Synthesis, characterization, and targeted chemotherapy of SCT200-linker-monomethyl auristatin E conjugates

Article abstract of DOI:10.1016/j.ejmech.2021.113297

Antibody-drug conjugates (ADCs) are currently among the most successful and important strategies for treating patients with solid tumors. ADCs are composed of a monoclonal antibody and warhead, which are conjugated via a linker. Currently, monomethyl auristatin E (MMAE) is the most widely applied warhead in the development of ADCs. However, MMAE-based ADCs are generally constructed using the MC-VC-PABC linker, and this design has limited structural diversity and some disadvantages. Accordingly, in this study, we generated three types of novel linker-MMAE (with alterations in the spacer, catabolizing area, and self-immolative compared with MC-VC-PABC-MMAE) in ADCs, termed SCT200-linker-MMAE conjugates, and then evaluated the linker-drug plasma stability and the rate of drug release by cathepsin B. The binding ability, internalization rates, and efficacy of all SCT200-linker-MMAE ADCs were systematically studied, and the expression of apoptosis-associated proteins and the therapeutic efficacies of SCT200-M-2, -C-2, and -C-4 were evaluated. The results showed that the activities of some of these ADCs were increased for epidermal growth factor receptor-positive tumors. Moreover, the novel linkers designed in this study can be linked with other antibodies to treat other types of cancer. Overall, these findings provide important insights into the application of SCT200-based linkers in ADCs.

Full text of DOI:10.1016/j.ejmech.2021.113297

European Journal of Medicinal Chemistry 216 (2021) 113297
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis, characterization, and targeted chemotherapy of SCT200-
linker-monomethyl auristatin E conjugates
a
,
b
,
1
a
,
1
a
a
a
a
Xinyue Hu
, Hailun Jiang , Weiqi Bai , Xiujun Liu , Qingfang Miao , Linlin Wang ,
a
a
a,
**
, Zhuorong Li a
, *
Jie Jin , Along Cui , Rui Liu
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
National Institutes for Food and Drug Control, Beijing, 102629, China
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 January 2021
Received in revised form
Antibody-drug conjugates (ADCs) are currently among the most successful and important strategies for
treating patients with solid tumors. ADCs are composed of a monoclonal antibody and warhead, which
are conjugated via a linker. Currently, monomethyl auristatin E (MMAE) is the most widely applied
warhead in the development of ADCs. However, MMAE-based ADCs are generally constructed using the
MC-VC-PABC linker, and this design has limited structural diversity and some disadvantages. Accordingly,
in this study, we generated three types of novel linker-MMAE (with alterations in the spacer, catabolizing
area, and self-immolative compared with MC-VC-PABC-MMAE) in ADCs, termed SCT200-linker-MMAE
conjugates, and then evaluated the linker-drug plasma stability and the rate of drug release by cathepsin
B. The binding ability, internalization rates, and efficacy of all SCT200-linker-MMAE ADCs were sys-
tematically studied, and the expression of apoptosis-associated proteins and the therapeutic efficacies of
SCT200-M-2, -C-2, and -C-4 were evaluated. The results showed that the activities of some of these ADCs
were increased for epidermal growth factor receptor-positive tumors. Moreover, the novel linkers
designed in this study can be linked with other antibodies to treat other types of cancer. Overall, these
findings provide important insights into the application of SCT200-based linkers in ADCs.
7
February 2021
Accepted 11 February 2021
Available online 24 February 2021
Keywords:
Antibody-drug conjugate
Solid tumor
Monomethyl auristatin E
Linker
Drug release
©
2021 Elsevier Masson SAS. All rights reserved.
1
. Introduction
effective warhead release in targeted tumor cells. In particular,
linker stability plays essential roles in the efficacy and safety/
tolerability, pharmacokinetic properties, and therapeutic indexes of
ADCs [4]. Linkers can be classified according to different categories,
e.g., cleavable and noncleavable linkers, according to their mecha-
nisms for releasing the drug payload from the antibody [5].
Cleavable linkers are widely used and can liberate the payload
based on the features of the physiological environment. Acid-
dependent cleavage, e.g., generation of hydrazine, yields products
that are relatively stable under serum-like conditions (pH 7.3e7.5)
but requires cleavage to occur at a low pH in order to release the
drug into the lysosomal compartment [6]. Another strategy for
development of a cleavable linker system is the disulfide-based
linker. Disulfide bonds within the linker are relatively stable at
low extracellular glutathione (GSH) concentrations but are reduced
by intracellular GSH to release the free drug. In a previous study, a
series of maytansine ADCs were prepared using a disulfide linker to
reduce the extent of payload release in the bloodstream [7]. The
combined application of hydrazone and disulfide linkers could
improve the efficiency of releasing cytotoxin. For example,
Antibody-drug conjugates (ADCs) use antibodies as carriers to
deliver potent cytotoxic agents as a warhead (or payload) to spe-
cifically target and kill tumor cells, thereby improving the selec-
tivity of cytotoxic agents and increasing the antitumor activity of
the antibody compared with the naked form [1]. ADCs have become
increasingly important in the treatment of cancer [2]. The number
of approved ADCs has grown in recent years, with seven ADCs
approved by the Food and Drug Administration and several more in
clinical trials [3].
ADCs are composed of a targeting antibody, a cytotoxic warhead,
and a linker that binds the antibody and warhead. The character-
istics of linkers contribute to the stability of ADCs in the serum and
*
Corresponding author.
*
* Corresponding author.
E-mail addresses: liurui@imb.pumc.edu.cn (R. Liu), lizhuorong@imb.pumc.edu.
cn (Z. Li).
1
XH and HJ contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113297
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
Mylotarg (an anti-CD33-calicheamicin conjugate) and Besponsa
2. Results and discussion
(an anti-CD22-calicheamicin conjugate) contain this bifunctional
linker [8,9]. Therefore, the linker and its chemical structure are
crucial for optimizing the therapeutic potential and safety profiles
of ADCs.
2.1. Chemistry
2.1.1. Generalized synthesis of linker-MMAE
Lysosomal protease-sensitive linkers have been incorporated
into Adcetris, Polivy, and Padcev (as monomethyl auristatin E
We generated a series of SCT200-linker-MMAE ADCs targeting
EGFR based on cysteine conjugation. Three types of linkers were
obtained by structural modification, including modification of the
spacer, catabolizing area (enzymatic hydrolysis peptides), and self-
immolative group. The general molecular formula of linker-MMAE
is illustrated in Table 1. Linking to antibody-derived cysteine is
commonly mediated through maleimide-type linkers [16]. The
most common linker-drug is MC-VC-PABC-MMAE, which conju-
gates MMAE via the MC-VC-PABC linker [17]. Structural differences
in M-1eM-3 were in the maleimidocaproyl functional group, un-
modified M-1 contained the MC spacer. This may increase linker-
drug plasma stability, in contrast to the MC spacer. The general
procedure for the synthesis of maleimidocaproyl active ester is
shown in Scheme S1. Structural differences in C-1eC-5 were in the
enzymatic hydrolysis peptide and involved modification of di-
peptides, tripeptides, and tetrapeptides as the catabolizing area
with the same maleimidocaproyl functional group, MCC. With this
design, linker-drug plasma stability and enzyme-mediated drug
release rates may increase. Linker-MMAE M-1eC-5 constructs were
prepared in five steps starting with the Fmoc-AA, which was
generated as previously described [18] (Scheme 1). Compounds 1a
and 1b were prepared in two steps with the starting material Fmoc-
Val-OH (Scheme S2). Compounds 1ce1f were prepared using a
solid-phase synthesis method employing 2-chlorotrityl chloride
resin (2-CTC resin) to synthesize Fmoc protective tripeptide and
tetrapeptide compounds. The Fmoc protective terminal amino acid
was anchored on the 2-CTC resin in the presence of N, N-diiso-
propylethylamine (DIEA) in dichloromethane anhydrous (DCM).
The linear peptide was synthesized using a consecutive Fmoc group
deprotection strategy, followed by an amino acid coupling reaction
(Schemes S3eS6). Then, a condensation reaction was performed
between 1ae1f and 4-aminobenzyl alcohol by treatment with 1-
[MMAE]-conjugated ADCs). These linkers are composed of three
parts: a spacer, a catabolizing area, and a self-immolative group.
The conventional conjugation method exploiting native cysteine
residues in the antibody involves using a maleimide spacer and
thiol coupling to reduced cysteines from the interchain disulfides,
which can theoretically yield 0, 2, 4, 6, or 8 payload molecules per
antibody [10]. The catabolizing area is a valine-citrulline (VC)
dipeptide, which can be cleaved by lysosomal enzymes (cathepsin
B) to release the free drug [11]. VC dipeptide was modified with the
drug MMAE, separated by the self-elimination group p-amino-
benzyoxycarbonyl (PABC). The PABC moiety undergoes a [1,6]-
elimination reaction to release the whole MMAE payload [12]. The
VC dipeptide linker has been widely used in various clinical ADCs.
Although this linker has been shown to have effective, novel linkers
based on the VC linker should also be explored. Notably,
maleimide-based linkers can undergo retro-Michael addition,
allowing the drug to be removed from the antibody during circu-
lation and undergo succinimidyl ring-opening hydrolysis, which
can retain the drug attachment [13]. Therefore, the maleimide-
based linker design strategy aims to increase the stability of the
ADC in the circulation. For the VC dipeptide, Dorywalska et al.
recently described an extracellular carboxylesterase (carbox-
ylesterase 1C) as a potential source of proteolytic activity for the
cleavage of VC linkers in the tumor extracellular milieu, presenting
a molecular design challenge [14].
Accordingly, in this study, we evaluated various linkers,
including the maleimide-based spacer maleimidocaproyl (MC),
maleimidomethyl cyclohexane-1-carboxylate (MCC), and N-phenyl
maleimide (MC-Ph), in the preparation of linker-drug conjugates
and evaluated their plasma stability. Furthermore, we sought to use
different dipeptides, tripeptides, and tetrapeptides as the catabo-
lizing area, and we replaced the peptide sequence for the self-
immolative group to induce cyclization and release MMAE [19].
These three linkers were designed to connect MMAE with SCT-200,
a fully-humanized anti-epidermal growth factor receptor (EGFR)
antibody, which may be less immunoreactive than the chimeric
antibody as the ADC carrier. This antibody is currently used to treat
metastatic colorectal cancer and some solid tumors in phase II
clinical trials in China [15]. The SCT200-linker-MMAE conjugates
ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in CH
3
OH/
0
CH
2
Cl
2
or O-(7-azabenzotriazol-1-yl)-N,N,N ,N’-tetramethyluro-
nium hexafluorophosphate (HATU) and DIEA in tetrahydrofurane
(THF), to yield 2ae2f. The Fmoc-groups of 2ae2f were deprotected
using diethylamine to obtain 3ae3f in dimethylformamide (DMF).
The surface-exposed N-terminal regions of 3ae3f were conjugated
with a maleimidocaproyl group to afford 4ae4h. The linkers 5ae5h
were finally obtained from 4ae4h with Bis(4-nitrophenyl) car-
bonate in DMF and were converted directly to the linker-MMAE (M-
1eC-5) using condensation agents in DMF.
(
Fig. 1) were evaluated in terms of internalization, binding ability,
apoptosis-related protein expression, and antitumor activity in vitro
and in vivo against EGFR-expressing models.
Structural modification of the self-elimination group PABC was
also evaluated. PABC is the most widely used self-immolative
group, and analyses have suggested that it may be necessary to
use an extended structure for the self-immolative group. Manabe
Table 1
Structures of the synthetic linker-MMAE conjugates.
Linker-MMAE
Spacer
Catabolizing area
Self-immolative group
M-1
M-2
M-3
C-1
C-2
C-3
C-4
C-5
S-1
MC
Val-Cit
Val-Cit
Val-Cit
Val-Ala
Ala-Val-Ala
Gly-Phe-Gly
Gly-Gly-Phe-Gly
Gly-Phe-Leu-Gly
Val-Cit
PABC
PABC
PABC
PABC
PABC
PABC
PABC
PABC
Gly-Pro
Ala-Pro
MCC
MC-Ph
MCC
MCC
MCC
MCC
MCC
MC
S-2
MC
Val-Cit
Fig. 1. Structures of linker systems for SCT200-linker-MMAE conjugates.
2

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
3 2 2
Scheme 1. General procedure for the synthesis of M-1-C-5. Reagents and conditions: a) 4-Aminobenzyl alcohol, EEDQ, CH OH/CH Cl for 2a; 4-Aminobenzyl alcohol, HATU, DIEA,
THF for 2b~2f; b) Et NH, DMF; c) DMF; d) Bis(4-Nitrophenyl) Carbonate, DIEA, DMF; e) MMAE, HOBt, DIEA, DMF.
2
et al. previously reported a Gly-Pro self-immolative group that
could form a diketopiperazine via a cyclization drug-release
mechanism [19]. Thus, we used this design for reference and hy-
pothesized that the conjugate could be cleaved between VC and
MMAE to release the parent drug completely. Therefore, the self-
immolative groups of S-1eS-2 were Gly-Pro and Gly-Ala di-
peptides, and the other two parts maintained the same basic
structure (MC-VC~). The generalized route for the synthesis of S-
treated with HATU and DIPEA in DMF to yield S-1 and S-2,
respectively (Scheme 2B).
2.1.2. Plasma stability and drug release of linker-MMAE
The rate of intracellular payload release is on the order of hours
[
20]. To determine the rate of drug loss from the linker-MMAE with
the production of free MMAE in plasma and the rate of drug release
with tumor-associated lysosomal cathepsin B, we detected the
concentration of free MMAE (nM) at various time points using
liquid chromatography (LC)-tandem mass spectrometry (MS/MS).
The curve of the slope (nM/h) was used to estimate the release rates
of MMAE. The data showed that the cleavage rates of linkers
differed in mouse plasma (Fig. 2A, Table 2). Specifically, the release
rate of MMAE for M-2 and M-3 was slower than that for M-1, which
contained an aliphatic maleimide spacer. Moreover, M-2 displayed
the lowest degree of instability, with a release rate of 4.0 nM/h.
These findings suggested that the structure modification of the
1
eS-2 is illustrated in Scheme 2. Compounds 15a and 15b were also
synthesized via standard solid-phase peptide synthesis. The first
amino acid Fmoc-Pro-OH was anchored on the 2-CTC resin, and
amino acids were then linked sequentially until the last amino acid,
Fmoc-Val-OH, was deprotected to obtain 13a and 13b. A conden-
sation reaction was then performed using MC-OSu in DMF for 3 h at
room temperature. The linkers of 15a and 15b were cleaved from
the resin using a solution of trifluoroacetic acid (TFA):H
2
O (95:5;
Scheme 2A). Finally, the linkers of 15a or 15b and MMAE were
3

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
Scheme 2. Synthetic Route of S-1 and S-2. Reagents and conditions: a) Fmoc-Pro-OH, DCM, DIEA, 3 h; b) 20% piperidine in DMF, 10 min and 10 min; c) Fmoc-Gly-OH or Fmoc-Ala-
OH, DMF, HCTU, DIEA, 1h; d) 20% piperidine in DMF, 10 min and 10 min; e) Fmoc-Cit-OH, DMF, HCTU, DIEA, 1h; f) 20% piperidine in DMF, 10 min and 10 min; g) Fmoc-Val-OH, DMF,
HCTU, DIEA, 1h; h) 20% piperidine in DMF, 10 min and 10 min; i) MC-OSu, DMF, 3h; j) 95% TFA/H O; h) MMAE, HATU, DIEA, DMF.
2
spacer contributed to the stability of the whole structure and then
affected MMAE release in plasma. In a comparison of the plasma
stability of C-1eC-5, we found that C-5 had a relatively lower de-
gree of stability, with a release rate of 97.9 nM/h. Notably, C-5
contained the GFLG tetrapeptide peptide sequence. Free MMAE
levels were comparable among C-1eC-4, and a release rate of less
than 22.6 nM/h (M-1) was observed. Interestingly, the release rate
was not correlated with the number of amino acids at the catabo-
lizing area. Both S-1 and S-2 were relatively stable in plasma.
Cathepsin B-mediated drug release of linker-MMAE (Fig. 2B,
4

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
Fig. 2. Plasma stability and drug release of linker-MMAE constructs. The horizontal axis represents the values of time points (h) and vertical axis represents the values of the
ꢀ
concentration of free MMAE (nM). A. Stability of the linker-MMAE constructs in plasma at 37 C. The linker-MMAE constructs were evaluated at the indicated time points (0, 30, 60,
ꢀ
1
80, and 360 min; n ¼ 2 each time point) via LC-MS. B. Cathepsin B reactivity for MMAE release. The linker-MMAE constructs were incubated with cathepsin B at 37 C and
evaluated at the indicated time points (0, 10, 60, 120, 240, or 360 min, n ¼ 2 each time point) via LC-MS, Error bars represent standard deviations from two parallel group ex-
periments. Data are shown as mean ± standard deviation.
Table 2
for the self-immolative group of S-1eS-2, the lower release rate of
MMAE may influence the activity of ADCs [25].
Average release rates of linker-MMAE constructs in plasma and cathepsin B.
Linker-MMAE
Stability in Plasma
K (nM/h)
Cathepsin B
K (nM/h)
2.1.3. Preparation of SCT200-linker-MMAE conjugates
ADCs were prepared via cysteine residues released by an anti-
M-1
M-2
M-3
C-1
C-2
C-3
C-4
C-5
S-1
22.6
4.0
10.9
8.6
13.2
8.77
14.7
97.9
8.8
/
body interchain disulfide bond reduction. SCT200 (>5 mg/mL) was
prepared in a buffer containing 50 mM potassium phosphate,
50 mM sodium chloride, and 1 mM DTPA (pH 8). Linker-MMAE M-
32.3
/
25.0
12.4
7.4
32.1
29.8
ND
ND
1
1
eS-2 constructs were prepared as solutions at a concentration of
0 mM in dimethylsulfoxide (DMSO). First, SCT-200 was partially
ꢀ
reduced by treatment with 3e4-fold TCEP for 2 h at 37 C under the
ꢀ
protection of nitrogen. The mixture was cooled to 4 C and quickly
S-2
1.31
dropped over linker-MMAE (6e8 M ratio), followed by incubation
ꢀ
ND: not detected.
for 1 h at 4 C under the protection of nitrogen. The reaction was
stopped by adding an excess amount of L-cysteine. Finally, SCT200-
linker-MMAE conjugates were purified using a Sephadex G25 col-
umn to remove excess linker-MMAE.
SCT200-linker-MMAE was characterized by hydrophobic inter-
action chromatography (HIC) [26,27], as shown in Fig. 3A and B. The
spectrum showed five major peaks, corresponding to zero, two,
four, six, and eight drugs per antibody. The drug-to-antibody ratios
Table 2) was nearly undetectable for S-1 and S-2. These findings
suggested that the amido bond between Pro and MMAE may be not
easy to cleave and could influence the self-immolative of the
cyclization drug-release mechanism. C-1eC-5 could be cleaved by
cathepsin B to release MMAE. In particular, GGFG and GFLG are
selectively cleaved by lysosomal enzymes [21,22], and the release
rates of C-4 (GGFG, 32.1 nM/h) and C-5 (GFLG, 29.8 nM/h) con-
taining the tetrapeptide peptide sequence were comparable to that
of M-2 (32.3 nM/h). Analysis of plasma stability and drug release by
cathepsin B, MMAE was observed by MS spectroscopy in three
different types of linker-MMAE in plasma, representing a small
amount of degradation by extracellular enzyme. It has been re-
ported that dipetide-based linkers (e.g. VC) can be hydrolyzed due
to their susceptibility to extracellular carboxylesterase (e.g. car-
boxylesterase 1C) [14,23,24]. In our study, the novel linker-MMAE
showed developed stability than M-1 expect C-5, the result indi-
cated that the structure modifications may reduce the degradation
from extracellular cleavage. Moreover, a good design strategy of
linker also depends on the sensitivity toward hydrolysis by
cathepsin B in tumor intracellular environment, linkers with
structural modifications in catabolizing area preserved suscepti-
bility towards cathepsin B. In conclusion, the MCC spacer could be a
suitable replaceable spacer. For the dipeptide sequence, M-2 was
slightly superior to C-1; for the tripeptide sequence, C-2 and C-3
were comparable; for tetrapeptides, C-4 was better than C-5; and
(DARs) are listed in Table 3. The average DAR was approximately 4.0
after integration of the observed peaks.
2
2
.2. Biological results
.2.1. Binding affinity and internalization rates of SCT200-linker-
MMAE ADCs
In order to test whether our new linker chemistry affected
binding ability, the binding affinity of SCT200-linker-MMAE ADCs
were evaluated by enzyme-linked immunosorbent assay (ELISA).
The ELISA-based binding assay revealed that both SCT200 and
SCT200-linker-MMAE ADCs could bind to the recombinant human
EGFR antigen in a concentration-dependent manner (Fig. 4BeD),
and the binding ability of SCT200-linker-MMAE ADCs showed
similar trends in binding affinity compared with naked antibody,
suggesting that conjugation of SCT200 with MMAE did not
decrease affinity and that the three different types of linkers did not
affect antigen recognition.
A successful ADC needs to bind specifically to the surface of
antigen-positive cells in the tumor, and the antigen-antibody
5

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
Fig. 3. Hydrophobic interaction chromatography (HIC) analysis of the SCT200-linker-MMAE conjugates with an average DAR of about 4. A. HIC analysis of the SCT200-M-1eM-3. B.
HIC analysis of the SCT200-C-1eC-5. C. HIC analysis of the SCT200-S-1eS-2.
Table 3
Average relative peak intensities for each DAR species of SCT200-M-1eM-3, -C-1eC-5, and -S-1eS-2.
ADC
D0
D2
D4
D6
D8
Average DAR
intensity
intensity
(%)
intensity
(%)
intensity
(%)
intensity
(%)
(%)
SCT200-M-1
SCT200-M-2
SCT200-M-3
SCT200-C-1
SCT200-C-2
SCT200-C-3
SCT200-C-4
SCT200-C-5
SCT200-S-1
SCT200-S-2
6.717
7.314
5.911
6.811
5.520
4.753
7.772
5.205
4.104
6.318
26.017
27.368
28.184
22.643
27.995
23.282
28.276
24.370
23.603
20.667
39.001
32.763
33.857
33.907
37.358
34.905
33.653
33.980
36.166
32.166
21.299
22.969
22.046
24.760
21.073
25.712
22.406
26.198
27.241
28.822
6.966
9.586
3.9
4.0
4.0
4.2
4.0
4.3
3.9
4.2
4.2
4.3
10.002
11.879
8.054
11.348
7.893
10.247
8.886
12.027
Average DAR ¼ [0 ꢂ D0 þ 2 ꢂ D2 þ 4 ꢂ D4 þ 6 ꢂ D6 þ 8 ꢂ D8]/100.
interaction generally has to result in internalization of the ADC [28].
Therefore, we developed a flow cytometry-based assay to measure
the internalization rates of three types of SCT200-linker-MMAE
ADCs. The degree of internalization was determined by the percent
decrease in the mean fluorescence intensity (MFI) of ADCs incu-
bated at 37 C compared with control ADCs incubated at 4 C in
KYSE450 cells (Fig. 4A, Table 4). The results revealed that the
endocytosis rates were similar for SCT200-linker-MMAE ADCs and
SCT200 (25.3% ± 3.2%, P > 0.05). Additionally, the novel antibody-
drug conjugate, which was composed of SCT-200 coupled
through different linkers, also had an internalization rate similar to
that of SCT200-M-1, which had a widely used linker (27.1% ± 5.4%).
Therefore, these findings showed that the three different linker
types did not affect the internalization ability of the ADCs.
effect (IC50 > 60 nM), implying that the cytotoxicity of SCT200-
linker-MMAE resulted from the delivery of MMAE rather than
from the efficacy of the antibody. We evaluated the MMAE conju-
gates (SCT200-ADCs) with SCT200-M-1 as a positive control and
found no significant changes in cytotoxic activity in linkers with
structural modifications in the spacer or catabolizing area. How-
ever, structural modifications to the self-immolative area of ADCs
SCT200-S-1 and SCT200-S-2 significantly reduced cytotoxic activ-
ity, yielding IC50 values that were approximately 100e1000 times
lower than that of SCT200-M-1. Based on the results of cathepsin B-
mediated drug release assays, the Pro-amine group self-immolative
linkers could not be completely cleaved by cathepsin B upon
internalization of ADCs by target cancer cells, resulting in the
traceless release of MMAE.
ꢀ
ꢀ
Based on our analysis of the plasma stability and drug release of
linker-MMAE and the toxicity of ADCs, we selected SCT200-M-2,
SCT200-C-2, and SCT200-C-4 for further analyses of the molecular
mechanisms of cell death by western blotting. p53 plays key roles
in the cell cycle and cell proliferation [29], cleaved poly (ADP-
ribose) polymerase (PARP) is a marker of apoptosis [30,31], and
caspase-3 is a frequently activated protease involved in pro-
grammed cell death [32]. Therefore, we performed immunoblot-
ting of these three targets to evaluate the effects of SCT200-M-2,
SCT200-C-2, and SCT200-C-4 on apoptosis in A431 cells (Fig. 5B).
There were no significant differences in the levels of phospho-p53
2
.2.2. Cytotoxicity of SCT200-linker-MMAE ADCs in cancer cells
The growth inhibitory activity of SCT200-linker-MMAE ADCs
þ
was evaluated in both EGFR cell lines (A431, FaDu, AsPc-1, HCC827,
and KYSE450) and EGFR cell lines (Daudi and Raji). Two types of
linkers for ADCs (M-1eM-3 and C-1eC-5 linker-MMAE conjugates)
showed significant cytotoxicity against EGFR tumor cells, with half
maximal inhibitory concentration (IC50) values in the nanomolar
range. In EGFR overexpressing cells (A431, KYSE450, and
HCC827 cells; Fig. 5A), the two types of ADC linkers inhibited cell
viability with IC50 values of approximately 0.2e2 nM. In contrast,
for cell lines with relatively low EGFR expression (AsPc-1 and FaDu
cells), the two types of ADC linkers inhibited cell viability with IC50
values of approximately 1e10 nM (Table 5). In contrast, no apparent
ꢁ
þ
or caspase-3 in A431 cells treated with 1 mg/mL SCT-200 (Fig. 5B,
P > 0.05). However, following treatment with SCT200-linker-
MMAE, p53 was upregulated, PARP was activated by caspase-3
cleavage, and procaspase-3 was proteolytically cleaved to produce
caspase-3 in a concentration-dependent manner (Fig. 5BeE,
P < 0.05e0.001). These findings indicated that SCT200-M-2,
ꢁ
activity was observed in EGFR cell lines (IC50 > 60 nM). These
results demonstrated the selective killing of EGFR tumor cells by
the novel antibody-drug conjugate. SCT-200 had a weak inhibitory
þ
6

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
Fig. 4. Internalization and binding ability of SCT200-linker-MMAE conjugates. A. Flow-based internalization assay of SCT200-linker-MMAE conjugates. B. Binding abilities of
SCT200-M-1eM-3 were assessed using ELISA. C. Binding abilities of SCT200-C-1eC-5 were assessed using ELISA. C. Binding abilities of SCT200-S-1eS-2 were assessed using ELISA.
Table 4
ADCs induced good antitumor responses, with 92.7%, 93.0%, 88.9%,
and 93.8% reductions in tumor volume for SCT200-M-1, SCT200-M-
Internalization percentage of SCT200-linker-MMAE conjugates.
2, SCT200-C-2, and SCT200-C-4, respectively, as compared with the
SCT200-linker-MMAE
Internalization percentage(%)
P
control group (P < 0.001). The ADCs were well tolerated without
any loss in body weight. Furthermore, there were no significant
differences between the novel ADCs (SCT200-M-2, SCT200-C-2,
and SCT200-C-4) and SCT200-M-1. Importantly, during the treat-
ment period and observation period after administration, no
weight loss was observed in all treatment groups, indicating that
the ADCs and naked antibody were all well-tolerated at the ther-
apeutic dose.
(
versus SCT-200)
SCT-200
25.3 ± 3.2
27.1 ± 5.4
27.8 ± 3.1
24.7 ± 4.2
35.4 ± 0.8
30.6 ± 3.2
33.3 ± 1.5
33.1 ± 7.2
31.4 ± 2.9
19.2 ± 3.2
19.6 ± 3.6
/
SCT200-M-1
SCT200-M-2
SCT200-M-3
SCT200-C-1
SCT200-C-2
SCT200-C-3
SCT200-C-4
SCT200-C-5
SCT200-S-1
SCT200-S-2
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
3. Conclusion
In conclusion, in this study, we developed and characterized a
stable controlled linker for effective release of MMAE in tumor cells.
The strategy with three types of novel linkers is based on modifi-
cation with three parts: a spacer, a catabolizing area, and a self-
immolative group. The resulted demonstrated that the MCC
spacer could be a suitable replaceable spacer in plasma and most
novel linker-MMAE undergoed rapid hydrolysis in cathepsin B.
Then, we generated three types of SCT-200-ADCs attached to
different linker-MMAE constructs. Novel SCT-200-ADCs retained
the receptor affinity and efficiency of internalization. Although the
SCT-200-S-1 and S-2 (modification of self-immolative group) were
not as well as expected, the other two types of which possessed
suitable chemical and biological characteristics as potential ADCs,
they also demonstrated their potency and antigen-selectivity
SCT200-C-2, and SCT200-C-4 could induce apoptosis in A431 cells
by increasing p53 expression, caspase-3 activation, and PARP
cleavage.
2.2.3. In vivo therapeutic efficacy
Four SCT200-linker-MMAE ADCs were evaluated in an A431
xenograft model. Mice (6 per group) received 4 mg/kg SCT200-M-1,
SCT200-M-2, SCT200-C-2, and SCT200-C-4 or 12 mg/kg SCT-200 by
intravenous injection every 4 days (total of four injections). As
shown in Fig. 6A, at 21 days post injection, the effects of SCT-200
(
tumor inhibition rate: 65.7%) on tumor growth were limited
compared with those of SCT200-linker-MMAE conjugates. The
7

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
Fig. 5. EGFR expression levels in different cancer cells and molecular mechanisms of SCT200-linker-MMAE-mediated apoptosis. A. Representative images of western blotting for
detection of EGFR in cancer cells. -Actin was used as a loading control. B. Representative images of western blotting for detection of phospho-p53, cleaved-caspase-3 (c-caspase-3),
and cleaved-PARP (c-PARP) in A431 cells exposed to the indicated concentrations of SCT200-ADCs (SCT200-M-2, -C-2, -C-4: 0.01, 0.1, and 1 g/mL, respectively), MMAE (1 nM), and
SCT200 (1 g/mL) for 24 h. GAPDH was used as a loading control. CeE. Quantitative analysis of phospho-p53, c-caspase-3, and c-PARP levels in A431 cells. Results are presented as
means ± standard errors of the means (n ¼ 4). *P < 0.05, **P < 0.01, ***P < 0.001 versus the control.
b
m
m
in vitro cytotoxicity. Especially, the M-2, C-2, and C-4 conjugated
with SCT200 were comparatively evaluated in vitro and in vivo. Our
findings showed that these ADCs showed potential anti-tumor
activity to EGFR-positive tumors, similar to the unmodified ADC
SCT200-MC-VC-PABC-MMAE (M-1). Thus, these SCT200-linker-
MMAE conjugates could have important applications and should be
characterized further. Moreover, the novel linkers designed in this
study may be used to conjugate with different antibodies for
treating other cancers.
4. Experimental section
4.1. General chemical procedures and reagents
4.1.1. Materials
Protected amino acids and condensation agents were purchased
from GL Biochem (Shanghai, China). Acetonitrile (high-perfor-
mance LC [HPLC] grade), TFA (HPLC grade), Et NH, THF, DCM, DMF,
2
and DIEA were purchased from J&K Scientific (Beijing, China).
Piperidine and diethyl ether anhydrous MeOH were purchased
from Beijing Chemical Works (Beijing, China). 4-Aminobezyl
8

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
Table 5
Cytotoxicity of the linker-MMAE-based SCT200-ADC in cancer cell lines.
ADCs
A431
KYSE450
IC50 (nM)
HCC827
AsPc-1
FaDu
Daudi
Raji
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
SCT200
>60
>60
>60
>60
>60
>60
15.82 ± 1.54
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
>60
SCT200-S-1
SCT200-S-2
SCT200-M-1
SCT200-M-2
SCT200-M-3
SCT200-C-1
SCT200-C-2
SCT200-C-3
SCT200-C-4
SCT200-C-5
15.05 ± 0.27
97.49 ± 1.37
0.17 ± 0.03
0.41 ± 0.05
0.36 ± 0.01
0.45 ± 0.01
0.71 ± 0.09
0.39 ± 0.07
0.62 ± 0.11
0.75 ± 0.03
28.26 ± 4.53
85.22 ± 9.22
0.64 ± 0.09
0.72 ± 0.09
0.33 ± 0.09
0.28 ± 0.03
0.63 ± 0.06
0.26 ± 0.11
0.81 ± 0.15
0.92 ± 0.13
0.21 ± 0.20
0.39 ± 0.06
0.49 ± 0.08
0.94 ± 0.11
0.60 ± 0.06
0.65 ± 0.01
0.42 ± 0.01
0.47 ± 0.05
2.54 ± 0.33
6.93 ± 0.53
6.21 ± 1.60
7.42 ± 0.54
3.07 ± 0.13
4.26 ± 1.00
5.13 ± 5.94
6.18 ± 0.47
4.80 ± 0.33
6.87 ± 1.13
7.27 ± 0.87
2.80 ± 0.13
2.13 ± 0.20
6.32 ± 0.20
3.59 ± 0.27
5.39 ± 1.06
The 50% inhibitory concentration (IC50) of the samples was calculated using Graphpad software. The data are presented as means ± standard deviations from three inde-
pendent experimental titrations.
Fig. 6. A431 xenograft studies of SCT200-linker-MMAE conjugates. A. Tumor growth curves for the A431 tumor xenograft model (n ¼ 6). Mice were treated with SCT200 (12 mg/kg),
SCT200-M-1 (4 mg/kg), SCT200-M-2 (4 mg/kg), SCT200-C-2 (4 mg/kg), or SCT200-C-4 (4 mg/kg) every 4 days (total of four injections). ***P < 0.001, compared with the control
group on day 21. B. Changes in the body weights of the mice during the observation period.
alcohol and Bis(4-nitrophenyl) carbonate were purchased from
Bidepharm Ltd. (Shanghai, China).
4.1.3. Antibody
The anti-EGFR antibody SCT-200 (~150 kDa) was kindly pro-
vided by Sinocelltech Ltd. (Beijing, China), the deconvoluted MS
spectra (ESI-MS) were consistent with the structure (Fig. S21).
4.1.2. Instruments
Semipreparative HPLC was performed on a Shimadzu Promi-
4.2. Synthetic procedures
nence HPLC LC-20AT system. HIC analysis was performed on a 1200
HPLC (Agilent Technologies) with the following parameters: TSKgel
4.2.1. Synthesis of linker
Butul-NPR column (4.6 ꢂ 35 mm, particle size 2.5
solvent A, 1.5 M ammonium sulfate and 25 mM phosphate (pH
.95); solvent B, 75% (v/v) 25 mM phosphate, 25% (v/v) isopropanol
m
m; TOSOH);
MC-VC-PABC-PNP (5a).
Compound 4a (0.69 g, 1.2 mmol) was dissolved in DMF (12 mL),
and (PNP) CO (1.1 g, 3.6 mmol) and DIEA (0.5 ml) were added. The
2
6
(
pH 6.95); gradient,100% A to 100% B over 15 min; flow rate, 0.5 mL/
reaction was stirred at room temperature for 16 h. After the
completion of the reaction, the solvent was removed under reduced
pressure to give a crude material, which was purified by column
ꢀ
min; column temperature, 25 C; ultraviolet detection wavelength,
2
2
quadrupole
(
solvent B over 5 min at a flow rate of 0.5 mL/min (solvent A: 0.1%
formic acid in water; solvent B: 0.1% formic acid in acetonitrile).
Mass spectra were acquired in the negative or positive ion mode
with a scan range of m/z 100e1500. High-resolution MS was per-
formed using an LTQ Orbitrap XLinstrument (Thermo Scientific,
USA). All final products were analyzed using a Bruker 500 MHz
spectrometer.
80 nm. LC-MS/MS analysis was conducted with a Shimadzu LC-MS
020 system using an electrospray ionization source and a single-
chromatography (CH
2
Cl
2
: CH
3
OH ¼ 20:1) to obtain compound 5a as
11, HRMS
[MþH] (ESI):found 738.3088, calcd 738.3093. H NMR (500 MHz,
DMSO‑d
mass
analyzer.
A
Shim-pack
VP-ODS
a light yellow solid (0.42g, 47% yield). C35H N O
43 7
þ
1
2.0 mm ꢂ 150 mm, 5
mm) was used with a gradient of 10e90%
6
)
d
(ppm): 10.09 (s, 1H), 8.32 (d, J ¼ 9.1 Hz, 2H), 8.14 (d,
J ¼ 7.1 Hz,1H), 7.83 (d, J ¼ 8.6 Hz,1H), 7.66 (d, J ¼ 8.4 Hz, 2H), 7.57 (d,
J ¼ 9.1 Hz, 2H), 7.41 (d, J ¼ 8.6 Hz, 2H), 7.01 (s, 2H), 6.00 (m, 1H), 5.44
(s, 2H), 5.24 (s, 2H), 4.39 (m,1H), 4.20 (m, 1H), 3.37 (m, 2H), 2.99 (m,
2H), 2.22e2.09 (m, 2H),1.96 (m,1H),1.72 (m,1H),1.65e1.56 (m,1H),
1.52e1.43 (m, 6H), 1.22e1.15 (m, 2H), 0.87e0.82 (m, 6H).
MCC-VC-PABC-PNP (5b).
9

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
Compound 5b was synthesized with the experimental protocols
2H), 1.65e1.58 (m, 2H), 1.51 (m, 1H), 1.31e1.21 (m, 2H), 0.93e0.85
(m, 2H).
described for compound 5a. A yellow solid powder was obtained in
þ
a yield of 66%. C37
calcd 764.3249. H NMR (400 MHz, DMSO‑d
H
45
N
7
O
11, HRMS (ESI): [MþH] found 764.3243,
MCC-GFLG-PABC-PNP (5h).
Compound 5h was synthesized with the experimental protocols
1
6
)
d
(ppm): 10.12 (s, 1H),
8
8
2
.38 (d, J ¼ 8.5 Hz, 1H), 8.33e8.31 (m, 2H), 8.29e8.25 (m, 1H),
described for compound 5a. A yellow solid powder was obtained in
þ
.13e8.08 (m,1H), 7.99 (d, J ¼ 8.3 Hz, 2H), 7.65 (s, 2H), 7.59e7.56 (m,
H), 7.47 (d, J ¼ 8.3 Hz, 2H), 7.42 (d, J ¼ 8.4 Hz, 2H), 7.22 (s, 2H), 6.88
a yield of 59%. C45
calcd 882.3668. H NMR (400 MHz, DMSO‑d
H
51
N
7
O
12, HRMS (ESI): [MþH] found 882.3698,
1
6
)
d
(ppm): 9.93 (s, 1H),
(
d, J ¼ 8.7 Hz, 1H), 6.00 (m, 2H), 5.43 (s, 2H), 5.25 (s, 2H), 4.44e4.36
8.32 (d, J ¼ 8.8 Hz, 2H), 8.19 (d, J ¼ 7.1 Hz, 2H), 8.00e7.92 (m, 2H),
7.67 (d, J ¼ 8.2 Hz, 2H), 7.57 (d, J ¼ 8.8 Hz, 2H), 7.43 (d, J ¼ 8.2 Hz,
2H), 7.25e7.17 (m, 5H), 7.01 (s, 2H), 5.26 (s, 2H), 4.54 (m, 1H), 4.30
(q, J ¼ 7.9, 7.5 Hz, 1H), 3.89 (d, J ¼ 6.0 Hz, 2H), 3.69 (m, 1H), 3.54 (m,
1H), 3.23 (d, J ¼ 7.0 Hz, 2H), 3.04 (m, 1H), 2.80 (m, 1H), 2.08 (m, 1H),
1.70 (m, 2H), 1.60 (m, 3H), 1.53 (m, 2H), 1.23 (m, 2H), 1.10 (t,
J ¼ 7.0 Hz, 1H), 0.91 (m, 4H), 0.86 (m, 4H).
(
m, 2H), 3.07e3.02 (m, 2H), 2.97 (m, 2H), 2.81 (s, 1H), 2.16 (m, 1H),
1
0
.72 (d, J ¼ 7.2 Hz, 1H), 1.64 (m, 1H), 1.51e1.44 (m, 2H), 1.41 (m, 2H),
.96 (m, 6H).
MC-Ph-VC-PABC-PNP (5c).
Compound 5c was synthesized with the experimental protocols
described for compound 5a. A yellow solid powder was obtained in
a yield of 59%. C36
calcd 744.2624. H NMR (400 MHz, DMSO‑d
þ
H
37
N
7
O
11, HRMS (ESI): [MþH] found 744.2620,
MC-VCGP-OH (15a).
1
6
)
d
(ppm): 10.09 (d,
The compound 15a was used solid-phase synthesis method
employing 2-chlorotrityl chloride resin (2-CTC resin). The first
amino acid Fmoc-Pro was anchored on the 2-CTC resin and then
linked the amino acid sequentially until the last amino acid Fmoc-
Val deprotected the Fmoc group to obtain 13a, followed by
condensation reaction using MC-OSu in DMF for 3h at room tem-
perature. The compound 15a was cleaved from the resin using a
J ¼ 10.6 Hz, 1H), 8.32 (d, J ¼ 8.6 Hz, 1H), 8.12e8.06 (m, 1H), 7.72 (d,
J ¼ 8.9 Hz, 1H), 7.66 (d, J ¼ 8.1 Hz, 2H), 7.58 (d, J ¼ 8.7 Hz, 2H), 7.42
(
d, J ¼ 8.1 Hz, 2H), 7.02 (s, 2H), 5.99 (d, J ¼ 6.1 Hz, 1H), 5.42 (s, 2H),
5
.25 (s, 1H), 4.39 (q, J ¼ 7.1 Hz, 1H), 4.17 (t, J ¼ 7.7 Hz, 1H), 3.25 (d,
J ¼ 7.0 Hz, 2H), 3.05e2.94 (m, 2H), 2.23 (m, 1H), 1.99 (m, 1H),
.72e1.60 (m, 5H), 1.28 (m, 2H), 0.84 (m, 6H).
1
MCC-VA-PABC-PNP (5d).
Compound 5d was synthesized with the experimental protocols
described for compound 5a. A yellow solid powder was obtained in
solution of TFA: H
2
O (95: 5). Yield was 73%, as a white solid.
þ
C
28
H
43
N
7
O
9
, HRMS (ESI): [MþH] found 622.3166, calcd 622.3195.
1
6
H NMR (400 MHz, DMSO‑d ) d(ppm): 12.41 (s, 1H), 8.01e7.91 (m,
þ
a yield of 65%. C34
calcd 678.2769. H NMR (500 MHz, DMSO‑d
H
39
N
5
O
10, HRMS (ESI): [MþH] found 678.2753,
2H), 7.78 (d, J ¼ 8.9 Hz, 1H), 7.00 (s, 2H), 6.00 (s, 1H), 4.30 (m, 1H),
4.24 (m, 1H), 4.17 (m, 1H), 3.80 (m, 1H), 3.50 (m, 2H), 3.37 (t,
J ¼ 7.1 Hz, 2H), 2.94 (t, J ¼ 6.8 Hz, 2H), 2.23e2.04 (m, 4H), 2.01e1.81
(m, 4H), 1.72e1.61 (m, 2H), 1.48 (m, 5H), 1.41e1.33 (m, 2H), 1.18 (m,
2H), 1.09 (m, 1H), 0.81 (m, 6H).
MC-VCAP-OH (15b).
Compound 15b was synthesized with the experimental pro-
tocols described for compound 15a. A white solid powder was
obtained in a yield of 67%.C29
1
6
)
d
(ppm): 10.04 (s,
1
1
(
H), 8.34e8.31 (m, 2H), 8.17 (d, J ¼ 6.8 Hz, 1H), 7.73 (d, J ¼ 8.6 Hz,
H), 7.66e7.63 (m, 2H), 7.59e7.56 (m, 2H), 7.43e7.40 (m, 2H), 7.02
s, 2H), 5.24 (s, 2H), 4.38 (t, J ¼ 7.0 Hz, 1H), 4.15 (m, 1H), 3.24 (d,
J ¼ 7.1 Hz, 2H), 2.23 (m, 1H), 1.97 (m, 1H), 1.75e1.67 (m, 2H), 1.62 (m,
2
H), 1.31 (m, 4H), 1.26e1.23 (m, 3H), 0.85 (m, 7H).
MCC-AVA-PABC-PNP (5e).
þ
Compound 5e was synthesized with the experimental protocols
described for compound 5a. A yellow solid powder was obtained in
45
H N
7
O
9
, HRMS (ESI): [MþH] found
1
6
636.3323, calcd 636.3352. H NMR (400 MHz, DMSO‑d ) d (ppm):
þ
a yield of 54%. C37
calcd 749.3141. H NMR (400 MHz, DMSO‑d
H
44
N
6
O
11, HRMS (ESI): [MþH] found 749.3128,
12.40 (s, 1H), 8.00 (d, J ¼ 7.2 Hz, 1H), 7.89 (d, J ¼ 7.8 Hz, 1H), 7.80 (d,
J ¼ 8.8 Hz, 1H), 7.01 (s, 2H), 6.16e5.93 (m, 1H), 4.48 (m, 1H),
4.31e4.11 (m, 3H), 3.62 (m, 1H), 3.50 (m, 1H), 3.37 (t, J ¼ 7.2 Hz, 2H),
2.94 (s, 2H), 2.13 (m, 3H), 1.89 (m, 4H), 1.61 (m, 1H), 1.54e1.30 (m,
7H), 1.27e1.06 (m, 7H), 0.81 (m, 6H).
1
6
)
d
(ppm): 10.03 (s, 1H),
8
7
1
(
1
1
.32 (d, J ¼ 8.8 Hz,1H), 8.21 (d, J ¼ 7.0 Hz,1H), 7.99 (d, J ¼ 7.4 Hz,1H),
.64 (d, J ¼ 8.2 Hz, 1H), 7.58 (m, 2H), 7.42 (d, J ¼ 8.1 Hz, 1H), 7.01 (s,
H), 5.25 (s, 1H), 4.41 (m, 2H), 4.28 (m, 1H), 4.23e4.16 (m, 1H), 3.24
d, J ¼ 7.2 Hz, 1H), 2.60 (s, 1H), 2.21e2.08 (m, 1H), 1.98 (m, 2H),
.77e1.56 (m, 4H), 1.48 (s, 1H), 1.37e1.22 (m, 8H), 1.19 (m, 3H),
.13e1.03 (m, 2H), 0.86 (m, 7H).
4.2.2. Synthesis of linker-MMAE
MC-VC-PABC-MMAE (M-1). To a solution of the 5a (93 mg,
0.125 mmol), MMAE (60 mg, 0.083 mmol), HOBt (10 mg,
MCC-GFG-PABC-PNP (5f).
Compound 5f was synthesized with the experimental protocols
0.074 mmol) in DMF (4 mL) was added DIEA (27 mL, 0.166 mmol).
described for compound 5a. A yellow solid powder was obtained in
The mixture was allowed to stand at rt overnight. and then poured
into water (15 mL). The mixture was extracted with ethyl acetate
(3 ꢂ 30 mL), the organic layers were combined, washed with brine,
þ
a yield of 51%. C39
calcd 769.2828. H NMR (600 MHz, DMSO‑d
H
40
N
6
O
11, HRMS (ESI): [MþH] found 769.2835,
1
6
)
d
(ppm): 9.84 (d,
J ¼ 8.7 Hz, 1H), 8.46 (m, 1H), 8.32 (d, J ¼ 8.6 Hz, 1H), 8.15 (d,
J ¼ 7.7 Hz, 1H), 7.94 (t, J ¼ 6.0 Hz, 1H), 7.67 (m, 2H), 7.57 (d,
J ¼ 8.6 Hz, 2H), 7.42 (d, J ¼ 8.5 Hz, 2H), 7.25 (d, J ¼ 5.0 Hz, 4H), 7.19 (s,
2 4
dried over anhydrous Na SO , and evaporated under reduced
pressure to give the crude product. The reaction mixture was
directly injected onto a semi-preparative chromatographic column
1
H), 7.00 (s, 1H), 5.25 (s, 1H), 4.48 (s, 1H), 3.92 (m, 1H), 3.88e3.83
(Shim-pack PREP-ODS, 20 ꢂ 250 mm ID, 10
mm), Solvent A: 0.08%
TFA/H2O; Solvent B: 0.08% TFA/acetonitrile. The gradient mode was
(
1
1
m, 1H), 3.73 (m, 1H), 3.56 (m, 1H), 3.22 (t, J ¼ 6.7 Hz, 2H), 3.06 (m,
H), 2.86e2.81 (m, 1H), 2.06 (m, 1H), 1.63 (m, 4H), 1.49 (s, 1H),
.26e1.18 (m, 3H), 1.09 (m, 1H), 0.87 (m, 3H).
20%e60% solvent B over 10 min, and 60% solvent B over 25 min.
Purification was performed by HPLC to give 80 mg (47%) of M-1 as a
þ
MCC-GGFG-PABC-PNP (5g).
Compound 5g was synthesized with the experimental protocols
white solid. C68
calcd 1316.7864. HPLC: t
(500 MHz, DMSO‑d
(ppm): 10.05 (s, 1H), 7.88 (d, J ¼ 7.6 Hz, 2H),
H
105
N
11
O
15, HRMS (ESI): [MþH] found 1316.7885,
1
R
¼ 5.838 min, purity 99.2%. H NMR
described for compound 5a. A yellow solid powder was obtained in
6
) d
þ
a yield of 59%. C41
calcd 826.3043. H NMR (500 MHz, DMSO‑d
H
43
N
7
O
12, HRMS (ESI): [MþH] found 826.3052,
7.73 (m, 2H), 7.56 (m, 2H), 7.41 (dd, J ¼ 7.6, 4.1 Hz, 2H), 7.37e7.21 (m,
7H), 7.16 (m, 2H), 5.16e4.95 (m, 4H), 4.77e4.69 (m, 1H), 4.69e4.59
(m, 1H), 4.55e4.49 (m, 2H), 4.46e4.38 (m, 2H), 4.37e4.17 (m, 5H),
4.09e3.94 (m, 3H), 3.93e3.89 (m, 1H), 3.80e3.75 (m, 1H),
3.34e3.29 (m,1H), 3.28e3.21 (m, 4H), 3.19 (m, 3H), 3.11 (s, 2H), 3.03
(m, 2H), 2.98e2.95 (m, 1H), 2.96e2.91 (m, 1H), 2.86 (m, 3H), 2.40
(m, 2H), 2.32e2.24 (m, 2H), 2.16e2.04 (m, 3H), 2.04e1.91 (m, 3H),
1
6
)
d
(ppm): 9.95 (s, 1H),
8
8
2
4
.43 (t, J ¼ 5.9 Hz, 1H), 8.34e8.31 (m, 2H), 8.22 (d, J ¼ 8.0 Hz, 1H),
.03 (m, 2H), 7.69e7.67 (m, 2H), 7.60e7.56 (m, 2H), 7.46e7.42 (m,
H), 7.27 (d, J ¼ 4.4 Hz, 4H), 7.19 (m, 1H), 7.01 (s, 2H), 5.26 (s, 2H),
.51 (m, 1H), 3.90 (m, 2H), 3.77 (m, 1H), 3.68e3.58 (m, 3H), 3.23 (d,
J ¼ 7.1 Hz, 2H), 3.08 (m,1H), 2.84 (m, 1H), 2.11 (m,1H),1.78e1.71 (m,
10

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
1
.86e1.65 (m, 5H), 1.63e1.43 (m, 5H), 1.41e1.27 (m, 3H), 1.09e0.94
(d, J ¼ 7.1 Hz, 2H), 1.08 (s, 1H), 1.04 (d, J ¼ 6.7 Hz, 2H), 1.01 (d,
J ¼ 6.6 Hz, 2H), 0.98 (d, J ¼ 6.6 Hz, 1H), 0.92e0.74 (m, 19H).
MCC-GFG-PABC-MMAE (C-3). Compound C-3 was synthesized
(
m, 6H), 0.94e0.69 (m, 21H).
MCC-VC-PABC-MMAE (M-2). Compound M-2 was synthesized
with the experimental protocols described for M-1. Yield was 41%,
with the experimental protocols described for M-1. Yield was 52%,
þ
þ
as a white solid. C70
342.8050, calcd 1342.8021. HPLC: t
NMR (600 MHz, DMSO‑d
(ppm): 9.94 (d, J ¼ 12.8 Hz, 1H),
.28e8.24 (m, 1H), 8.00 (d, J ¼ 7.5 Hz, 1H), 7.86 (d, J ¼ 8.7 Hz, 1H),
.67 (m,1H), 7.61e7.59 (m,1H), 7.54 (m, 2H), 7.33e7.26 (m, 3H), 7.23
m, 2H), 7.14 (m, 1H), 6.97 (d, J ¼ 4.4 Hz, 2H), 6.15e5.95 (m, 2H),
.08e4.93 (m, 3H), 4.74e4.68 (m, 1H), 4.63e4.57 (m, 1H), 4.46 (d,
H
107
N
11
O
15, HRMS (ESI): [MþH] found
as a white solid. C72
1347.7619, calcd 1347.7599. HPLC: t
NMR (600 MHz, DMSO‑d
(ppm): 9.79 (d, J ¼ 5.8 Hz, 1H), 8.45 (d,
H
102
N
10
O
15, HRMS (ESI): [MþH] found
1
1
1
R
¼ 5.964 min, purity 97.2%. H
R
¼ 8.957 min, purity 99.8%. H
6
)
d
6
) d
8
7
(
5
J ¼ 5.9 Hz, 1H), 8.31 (d, J ¼ 9.0 Hz, 1H), 8.13 (d, J ¼ 8.1 Hz, 1H), 8.05
(d, J ¼ 9.2 Hz, 1H), 7.93 (d, J ¼ 6.1 Hz, 1H), 7.89 (d, J ¼ 8.6 Hz, 1H),
7.64e7.57 (m, 2H), 7.31 (d, J ¼ 7.8 Hz, 3H), 7.28e7.22 (m, 5H), 7.17
(m, 2H), 7.00 (s, 1H), 5.40 (s, 1H), 5.05 (m, 3H), 4.74 (s, 1H), 4.63 (s,
1H), 4.48 (t, J ¼ 7.1 Hz, 2H), 4.42 (m, 1H), 4.27 (m, 1H), 3.99 (m, 2H),
3.87 (m, 2H), 3.72 (m, 2H), 3.56 (m, 2H), 3.51e3.34 (m, 5H), 3.31 (m,
1H), 3.24 (s, 2H), 3.22 (d, J ¼ 6.8 Hz, 3H), 3.20 (s, 1H), 3.17 (s, 1H),
3.12 (s, 1H), 3.06 (m, 2H), 2.97 (s, 1H), 2.90e2.79 (m, 4H), 2.41 (m,
1H), 2.26 (m, 1H), 2.15e2.03 (m, 3H), 1.98 (m, 2H), 1.83e1.71 (m,
3H), 1.71e1.65 (m, 2H), 1.61e1.55 (m, 2H), 1.54e1.47 (m, 2H), 1.30
(m, 1H), 1.27e1.17 (m, 2H), 1.09 (t, J ¼ 7.0 Hz, 1H), 1.04 (d, J ¼ 6.6 Hz,
1H), 0.99 (m, 4H), 0.88 (d, J ¼ 6.1 Hz, 2H), 0.81 (m, 16H).
J ¼ 5.9 Hz,1H), 4.40 (m, 1H), 4.35 (m, 2H), 4.23 (m, 2H), 4.12 (m, 2H),
3.95 (m, 4H), 3.76e3.74 (m, 1H), 3.59e3.49 (m, 2H), 3.4e3.41 (m,
1H), 3.28 (d, J ¼ 10.1 Hz, 1H), 3.21 (d, J ¼ 8.4 Hz, 4H), 3.16 (m, 3H),
3.09 (s, 2H), 3.04e2.90 (m, 4H), 2.88e2.79 (m, 3H), 2.38 (m, 1H),
2.22 (m, 2H), 2.09 (m, 2H), 1.95 (m, 2H), 1.77 (m, 2H), 1.72e1.62 (m,
4H), 1.61e1.55 (m, 2H), 1.54e1.45 (m, 3H), 1.41e1.39 (m, 1H),
1
.34e1.31 (m, 1H),1.29e1.20 (m, 3H),1.01 (d, J ¼ 6.6 Hz, 2H), 0.98 (d,
J ¼ 6.6 Hz, 2H), 0.95 (d, J ¼ 6.6 Hz, 1H), 0.90 (m, 2H), 0.88e0.66 (m,
1H).
MC-Ph-VC-PABC-MMAE (M-3). Compound M-3 was synthe-
2
MCC-GGFG-PABC-MMAE (C-4). Compound C-4 was synthesized
with the experimental protocols described for M-1. Yield was 43%,
þ
sized with the experimental protocols described for M-1. Yield was
as a white solid. C74
1404.7839, calcd 1404.7814. HPLC: t
NMR (600 MHz, DMSO‑d
(ppm): 9.82 (d, J ¼ 5.4 Hz, 1H), 8.35 (d,
H
105
N
11
O
16, HRMS (ESI): [MþH] found
þ
1
3
1
5%, as a white solid. C69
322.7418, calcd 1322.7395. HPLC: t
NMR (600 MHz, DMSO‑d
(ppm): 10.01 (d, J ¼ 11.6 Hz, 1H), 8.35
d, J ¼ 8.4 Hz, 1H), 8.23 (d, J ¼ 7.5 Hz, 1H), 7.95 (d, J ¼ 8.2 Hz, 2H),
H
99
N
11
O
15, HRMS (ESI): [MþH] found
R
¼ 8.618 min, purity 99.2%. H
1
R
¼ 5.853 min, purity 98.7%. H
6
) d
6
)
d
J ¼ 5.9 Hz, 1H), 8.14 (d, J ¼ 7.1 Hz, 1H), 7.97 (m, 2H), 7.86 (d,
J ¼ 8.6 Hz, 1H), 7.58 (m, 2H), 7.28 (d, J ¼ 7.6 Hz, 3H), 7.22 (d,
J ¼ 4.5 Hz, 5H), 7.14 (m, 2H), 6.97 (s, 2H), 5.03 (m, 3H), 4.72 (s, 1H),
4.61 (s, 1H), 4.52e4.35 (m, 4H), 4.24 (m, 2H), 4.03e3.89 (m, 3H),
3.84 (m, 2H), 3.77e3.70 (m, 2H), 3.65e3.51 (m, 4H), 3.44 (q,
J ¼ 7.8 Hz, 1H), 3.29 (m, 1H), 3.21 (d, J ¼ 6.3 Hz, 5H), 3.16 (m, 3H),
3.11e3.01 (m, 3H), 2.95 (s, 1H), 2.86 (d, J ¼ 8.8 Hz, 2H), 2.82 (m, 2H),
2.38 (m, 1H), 2.24 (m, 1H), 2.09 (m, 3H), 1.95 (m, 2H), 1.75 (m, 5H),
1.58 (m, 2H), 1.54e1.43 (m, 3H), 1.30e1.18 (m, 4H), 0.98 (m, 6H),
0.90e0.69 (m, 18H).
(
7
5
4
2
2
.56 (m, 2H), 7.43 (d, J ¼ 8.1 Hz, 2H), 7.33e7.22 (m, 5H), 7.19 (s, 1H),
.96 (s, 1H), 5.39 (s, 2H), 5.10e4.92 (m, 3H), 4.49e4.33 (m, 4H),
.27e4.18 (m, 2H), 3.96 (m, 3H), 3.55 (m, 3H), 3.18 (m, 7H), 3.09 (s,
H), 3.04e2.90 (m, 4H), 2.83 (m, 3H), 2.38 (m, 1H), 2.23 (m, 1H),
.10 (m, 3H), 1.95 (m, 2H), 1.83e1.65 (m, 5H), 1.59 (m, 2H), 1.55e1.41
(
(
m, 3H), 1.35 (m, 2H), 1.24 (m, 3H), 1.04e0.90 (m, 12H), 0.87e0.71
m, 16H).
MCC-VA-PABC-MMAE (C-1). Compound C-1 was synthesized
with the experimental protocols described for M-1. Yield was 40%,
MCC-GFLG-PABC-MMAE (C-5). Compound C-5 was synthesized
þ
as a white solid. C67
256.7564, calcd 1256.7541. HPLC: t
NMR (600 MHz, DMSO‑d
(ppm): 9.89 (d, J ¼ 12.3 Hz, 1H),
.26e8.24 (m, 1H), 8.08 (d, 6.9 Hz, 1H), 8.01e7.99
m,1H),7.88e7.86 (m, 1H), 7.67 (d, J ¼ 8.3 Hz, 1H), 7.61e7.59 (m, 1H),
.54 (d, J ¼ 7.4 Hz, 2H), 7.29 (m, 3H), 7.23 (m, 2H), 7.14 (q, J ¼ 7.8 Hz,
H), 6.97 (d, J ¼ 3.5 Hz, 2H), 5.00 (m, 3H), 4.71 (m, 1H), 4.48e4.44
m, 1H), 4.40 (d, J ¼ 6.7 Hz, 1H), 4.35 (m, 1H), 4.23 (m, 2H), 4.11 (t,
J ¼ 7.7 Hz, 1H), 3.96 (m, 4H), 3.76e3.74 (m, 1H), 3.59e3.49 (m, 6H),
.45e3.43 (m, 1H), 3.31e3.28 (m, 1H), 3.21 (d, J ¼ 8.4 Hz, 4H), 3.16
m, 3H), 3.09 (s, 1H), 3.01 (m, 1H), 2.94 (s, 1H), 2.85 (d, J ¼ 6.4 Hz,
H), 2.81 (d, J ¼ 6.2 Hz, 1H), 2.38 (m, 1H), 2.22 (m, 2H), 2.11e2.05
m, 2H), 1.93 (m, 2H), 1.82e1.73 (m, 2H), 1.67 (m, 3H), 1.61e1.55 (m,
H
101
N
9
O
14, HRMS (ESI): [MþH] found
with the experimental protocols described for M-1. Yield was 41%,
1
þ
1
R
¼ 9.658 min, purity 99.9%. H
as a white solid. C78
1460.84619, calcd 1460.84395. HPLC: t
H NMR (500 MHz, DMSO‑d ) d
6
H
113
N
11
O
16, HRMS (ESI): [MþH] found
6
)
d
R
¼ 9.837 min, purity 96.6%.
(ppm): 9.88 (d, J ¼ 4.2 Hz, 1H), 8.19
1
8
(
J
¼
(t, J ¼ 8.7 Hz, 3H), 8.02e7.87 (m, 3H), 7.60 (d, J ¼ 9.1 Hz, 3H),
7.38e7.13 (m, 12H), 5.04 (m, 2H), 4.69 (m, 1H), 4.51 (m, 3H),
4.34e4.23 (m, 2H), 3.99 (m, 3H), 3.87 (d, J ¼ 5.8 Hz, 6H), 3.82e3.62
(m, 2H), 3.53 (m, 3H), 3.31e3.17 (m, 9H), 3.12 (s, 2H), 3.08e2.93 (m,
3H), 2.83 (m, 4H), 2.41 (m, 1H), 2.26 (m, 1H), 2.16e1.89 (m, 5H),
1.85e1.65 (m, 4H), 1.63e1.44 (m, 8H), 1.34e1.18 (m, 4H), 1.01 (m,
6H), 0.90e0.76 (m, 22H).
7
1
(
3
(
1
(
MC-VCGP-MMAE (S-1). To a solution of the 15a (108.6 mg,
0.175 mmol), MMAE (104 mg, 0.145 mmol), HATU (66.4 mg,
2
H), 1.52e1.45 (m, 2H), 1.26 (d, J ¼ 7.4 Hz, 4H), 1.22 (m, 2H), 0.98 (m,
0.175 mmol) in DMF (4 mL) was added DIEA (52 mL, 0.290 mmol).
6
H), 0.86e0.72 (m, 21H).
The mixture was allowed to stand at rt overnight. The mixture was
allowed to stand at rt overnight. and then poured into water
(15 mL). The mixture was extracted with ethyl acetate (3 ꢂ 30 mL),
the organic layers were combined, washed with brine, dried over
anhydrous Na SO , and evaporated under reduced pressure to give
2 4
the crude product. Purification was performed by HPLC (HPLC
MCC-AVA-PABC-MMAE (C-2). Compound C-2 was synthesized
with the experimental protocols described for M-1. Yield was 43%,
as a white solid. C70
þ
H
106
N
10
O
15, HRMS (ESI): [MþH] found
1
327.79309, calcd 1327.79119. HPLC: t
R
¼ 8.952 min, purity 99.4%.
1
H NMR (600 MHz, DMSO‑d
6
)
d
(ppm): 9.94 (d, J ¼ 11.8 Hz, 1H), 8.29
(
(
7
t, J ¼ 9.5 Hz, 1H), 8.18 (d, J ¼ 6.9 Hz, 1H), 8.05 (t, J ¼ 7.7 Hz, 1H), 7.97
d, J ¼ 7.4 Hz, 1H), 7.89 (d, J ¼ 8.7 Hz, 1H), 7.62 (d, J ¼ 8.6 Hz, 1H),
method was same as M-1) to give 67 mg (35%) of S-1 as a white
þ
solid. C67
1321.8129. HPLC: t
DMSO‑d
H
108
N
12
O
15, HRMS (ESI): [MþH] found 1321.8131, calcd
1
.59e7.52 (m, 2H), 7.36e7.24 (m, 5H), 7.19e7.14 (m, 1H), 7.01 (s, 1H),
R
¼ 7.535 min, purity 97.0%. H NMR (600 MHz,
5
.14e4.92 (m, 3H), 4.74 (s, 1H), 4.63 (s, 1H), 4.50 (m, 1H), 4.46e4.35
m, 2H), 4.30e4.23 (m, 2H), 4.17 (t, J ¼ 7.5 Hz, 1H), 3.98 (m, 3H),
.91e3.62 (m, 9H), 3.57 (m, 2H), 3.49e3.45 (m, 1H), 3.38 (q,
J ¼ 7.1 Hz, 1H), 3.31 (m, 1H), 3.26e3.22 (m, 4H), 3.19 (m, 3H), 3.12 (s,
H), 3.04 (m, 1H), 2.97 (s, 1H), 2.88 (d, J ¼ 7.5 Hz, 1H), 2.84 (d,
J ¼ 7.2 Hz, 1H), 2.41 (m, 1H), 2.26 (m, 1H), 2.11 (m, 4H), 1.96 (m, 2H),
.84e1.67 (m, 5H), 1.62 (m, 2H), 1.58e1.45 (m, 3H), 1.27 (m, 5H), 1.18
6
) d
(ppm): 8.51 (d, J ¼ 9.9 Hz, 1H), 7.97e7.81 (m, 3H), 7.77
(
3
(t, J ¼ 5.9 Hz,1H), 7.62 (d, J ¼ 8.4 Hz, 1H), 7.28 (d, J ¼ 7.7 Hz, 2H), 7.24
(d, J ¼ 7.7 Hz, 2H), 7.15 (t, J ¼ 7.8 Hz, 1H), 6.98 (d, J ¼ 4.5 Hz, 1H), 5.86
(m, 1H), 5.44e5.29 (m, 3H), 4.92 (m, 1H), 4.76e4.68 (m, 1H),
4.66e4.55 (m, 1H), 4.49 (m, 2H), 4.40 (m, 1H), 4.26 (m, 1H), 4.15 (m,
1H), 4.09e3.89 (m, 4H), 3.72 (m, 2H), 3.52 (m, 3H), 3.42 (m, 1H),
3.35 (d, J ¼ 7.2 Hz, 3H), 3.29 (s, 1H), 3.25e3.14 (m, 6H), 3.10 (d,
1
1
11

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
J ¼ 3.6 Hz, 1H), 3.05e2.98 (m, 2H), 2.95 (t, J ¼ 8.9 Hz, 2H), 2.89 (m,
antibody was determined by the percent decrease in the mean
ꢀ
2
5
3
H), 2.79 (m, 1H), 2.42e2.35 (m, 1H), 2.28e2.21 (m, 1H), 2.12 (m,
H), 1.94 (m, 3H), 1.74 (m, 4H), 1.61 (m, 2H), 1.46 (m, 7H), 1.31 (m,
H), 1.15 (m, 2H), 1.06 (m, 2H), 0.98 (m, 5H), 0.78 (m, 21H).
MC-VCAP-MMAE (S-2). Compound S-2 was synthesized with
fluorescence intensity (MFI) of samples incubated at 37
C
ꢀ
compared with control samples incubated at 4 C. The formulas for
calculating the internalization percentage and the percentage of
ꢀ
cell surface-bound antibodies (% of MFI) after incubation at 37 C at
the experimental protocols described for S-1. Yield was 30%, as a
each time point (t
x
) compared with control samples incubated at
þ
ꢀ
white solid. C68
calcd 1335.8286. HPLC: t
600 MHz, DMSO‑d
H
110
N
12
O
15, HRMS (ESI): [MþH] found 1335.8256,
4 C were as follows: (a) % of MFI t
x
¼ MFI of sample incubated at
1
ꢀ
ꢀ
R
¼ 7.621 min, purity 98.9%. H NMR
37 C/MFI of control sample incubated at 4 C ꢂ 100; (b) internal-
(
6
)
d
(ppm): 8.53 (d, J ¼ 8.4 Hz, 1H), 7.99 (q,
ization percentage (% t
of MFI t
x
) of cell surface-bound antibodies ¼ 100 e %
J ¼ 6.7 Hz, 1H), 7.94e7.89 (m, 1H), 7.87 (d, J ¼ 7.0 Hz, 1H), 7.80 (d,
J ¼ 8.8 Hz,1H), 7.65 (d, J ¼ 8.4 Hz,1H), 7.31 (t, J ¼ 5.0 Hz, 2H), 7.27 (d,
J ¼ 7.5 Hz, 2H), 7.18 (t, J ¼ 7.6 Hz, 1H), 7.01 (s, 1H), 5.89 (d, J ¼ 6.7 Hz,
x
.
4.4.3. Evaluation of ADCs for cancer cell killing in vitro
Human cell lines (A431, KYSE450, HCC827, AsPc-1, FaDu, Daudi,
and Raji) were purchased from American Type Culture Collection
1H), 5.76 (s, 1H), 5.40 (m, 3H), 4.96 (m, 1H), 4.74 (m, 1H), 4.64 (m,
1H), 4.58e4.38 (m, 4H), 4.27e4.21 (m, 1H), 4.16 (m, 1H), 3.99 (m,
2H), 3.76 (m, 1H), 3.66 (m, 1H), 3.57 (m, 1H), 3.49 (m, 2H), 3.38 (d,
3
(Manassas, VA, USA). All cells (1e3 ꢂ 10 ) were cultured in an
ꢀ
J ¼ 6.5 Hz, 3H), 3.31 (m, 1H), 3.24 (m, 3H), 3.20 (m, 2H), 3.16 (m, 2H),
incubator and maintained at 37 C with 5% CO
2
. The cells were
3.12 (s, 1H), 3.07e3.01 (m, 2H), 2.97 (m, 1H), 2.92 (d, J ¼ 6.5 Hz, 2H),
2.76 (m, 1H), 2.45e2.38 (m, 1H), 2.30e2.23 (m, 1H), 2.22e2.06 (m,
5H), 1.99 (m, 3H), 1.91e1.68 (m, 5H), 1.63 (m, 2H), 1.49 (m, 7H), 1.32
added at various concentrations (10, 1, 0.1, 0.01, 0.001, and
0.0001 g/mL) with 100 L medium for 72 h, and 10 L of MTS
m
m
m
reagent was added to measure cell viability. The optical density was
read at 490 nm using a microplate reader (Thermo Fisher Scienti-
fic), and the cell viability ratio (%) was calculated using the
following formula: ([A sample e A blank]/[A control e A
blank]) ꢂ 100%. The IC50 of the samples was calculated using
GraphPad Prism software (GraphPad, Inc., La Jolla, CA, USA).
(
m, 3H), 1.17 (m, 4H), 1.09 (m, 2H), 1.06e0.97 (m, 5H), 0.81 (m, 21H).
4.3. In vitro stability and mediated drug release of Linker-MMAE
For analysis of stability and drug release, 0.004 mL of 10 mM
linker-MMAE stock of the substrate in DMSO was injected into
ꢀ
freshly collected rat plasma (1 mL, incubated at 37 C). Aliquots
4.4.4. Western blotting analysis
(
5
m
L) were evaluated at 0, 30, 60, 180, and 360 min (n ¼ 2 at each
Changes in protein expression levels in A431 cells were deter-
mined by Western blot analysis. Briefly, cells were treated with the
time point) using LC-MS/MS. The amount of released MMAE (nM)
was quantified using the standard curve method.
indicated drugs (SCT200-ADCs: 0.01, 0.1, and 1
mg/mL; SCT200:
The linker-MMAE stock solution (10 mM) was diluted into
1 mg/mL; MMAE: 1 nM) for 24 h, washed twice with ice-cold PBS,
0
(
~
.04 mM solution using 100 mM L-lys (pH ¼ 6). Next, cathepsin B
Sigma, St. Louis, MO, USA; cat. no. C8571, ꢃ1500 units/mg protein,
15.7 M) was diluted into 4
linker-drug (0.04 mM, 1000
and lysed in RIPA tissue/cell lysis buffer (New Cell & Molecular
Biotech Co., Ltd., Suzhou, China). The concentrations of protein
samples were quantified using a BCA protein assay kit (Thermo
Fisher Scientific). The samples were electrophoretically separated
using sodium dodecyl sulfate polyacrylamide gel electrophoresis
and transferred to polyvinylidene difluoride membranes. After
blocking, the membranes were probed with specific antibodies. The
following antibodies were used: anti-EGFR (Abcam, Cambridge,
MA, USA; dilution 1:1000), anti-cleaved PARP (Abcam; dilution
1:1000), anti-PARP (Cell Signaling Technology, Danvers, MA, USA;
dilution 1:1000), anti-phospho-p53 (Ser15; Cell Signaling Tech-
nology; dilution 1:500), anti-p53 (Abcam; dilution 1:1000), anti-
cleaved caspase-3 (Cell Signaling Technology; dilution 1:500),
anti-caspase-3 (Cell Signaling Technology; dilution 1:500), anti-
glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech,
m
m
M solution using PBS (pH ¼ 6). The
mL) was added to 10
m
L of the 4
mM
ꢀ
solution, and the reaction mixture was incubated at 37 C. Aliquots
(
5
mL) were evaluated at 0, 10, 60, 120, 240, and 360 min (n ¼ 2 at
each time point) using LC-MS/MS. Concentrations of released
MMAE (nM) were quantified using the standard curve method.
Samples were analyzed using LC-MS/MS with the following
parameters: instrument, Agilent 1100 and Thermo TSQ Ultra; col-
umn, Venusil ASB C18 3
mm, 150 Å, 4.6 ꢂ 50 mm; flow rate, 0.8 mL/
min; linear gradient, water as mobile phase A and acetonitrile as
mobile phase B, both in 0.1% formic acid. The gradient elution used
was as follows: 0e0.3 min, 5% B; 0.3e0.6 min, 5e95% B;
0
.6e2.5 min, 95% B; 2.5e3.5 min, 5% B. MMAE was detected in
positive ion mode (m/z 718.5; daughter ions used for quantitation:
m/z 686.7).
Rosemont, IL, USA; dilution 1:5000), or anti-b-actin (Proteintech;
dilution 1:5000). Subsequently, membranes were washed three
times in TBST, followed by incubation with horseradish peroxidase-
conjugated goat anti-rabbit or anti-mouse IgG secondary antibody
4
4
.4. Biological evaluation
(EarthOx Life Sciences, Millbrae, CA, USA; dilution 1:10,000) for
.4.1. Binding affinity
1 h at room temperature. Membranes were washed three times in
TBST, and proteins were visualized by chemiluminescent detection
using an ECL detection kit (Thermo Fisher Scientific). Images and
the densities of protein bands were acquired on a Fusion-FX6 im-
High-binding 96-well plates were coated with 1 mg/mL recom-
ꢀ
binant EGFR protein (ACRO Biosystems, Beijing) at 4 C overnight.
ꢀ
After blocking for 1 h at 37 C with 1% bovine serum albumin in PBS,
ꢀ
various concentrations of ADCs were incubated at 37 C for 1 h. The
aging system (Vilber Lourmat). GAPDH or
ternal control.
b-actin served as an in-
wells were washed three times with PBS containing Tween 20 and
were incubated with goat anti-human IgG-alkaline phosphatase
ꢀ
antibodies at 37 C for 2 h. TMB was then added to the wells for
4.4.5. In vivo antitumor activity in A431 xenograft tumors
color development, and the reaction was stopped using 2 M H
The optical density was read at 450 nm using a microplate reader
Thermo Fisher Scientific, Carlsbad, CA, USA).
2
SO
4
.
BALB/c nude mice (6e8 weeks old, 18e20 g, female) were pur-
chased from Beijing SPF Biotechnology Co., Ltd (Beijing, China). The
mice were acclimated for 1 week prior to use in experiments, and
all experiments were conducted according to protocols approved
(
4
.4.2. Flow-based internalization assay
The specific method for analysis of internalization was reported
by the Animal Care and Use Committee of the People’s Republic of
6
China. In the A431 model, 5 ꢂ 10 A431 tumor cells/100
mL were
previously [33]. The degree of internalization of cell surface-bound
implanted subcutaneously into the right armpit of BALB/c nude
12

X. Hu, H. Jiang, W. Bai et al.
European Journal of Medicinal Chemistry 216 (2021) 113297
mice. When tumors reached a volume of approximately 110 mm3,
mice were randomly divided into treatment and vehicle control
groups (n ¼ 6 mice/group). The mice were treated with SCT200
J.A. Francisco, Effects of drug loading on the antitumor activity of a mono-
clonal antibody drug conjugate, Clin. Canc. Res. 10 (2004) 7063e7070.
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[
(
12 mg/kg), SCT200-M-1 (4 mg/kg), SCT200-M-2 (4 mg/kg),
SCT200-C-2 (4 mg/kg), and SCT200-C-4 (4 mg/kg) every 4 days
total of four injections). Tumor volume and body weight were
855e869.
(
[
[
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measured 2e3 times per week during the treatment period. Tumor
2
volumes were calculated according to the formula: TV ¼ a ꢂ b /2,
where “a” and “b” are long and short diameters of a tumor,
respectively. The statistical analysis and graphic presentations were
performed with GraphPad Prism 5 software. The statistical signif-
icance of the differences between the two groups was determined
using the Student’s t-test, and multiple groups were compared
using a one-way analysis of variance (ANOVA) test. P values of less
than 0.05 were accepted as a significant difference. (*P < 0.05;
14] M. Dorywalska, R. Dushin, L. Moine, S.E. Farias, D. Zhou, T. Navaratnam, V. Lui,
A. Hasa-Moreno, M.G. Casas, T.T. Tran, K. Delaria, S.H. Liu, D. Foletti,
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*
*P < 0.01, ***P < 0.001). At the end of the experiment, the mice
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Declaration of competing interest
[
18] X.Y. Hu, R. Wang, J. Jin, X.J. Liu, A.L. Cui, L.Q. Sun, Y.P. Li, Y. Li, Y.C. Wang,
Y.S. Zhen, Q.F. Miao, Z.R. Li, An EGFR-targeting antibody-drug conjugate
LR004-VC-MMAE: potential in esophageal squamous cell carcinoma and other
malignancies, Mol Oncol (13) (2019) 246e263.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[
[
19] S. Manabe, H. Machida, Y. Aihara, M. Yasunaga, Y. Ito, Y. Matsumura, Devel-
opment of a diketopiperazine-forming dipeptidyl Gly-Pro spacer for prepa-
ration of an antibodyedrug conjugate, MedChemComm (4) (2013) 792e796.
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Rosario, S.F. Yu, H. Erickson, R. Vandlen, FRET reagent reveals the intracellular
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Acknowledgments
The work was supported by CAMS Innovation Fund for Medical
Sciences (CIFMS, grant no. 2016-I2M-3e013) and Science Founda-
tion for Young Scientists of PUMC (grant no. 2017350021). The
authors thank Dr. Liang-Zhi Xie, who provided the SCT-200 anti-
body (Sinocelltech Ltd., Beijing, China).
(29) (2018) 2468e2477.
[21] T. Nakada, T. Masuda, H. Naito, M. Yoshida, S. Ashida, K. Morita, H. Miyazaki,
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22] Y.J. Zhong, L.H. Shao, Y. Li, Cathepsin B-cleavable doxorubicin prodrugs for
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23] Y. Anami, C.M. Yamazaki, W. xiong, X. Gui, N. Zhang, Z. An, K. Tsuchikama,
Glutamic acid-valine-citrulline linkers ensure stability and efficacy of
antibody-drug conjugates in mice, Nat. Commun. (1) (2018) 2512.
Appendix A. Supplementary data
[
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113297.
[24] A. Dal Corso, S. Cazzamalli, R. G eꢀ bleux, M. Mattarella, D. Neri, Protease-
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