity. The cell death mechanism is predominantly necrosis in lution was added to the resin loaded with deprotected amino acid
the early stage of cancer development and predominantly apoptosis in from part “b” and shaken for 2 h at room temperature. The resin was
the late stage of cancer development. washed twice with DCM/DMF (1:1) and twice with DCM to yield
We anticipate, therefore, that the Cy5-Ab-SS-SN38 ADC can expand resin loaded with 10-O-Boc-SN38-SS-CO2H.
the scope of efficient tools for theranostic applications aimed at HER2+ d) Cleavage of SN38-SS-CO2H from the resin with simultaneous Boc
breast cancer. deprotection: To the dried resin loaded with 10-O-Boc-SN38-SS-
CO2H (from part “c”) was added a cold 30% solution of TFA in DCM
4. Experimental section (2 mL). After shaking for 1 h, the solution was filtered; the filtrate
was collected and evaporated under a N2 stream. The crude residue
4.1. General was purified by preparative HPLC (Phenomenex Gemini® 10 μm
RP18 110 Å, LC 250 × 21.2 mm column, 0–100% ACN–water + 0.1%
All chemicals were supplied by Alfa Aesar Israel and Sigma‒Aldrich. FA, flow rate of 30 mL/min, 25 ◦ C) to afford pure SN38-SS-CO2H as a
Solvents were purchased from Bio-Lab Israel and used as is. Chemical white solid (53 mg, 0.09 mmol, 36% overall yield). 1H NMR (400
reactions were monitored by TLC (Silica gel 60 F-254, Merck) and LC‒ MHz, DMSO‑d6, ppm): δ 12.33 (br. s., 1H), 10.31 (br. s., 1H), 8.06 (d,
MS. LC‒MS analysis was performed using an Agilent Technologies 1260 J = 9.7 Hz, 1H), 7.39–7.43 (m, 2H), 7.28 (s, 1H), 5.27 (s, 2H),
Infinity (LC) 6120 quadruple (MS) column Agilent SB-C18, 1.8 mm, 2.1 4.70–4.94 (m, 3H), 3.74–3.89 (m, 2H), 3.09 (q, J = 7.5 Hz, 2H), 2.98
× 50 mm, column temperature 50 ◦ C, eluent acetonitrile (ACN)–water (t, J = 7.0 Hz, 2H), 2.88 (t, J = 7.0 Hz, 2H), 2.59 (t, J = 8.0 Hz, 2H),
+ 0.1% formic acid (FA). HPLC purifications were carried out on an 2.30–2.41 (m, 1H), 1.94–2.07 (m, 1H), 1.30 (t, J = 7.6 Hz, 3H), 0.95
ECOM preparative system, with dual UV detection at 254 nm and 214 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6, ppm): δ 172.49,
nm. HRMS was performed in ESI positive mode by using an Agilent 6550 172.34, 160.46, 156.68, 153.59, 148.64, 144.85, 144.06, 143.55,
iFunnel Q-TOF LC‒MS instrument. 1H NMR and 13C NMR spectra were 142.7, 131.46, 128.56, 128.15, 122.3, 104.69, 95.98, 88.51, 74.68,
measured in DMSO‑d6 at 300 K on a Bruker AvanceIII HD (1H 400 MHz 54.76, 49.68, 37.11, 33.83, 33.49, 32.75, 31.14, 22.21, 13.26, 7.53.
and 13C 100 MHz) spectrometer and a BBO probe equipped with a Z HRMS (ESI) m/z calcd for C28H29N3O8S2 [M+H]+ 600.1467, found:
gradient coil. The Ab-conjugate was purified by Sephadex G50 gel 600.1442.
permeation chromatography, and its structure was confirmed by MALDI
(Agilent 6545A-Q-TOF).
4.2.1. Synthesis of Cy5-Ab–SS–SN38 conjugate
4.2. Synthesis SN38-SS-CO2H (0.5 mg, 0.83 μmol), 1-ethyl-3-(3-dimethylamino­
propyl)carbodiimide (EDC, 1.6 mg, 0.83 μmol) and N-hydrox­
4.2.1. a) tert-Butyl ((4S)-4,11-diethyl-3,14-dioxo-3,4,6,11,12,14- ysuccinimide (NHS, 2.4 mg, 20.8 μmol) were dissolved in DMF (107 μL).
hexahydro-1H-pyrano[3′ ,4’:6,7]indolizino [1,2-b]quinoline-4,9-diyl) The mixture was stirred at room temperature for 13 h to give SN38-SS-
(4-nitrophenyl) bis(carbonate) (10-O-Boc-SN38)-20-O-PNC) was syn­ NHS solution (0.58 mg in 107 μL DMF). The obtained solution was used
thesized from commercially available SN38 in 78% yield according to for conjugation with antibody.
the procedure [42]. HRMS (ESI) m/z calcd for C34H31N3O11 [M+H]+ An aliquot of the commercially available trastuzumab solution (13.0
658.2032, found: 658.2003. mg, 86.6 nmol, 619 μL) was diluted with 0.1 M PBS pH 7.4 (1.38 mL) to a
b) Fmoc-protected 3-((2-aminoethyl)disulfanyl)propanoic acid final concentration of 6.5 mg/mL. Then, an aliquot (0.42 mg, 0.6 μmol,
(Fmoc–SS–CO2H) was synthesized in 47% overall yield according to the 77.9 μL) of the prepared SN38-SS-NHS solution was added dropwise to

9
D. Kobzev et al. European Journal of Medicinal Chemistry 252 (2023) 115298

the above trastuzumab solution and stirred for 1 h at 25 ◦ C to form the 4.5. Self-immolative release of SN38
Ab-SN38-SS conjugate. The obtained conjugate was separated from the
unbound SN38-SS-NHS by using gel-permeation chromatography Self-immolative release of SN38 was measured according to a pre­
(Sephadex G50, PBS pH 7.4). The first pale yellow fraction was collected viously described procedure [31]. SN38-SS-CO2H (2 mg) was dissolved
to yield the Ab-SS-SN38 solution (11.2 mg, 74.6 nmole, 9.2 mL). The in 1 mL PBS, DTT was added at a final concentration of 10 mM, and the
obtained Ab-SS-SN38 solution was used for further conjugation with mixture was incubated at 37 ◦ C. Aliquots (10 μL) of the solution were
Cy5. taken over 50 h, transferred to LC‒MS vials by a filtering syringe,
Cy5 (0.5 mg, 0.78 μmol) and N,N,N′ ,N′ -tetramethyl-O-(N-succini­ diluted with ACN–water (50:50, 150 μL), and analyzed using LC‒MS at
midyl)uronium tetrafluoroborate (TSTU, 0.35 mg, 1.2 μmol) were dis­ 254 nm [31]. Then, the kinetic curves of the relative concentrations of
solved in DMF (100 μL), and N,N-diisopropylethylamine (DIPEA) (0.8 SN38-SS-CO2H, SN38-SS, and SN38 over time were drawn, and the
μL, 4.67 μmol) was added. The mixture was stirred at 25 ◦ C for 20 min to reaction rates were quantified through the half-lives (τ1/2).
give a Cy5-NHS solution (0.69 mg in 100 μL DMF). The obtained solu­
tion was used for conjugation with antibody. 4.6. Accumulation of Cy5-Ab–SS–SN38 on cells
An aliquot (0.26 mg, 0.3 μmol, 37.7 μL) of the above Cy5-NHS so­
lution was added dropwise to the Ab-SS-SN38 solution and stirred for 1 The breast cancer cell lines BT474, SKBR3 and MDA-MB-231 were
h at 25 ◦ C. The obtained conjugate was column separated from the plated in poly-D-lysine (PDL)-coated 0.8 cm2/well chamber slides
unbound Cy5 (Sephadex G50, PBS pH 7.4). The first blue fraction was (Thermo Fisher Scientific) at 1 × 104 cells/well. The cells were allowed
collected to yield a Cy5-Ab-SS-SN38 solution (11.0 mg, 10.6 mL). to grow overnight in the corresponding medium (see Section 2.4). Wells
MALDI-TOF: MW (trastuzumab) found: 148,225 Da (Fig. S1); MW were then decanted and filled with the corresponding medium (see
(Cy5-Ab-SS-SN38) calcd: 150,011 Da, found: 150,018 Da (Fig. S2). The Section 2.4) containing 50 nM Cy5-Ab-SS-SN38 and incubated for 0, 1,
molar ratios of the components in Cy5/Ab/SS-SN38 were approxi­ 3, and 6 h. After incubation, the cells were washed 3 times with phenol
mately 1:1:2 (MALDI-TOF). red− free RPMI-1640-based medium containing penicillin/streptomycin
Spectrophotometry: The dye-to-antibody ratio (Cy5/Ab) for Cy5- and 10% FBS and then analyzed by fluorescence microscopy.
Ab-SS-SN38 was 0.7, and the drug-to-antibody ratio (SN38/Ab) was Then, imaging experiments were carried out at 37 ◦ C in a humidified
2.0. The obtained amount of the conjugate (11.0 mg) was also deter­ atmosphere containing 5% CO2. The bright field and fluorescence im­
mined spectrophotometrically. ages were acquired by a Photometrics CoolSNAP HQ2 camera mounted
The Cy5-Ab-SS-SN38 solution was diluted with PBS to a final con­ on an Olympus iX81 fluorescence microscope. The microscope was
centration of 1 mg/mL, kept at 4 ◦ C, and used for further experiments. equipped with a 100 W halogen lamp for phase contrast observations
and a 120 W metal halide discharge lamp for fluorescence imaging. For
4.3. Dye-to-antibody (D/P) and drug-to-antibody (DAR) ratios fluorescence imaging, a T495lpxr dichroic filter and a Cy5 (red) cube
comprising an ET620/60x bandpass excitation filter, ET700/75 m
Dye-to-antibody (Cy5/Ab) and drug-to-antibody (SN38/Ab) ratios bandpass emission filter, and T660lpxr dichroic filter were used. All the
were measured by the spectrophotometric method and calculated images were taken with the same instrument settings for each channel.
similar to Ref. [49], according to Equations (1) and (2). The light source intensity was 12%, and the gain was 20.5 dB. The
/ magnification was × 20. The exposure time was 200 ms for the bright
A650 × εAb
Cy5 Ab = , (1) field and 500 ms for the fluorescence red channel. The integrated fluo­
[(A280 − x1 × (A379 − x2 × A650 ) − x3 × A650 ] × εCy5
rescence intensities were quantified by using ImageJ software [50]
/ through the integrated densities. Then, the integrated fluorescence in­
(A379 − x3 × A650 ) × εAb
SN38 Ab = , (2) tensities of the Cy5-Ab-SS-SN38-treated cells over time were plotted
[(A280 − x1 × (A379 − x2 × A650 ) − x3 × A650 ] × εSN38−
and compared to evaluate ADC accumulation.
SS

where A650 is the absorbance of the Cy5-Ab-SS-SN38 conjugate at the

Cy5 absorption maximum; A280 is the absorbance of the Cy5-Ab-SS- 4.7. Cytotoxicity of Cy5-Ab–SS–SN38
SN38 conjugate at the Ab absorption maximum (280 nm); A379 is the
absorbance of the Cy5-Ab-SS-SN38 conjugate at the SN38-SS absorp­ Cytotoxicity was quantified through the cell viability that was
tion maximum (379 nm); εCy5, εAb, and εSN38-SS are the extinction co­ measured by a commercial CCK-8 assay kit (Cell Counting Kit-8, Merck,
efficients of Cy5 (250,000 M− 1cm− 1), Ab (201,700 M− 1cm− 1), and Israel) according to the following procedure [51]. BT474, SKBR3 and
SN38-SS (8500 M− 1cm− 1), respectively; x1–x3 are the correction factors MDA-MB-231 cells were seeded in 96-well plates (1 × 104 cells/well)
for the absorbances: x1 = A280/Amax(SN38-SS), x2 = A379/Amax(Cy5), and incubated overnight (37 ◦ C, 5% CO2 atmosphere) in the corre­
and x3 = A280/Amax (Cy5), where Amax(SN38-SS), A379/Amax(Cy5), and sponding growth medium (see Section 2.4). Then, the cells were washed
A280/Amax(Cy5) are the absorbances for the unconjugated SN38-SS and with 0.1 M PBS pH 7.4 and recultured in 100 mL of the corresponding
Cy5, respectively. medium, incubated for 6 h and 16 h with a range of concentrations
(0.04, 0.4, 4, 40, 400, and 1000 nM) of Cy5-Ab-SS-SN38, Ab, SN38, and
4.4. Cell culture an equimolar mixture of Ab with SN38 (Ab + SN38). After washing with
PBS, the wells were filled with the corresponding medium (see Section
All human breast cancer cell lines were obtained from ATCC (Man­ 2.4) and incubated (37 ◦ C, under 5% CO2) for an additional 72 h. Then,
assas, VA). MDA-MB-231 cells were cultured in DMEM (high glucose), 10 μl of CCK-8 solution was added to each well, and the plate was further
SKBR3 cells in McCoy’s 5A medium and BT474 cells in DMEM/F12 incubated for 2.5 h at 37 ◦ C. Production of formazan from WST8 (con­
medium. All the culture media were supplemented with 10% fetal tained in CCK-8) in the presence of live cells was quantified by a TECAN
bovine serum, 1% penicillin‒streptomycin, 1% glutamine, and 1% so­ Infinite M200 ELISA plate reader through the absorbances at 460 nm.
dium pyruvate. DMEM/F12 medium was additionally supplemented Then, the cell viability (the number of living cells), which was directly
with 0.1% (v/v) insulin. Culture conditions were maintained at 37 ◦ C in proportional to absorbance at 460 nm, was determined, and a corre­
a humidified atmosphere containing 5% CO2. sponding plot for the cell viability vs. concentration of a drug under
examination was built. All tests were performed in triplicate, and each
experiment was repeated three times. The results are expressed as the
mean ± standard deviation. Data were analyzed using GraphPad Prism
6.0 (GraphPad Software, Inc., San Diego, CA, USA) [52]. Statistical

10
D. Kobzev et al. European Journal of Medicinal Chemistry 252 (2023) 115298

analysis was performed by two-way ANOVA. A p value < 0.05 indicated Author’s contributions
a statistically significant difference.
D.K. was involved in the investigation, ADC synthesis, spectral
characterization, mouse experiments including mouse imaging, and
4.8. Flow cytometry writing of the original draft. C.P. provided in vitro cytotoxicity and cell
uptake experiments, flow cytometry, and microscopy. D.W. and A.B.
Preparation of cells after in vivo experiment. Tumor-bearing mice were involved in the synthesis of the dye and SN38 with linkers and
were sacrificed, the tumors were resected, and single-cell suspensions structure confirmation. H.G. performed the investigation of SN38
(0.5–1.0 × 106 cells) were prepared from mouse tumors according to a release from ADC. O.S. provided spectroscopic and fluorescence char­
previously described procedure [48]. Isolated cells were washed and acterization. L.P. and G.G. designed the study, reviewed experiments,
then fixed with paraformaldehyde to prevent further degradation. and drafted, edited and reviewed the manuscript. All authors reviewed
Flow cytometry analysis. The cells were stained with the Annexin V- the manuscript and approved the final version.
FITC/PI apoptosis detection kit according to the manufacturer’s proto­
col [53]. The data were acquired by a CytoFLEX (Beckman Coulter) flow
cytometer and analyzed using FlowJo software to quantify the number
Declaration of competing interest
of live, apoptotic and necrotic cells.
The authors declare that they have no known competing financial
4.9. Animal experiments interests or personal relationships that could have appeared to influence
the work reported in this paper.
All animal experiments were carried out in compliance with the
Israel Council on Animal Care regulations and were approved by the Data availability
Animal Care Committee of Ariel University (Authorization number IL-
179-06-19). Twenty six-week-old athymic BALB/c female nude mice Data will be made available on request.
(Harlan Labs, Nes Ziona, Israel) were subcutaneously inoculated in the
dorsal right side with the human breast cancer cell lines MDA-MB-231 Acknowledgements
(MDA, 10 mice) and SKBR3 (10 mice), 1 × 106 cells in 0.1 M PBS pH
7.4 into nu/nu mice, 100 μL per mouse. Similarly, 5 mice were inocu­ The authors are grateful to Ms. Helena Tuchinsky and Dr. Galia
lated simultaneously on the dorsal right side with SKBR3 cells and on the Luboshits (Ariel University, Israel) for their kind help with cell and
dorsal left side with MDA-MB-231 cells. Tumors were allowed to mouse imaging experiments.
establish until the tumor sizes reached approximately 30 mm3 (11 days).
Then, the mice were randomly separated into five groups (5 mice per Appendix B. Supplementary data
group). Group 1 (MDA-MB-231) and group 3 (SKBR3) were used as the
controls for tumor growth and background autofluorescence in imaging Supplementary data to this article can be found online at https://doi.
experiments. After 11 days of tumor development, groups 2, 4 and 5 org/10.1016/j.ejmech.2023.115298.
were intravenously (IV) administered (tail) every 7 days (until the end of
the experiment) the Cy5-Ab-SS-SN38 conjugate (200 μg in 200 μL PBS,
Appendix A. Supplementary data
10 mg/kg). The tumor sizes were measured in vivo over time by using the
hands-on digital caliper method, and the tumor volumes were calculated
Supplementary data for this article can be found online at https
according to Equation (3) [47]:
://doi.org/10.1016/j.ejmech.2023.115298.
( )
Tumor volume mm3 = 0.5 × Tumor Length × Tumor Width2 (3)
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