Novel silyl ether-based acid-cleavable antibody-MMAE conjugates with appropriate stability and efficacy

Article abstract of DOI:10.3390/cancers11070957

Antibody-drug conjugate (ADC) is a novel efficient drug delivery system that has been successfully used in clinical practice, and it has become a research hotspot in the anti-tumor drug field. Acid-cleavable linkers were first used in clinical ADCs, but their structural variety (e.g., hydrazone and carbonate) is still limited, and their stability is usually insufficient. Designing novel acid-cleavable linkers for the conjugation of the popular cytotoxin monomethyl auristatin E (MMAE) has always been a significant topic. In this paper, we generate a novel, silyl ether-based acid-cleavable antibody-MMAE conjugate, which skillfully achieves efficient combination of amino-conjugated MMAE with the acid-triggered silyl ether group by introducing p-hydroxybenzyl alcohol (PHB). The stability, acid-dependence cleavage, effective mechanism, efficacy and safety of the resulting ADC were systematically studied; the results show that it exhibits a significant improvement in stability, while maintaining appropriate efficacy and controlled therapeutic toxicity. This strategy is expected to expand a new type of acid-cleavable linkers for the development of ADCs with highly potent payloads.

Full text of DOI:10.3390/cancers11070957

cancers
Article
Novel Silyl Ether-Based Acid-Cleavable
Antibody-MMAE Conjugates with Appropriate
Stability and Efficacy
Yanming Wang † , Shiyong Fan †, Dian Xiao, Fei Xie, Wei Li, Wu Zhong * and Xinbo Zhou *
National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and
Toxicology, Beijing 100850, China
* Correspondence: zhongwu@bmi.ac.cn (W.Z.); zhouxinbo@bmi.ac.cn (X.Z.); Tel.: +86-010-6693-0674 (X.Z.)
† Contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 20 May 2019; Accepted: 4 July 2019; Published: 8 July 2019
Abstract: Antibody-drug conjugate (ADC) is a novel efficient drug delivery system that has been
successfully used in clinical practice, and it has become a research hotspot in the anti-tumor drug
field. Acid-cleavable linkers were first used in clinical ADCs, but their structural variety (e.g.,
hydrazone and carbonate) is still limited, and their stability is usually insufficient. Designing novel
acid-cleavable linkers for the conjugation of the popular cytotoxin monomethyl auristatin E (MMAE)
has always been a significant topic. In this paper, we generate a novel, silyl ether-based acid-cleavable
antibody-MMAE conjugate, which skillfully achieves efficient combination of amino-conjugated
MMAE with the acid-triggered silyl ether group by introducing p-hydroxybenzyl alcohol (PHB).
The stability, acid-dependence cleavage, effective mechanism, efficacy and safety of the resulting
ADC were systematically studied; the results show that it exhibits a significant improvement in
stability, while maintaining appropriate efficacy and controlled therapeutic toxicity. This strategy is
expected to expand a new type of acid-cleavable linkers for the development of ADCs with highly
potent payloads.
Keywords: antibody-drug conjugate; linker; acid-cleavable; silyl ether; monomethyl auristatin E
1. Introduction
Antibody-drug conjugate (ADC), comprised of an antibody, a cytotoxin and an appropriate
linker [
1
,
2
], achieves efficient delivery of a cytotoxin by a monoclonal antibody, greatly enhancing the
]. Recently, ADCs
], with more than 80 ADCs in
therapeutic index of cytotoxic drug and potentially reducing systemic toxicity [
have shown great promise in the treatment of various types of cancer [6,7
3–5
different stages of clinical development [8].
There are many important factors involved in the successful development of ADC, such as the
selection of suitable targets and antibodies, efficient cytotoxins, and appropriate linkers [ 11]. Among
9–
them, the linker is one of the core factors for the success of the ADC design, which needs to maintain
the stability of the ADC in systemic circulation and release the cytotoxin after internalization at the
target site [12]. Based on the release mechanism, linkers are generally divided between acid-cleavable
linkers, reduction-cleavable linkers, enzyme-cleavable linkers, and non-cleavable linkers [13,14].
Acid-cleavable linker was the first to be used in early ADC constructs, which cleverly utilized the pH
difference between tumor tissue (4.0–5.0) and plasma (~7.4) [14,15]. The hydrazone-based acid-cleavable
linkers (Figure 1A,B) are not only the first ones that were successfully used in ADCs, but they were also
used in two of the four currently approved ADCs [16]. Additionally, a carbonate-based acid-cleavable
linker has been tried to conjugate cytotoxin SN-38 (Figure 1C) [17]; for example, a phase III trial for
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the resulting sacituzumab govitecan has been initiated, and it has earned the Breakthrough Therapy
designation from the U.S. Food and Drug Administration (FDA) [18]. However, the lack of stability
of current acid-cleavable linkers could potentially lead to toxicity during clinical applications [14,19].
The first approved ADC, Mylotarg® (Pfizer Inc., Brooklyn, NY, USA), was voluntarily withdrawn
in 2010 mainly because of the instability of the hydrazone linker, and it still has dose-limiting
hepatotoxicity, although it was subsequently re-marketed [20,21]. It is also generally believed that
existing acid-cleavable linkers are not suitable for conjugating highly cytotoxic drugs, just as IMMU-132
improved efficacy by improving drug/antibody ratio (DAR) to 7.6 due to the insufficient effectiveness of
SN-38 (IC50 = 10−7–10−8 mol/L). However, high DAR values might lead to faster clearance or potential
immunogenic reactions in some cases [22,23]. Overall, the structural category of acid-cleavable linkers
is still limited, and insufficient stability greatly restricts its wide application in constructing ADCs.
Figure 1. Structures of the chemically labile antibody-drug conjugates (ADCs) with acid-cleavable
linkers, and the drug release mechanism of ADCs with monomethyl auristatin E (MMAE) as the payload.
(
A
) The doxorubicin-based ADC with the pH-sensitive hydrazone linker; (
ADC with the pH-sensitive hydrazone linker; ( ) the SN-38-based ADC with the pH-sensitive carbonate
linker; ( ) the designed MMAE-based ADC with the pH-sensitive silyl ether linker, and its drug
release mechanism.
B) the calicheamicin-based
C
D
For the most popular, highly cytotoxic, monomethyl auristatin E (MMAE), which accounts for
nearly one-third of the clinical ADCs [18], it has always been designed to employ enzyme-cleavable
linkers, such as the commonly used Val-Cit (VC)-based cathepsin B-cleavable linker [24
Nevertheless, due to susceptibility to an extracellular carboxylesterase [29], a number of ADCs
failed during development due to tolerability reasons [30 31]. Overall, the current linker design for
–28].
,
ADCs is not yet perfect, and the design of acid-cleavable linkers for the conjugation of MMAE has
always been a topic worth exploring.

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In this paper, a novel, silyl ether-based acid-cleavable ADC was generated with MMAE as the potent
payload. Methylsilyl ether groups have characteristics that are sensitive to acidity, and have been used
as protective bases for organic synthesis and drug release groups for nanomaterials [32
these groups have also been used in the design of prodrugs, mainly limited to hydroxyl-conjugated drugs
(e.g., acetaminophen, and docetaxel) due to the limitation of the silyl ether structure [35 36]. Although
–34]. In addition,
,
this type of linker has been tried for model ADC construction, which exhibits the characteristics of
stability and acid-dependent cleavage, gemcitabine does not meet the high-efficiency requirements for
ADC’s payload and it has not been evaluated for its efficacy [37]. By introducing p-hydroxybenzyl
alcohol (PHB), the ADC skillfully achieves the efficient combination of amino-conjugated MMAE with
acid-triggered silyl ether groups (Figure 1D). The resulting ADC exhibits improved stability, efficient
release of MMAE just like enzyme-cleavable ADCs, the appropriate efficacy, and controlled toxicity.
This linker system is expected to deepen the cognition of acid-cleavable linkers and provide a novel
conjugating model for MMAE. As an addition to the existing acid-cleavable linkers, such as hydrazone
and carbonate, this strategy may provide an alternative opition for the development of ADCs.
2. Results
2.1. Design and Synthesis of Silyl Ether-Based ADC Payload
In the paper, we have attempted to introduce a novel, acid-cleavable mechanism for the design of
ADC with MMAE as the potent payload. We designed the target ADCs using silyl ether groups that
have good acid-cleavable properties. Additionally, inserting PHB between MMAE and the silyl ether
group achieved an efficient combination of amino-containing cytotoxin. Various substituted silyl ethers
were designed as new linkers, and evaluated for their stability and acid-cleavable properties. However,
these silyl ethers-based linkers were found to have different stabilities during the synthesis process. The
dimethyl, diethyl and diphenyl substitution modes were not feasible due to their inherent instability
(Figure S1). In addition, larger alkyl substituents will undoubtedly increase the hydrophobicity of the
linker, which is detrimental to the properties of the ADC. Thus, a diisopropyl-silyl ether-based linker
was prepared (Figure 2), and further used for the conjugation of MMAE to an anti-human epithelial
growth factor receptor 2 (HER2) antibody to develop a novel acid-cleavable ADC.
Figure 2. General synthetic route for the linker-MMAE conjugate (compound 6). Reagents and
conditions: (
temperature (rt), 1 h, 57%; (
2 h, 76%; ( ) NaBH4, THF,
72%; (f) MMAE, HOBt, DIPEA, DMF, rt, overnight, 62%.
a b
) NMM, EA, 0 ◦C, 2 h, 99%; ( ) 2-(2-aminoethoxy)ethan-1-ol, NaHCO3, H2O, 0 ◦C ~ room
c
−
) dichlorodiisopropylsilane, 4-hydroxybenzaldehyde, TEA, DCM, 0 ◦C ~ rt,
5 ◦C–0 ◦C, 3 h, 23%; (
) Bis(4-Nitrophenyl) Carbonate, DIPEA, DMF, rt, 5 h,
d
e

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2.2. Antibody Conjugation
The reaction of maleimide compound with antibody thiols was evaluated using a humanized
anti-HER2 antibody mil40, which is a biosimilar of trastuzumab. The cytotoxin payload was typically
conjugated to the antibody through cysteine, obtained from the reduction of inter-molecule chain
disulfide bonds. By controlling the equivalent of the reducing agent tris(2-carboxyethyl)phosphine
hydrochloride (TCEP), a defined number of antibody payload modifications can be achieved, which
were in a relatively fixed position. Hydrophobic interaction chromatography (HIC) analysis of the
final ADC product allowed for resolution of the conjugates into several major peaks with an average
DAR of about 5.5 (Figure S14) [38].
2.3. The Stability Assays of the Conjugate in Plasma
The silyl ether-based linker-MMAE conjugate and the prepared ADC (mil40-6) was initially
evaluated for their in vitro stability in human plasma. In this experiment, the ADC of DAR ~5.5
was selected, and the silyl ether-based linker-MMAE conjugate was added to a solution of excess
N-acetyl-L-cysteine (NAC) to complete conversion to NAC-linker-MMAE (NAC-6) before test [38].
The above mixtures were incubated in 50% plasma at 37 ◦C for a period of 7 days. Aliquots were
taken at the indicated time points and analyzed by LC-MS/MS for the release of free cytotoxin MMAE.
As shown in Figure 3, less than 3% of the total MMAE in the NAC-linker-MMAE conjugate was
released during 7 days in human plasma. Correspondingly, about 24% of the total MMAE in ADC was
off-targeted. Overall, this type of acid-cleavable linker system displayed acceptable in vitro plasma
stability for the initial study.
100%
ADC
NAC-6
80%
60%
40%
20%
0%
0
1
2
3
4
5
6
7
Time (day)
Figure 3. Stability assays of silyl ether-based ADC (mil40-6) and its payload conjugate (NAC-6) in
human plasma. ADC and NAC-6 were incubated at 37 ◦C for 7 days. Error bars represent standard
deviation from three independent experiments (triplicates). Results are shown as the mean
N-acetyl-L-cysteine.
±
SD. NAC:
2.4. Effect of Acidity on ADC Stability
Further, the pH-dependence of the cytotoxin release process of the silyl ether-based acid-cleavable
ADC (mil40-6) was evaluated. Under three selected pH values (pH 7.4, 5.5, and 4.5), this novel ADC
(DAR ~ 5.5) was incubated for 7 days at 37 ◦C. As showed in Figure 4, the release rate of MMAE exhibited
significant pH-dependence, with approximately one-third of the total MMAE in ADC being released
after 7 days under neutral conditions (pH = 7.4), whereas the loaded MMAE rarely been detected
under acidic test conditions (pH = 4.5). This result demonstrated that the diisopropyl-substituted silyl
ether has a certain acid-dependent acid-cleavable property in this model [37].

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6
5
4
3
2
1
0
pH=7.4
pH=5.5
pH=4.5
0
24
48
72
96 120 144 168
Time (h)
Figure 4. pH-dependent drug release of acid-sensitive ADC (mil40-6). Typical time course of MMAE
cleaved from ADC in PBS that pH 7.4, pH 5.5, and pH 4.5. Error bars represent standard deviation from
three independent experiments (triplicates). Data are shown as mean
±
SD. DAR: drug/antibody ratio.
2.5. In Vitro Potency Assay
The prepared ADC with an average DAR of 5.5 was evaluated for its cytotoxicity against
HER2-positive cell lines (NCI-N87, BT-474, and MDA-MB-453) [39], and the HER2-negative cell lines
(MCF-7 and MDA-MB-231) [40], along with the naked antibody (mil40) and cytotoxin (MMAE) as
controls [41]. The ADC displayed excellent anti-tumor activity against all HER2-positive cell lines
(IC50, 0.028–0.170 nM), whereas exhibited low activity against HER2-negative cell lines (IC50, 7.742 ~
>1000 nM). The ADC exhibited a similar maximum inhibition rate to naked antibody in BT-474 cells,
mainly because the antibody itself was very sensitive to BT-474 cells just as we reported previously [38].
As a comparison, MMAE showed potent anti-tumor activity in both HER2-positive and -negative cell
lines with almost no antigenic selectivity (Table 1).
Table 1. Cytotoxicity of ADC, antibody and cytotoxin in cancer cell lines in vitro.
ADC
mil40
MMAE
HER2
Status
Cell Lines
Max.
Max.
Max.
IC50 (nM)
IC50 (nM)
IC50 (nM)
Inhibition
Inhibition
Inhibition
BT-474
NCI-N87
MDA-MB-453
MCF-7
HER2+++
HER2+++
HER2++
HER2−
0.170
0.028
0.101
7.742
>1000
50.50%
93.54%
87.30%
47.8%
-
0.323
1.157
0.315
>1000
>1000
61.25%
45.80%
34.80%
0.400
0.369
0.295
0.688
1.156
40.65%
68.22%
83.79%
77.64%
53.4%
-
-
MDA-MB-231
HER2−
Note: the concentration of the ADC was calculated as the concentration of the antibody.
2.6. Trafficking Assay by Fluorescence Microscopy
To release cytotoxin inside cancer cells, ADC needs to be internalized and exposured in the
lysosomal compartments. Thus, the internalization and trafficking of the ADC mil40-6 was detected
via fluorescently labeled antibody in HER2+ BT-474 cell line with a laser scanning confocal microscope.
Lysosomal compartments were visualized by staining with lysosomal-associated membrane protein 1
(LAMP-1). As shown in Figure 5, the mil40 and mil40-6 were incubated with the lysosomal labeled
BT-474 cells at 4 ◦C, under which the antibody was not been internalized (no obvious red). However,
after incubation at 37 ◦C for 16 h, the intracellular antibody signals colocalized with the signals of
LAMP-1 (green), indicating that the mil40-6 (red) can be efficiently internalized in cancer cells through
endocytosis (Pearson’s correlation = 0.467; Mander’s overlap = 0.567), which is very similar to the
naked antibody mil40 (Pearson’s correlation = 0.538; Mander’s overlap = 0.525).

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Figure 5. Receptor-mediated internalization of ADC mil40-6 by HER2+ breast cancer cell line BT-474.
Red fluorescence-labeled mil40-6 (1
g/mL) was incubated with GFP-labeled BT-474 cells at 37 ◦C for
µ
16 h while mil40-6 was endocytosed into the cell and located in lysosomes. The cell nuclei were stained
with DAPI (blue), the lysosomes were labeled with a lysosomes-GFP (green), and the antobody were
labeled with pHrodo Red (red). GFP: green fluorescent protein; DAPI: 40,6-diamidino-2-phenylindole.
Scale bar = 50 µm.
2.7. Linker Composition on Inhibition of Microtubule Polymerization
As an auxiliary verification and interpretation of the in vitro cytotoxicity, an in vitro microtubule
polymerization assay was performed to confirm that the incorporated structural variations and
elaborations to MMAE did not impair the drug’s ability to inhibit microtubule polymerization
(Figure 6). The MMAE and corresponding Cys-linker-MMAE conjugate (Cys-6) were used at 3 µM
and 0.3 µM.
120,000
vehicle(1% DMSO)
paclitaxel: 3 μM
CaCl2: 5 mM
MMAE: 3 μM
MMAE: 0.3 μM
Cys-6: 3 μM
100,000
80,000
60,000
40,000
20,000
Cys-6: 0.3 μM
0
10
20
30
40
50
60
Time (min)
Figure 6. The inhibition tests of microtubule polymerization. The cytotoxin MMAE and the
Cys-linker-MMAE (Cys-6) were tested in a microtubule polymerization assay. Free MMAE was
included as a positive control. Paclitaxel and CaCl2 were included as a promoter and inhibitor,
respectively, of microtubule polymerization. The buffer control indicates the polymerization rate of
untreated microtubules.
The conjugate and free MMAE showed similar inhibition rates of tubulin polymerization at
various concentrations. As expected, the panel of drug/linkers resulted in microtubule polymerization
inhibition similar to unmodified cytotoxin MMAE. This result may provide some basis for the high
in vitro cytotoxicity of this novel ADC.
2.8. Cell Cycle Effects
The anti-tumor mechanisms of the acid-cleavable ADC was further evaluated with respect to
cell cycle arrest. MMAE is a synthetic analogue of the natural product dolastatin 10, which can bind

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to tubulin and block cell division by inhibiting microtubule assembly [28
,
42,
43]. Normally, ADCs
consisting of MMAE induce G2/M phase arrest in target cells preceding the onset of apoptosis [44,45].
As showed in Figure 7, the in vitro effects of mil40-6 (10 ng/mL, 50 ng/mL, and 100 ng/mL) on BT-474
cell cycle arrest after 6 days’ treatment were presented. Compared to the vehicle, the ADC showed no
cell cycle inhibition at the low dose group (10 ng/mL). While, few cells treated with mil40-6 remained
in the G0/G1 phase when the dose is increased, with most were either in the S or G2/M phases. At the
dose of 100 ng/mL, the proportion of G2/M phase in ADC-treated cells was 34.04% higher than vehicle,
indicating that this ADC can suppress the cell cycle to the G2/M phase.
10 ng/mL
50 ng/mL
100 ng/mL
80
60
40
20
0
80
60
40
20
0
80
60
40
20
0
Vehicle
ADC
Vehicle
ADC
Vehicle
ADC
Figure 7. The effects of the ADC (mil40-6) on cell cycle arrest. The BT-474 cells cycle arrest results
analyzed by flow cytometry (FCM). ADC was tested in three doses of 10 ng/mL, 50 ng/mL, and 100
ng/mL. The concentration of the ADC is calculated as the concentration of the antibody.
2.9. In Vivo Potency in Xenografted Nude Mice
To evaluate the efficacy of the acid-cleavable ADC (mil40-6) in vivo, we established NCI-N87
xenografts in nude mice and then treated these BALB/c mice with the naked antibody (mil40), ADC,
and vehicle [46]. As shown in Figure 8A, the animals were given vehicle, mil40, and ADC on days
0, 7, 14, and 21, and 4 intravenous injections of ADC at doses of 2.5 and 5 mg/kg. Both doses of the
ADC treatment group produced significant and sustained anti-tumor effects and showed significant
dose-efficacy dependence. As a comparison, 4 intravenous injections of mil40 at doses of 5 mg/kg
resulted in almost no tumor suppression. Compared with mil40, the ADC had a significant increase in
potency with the same dose (p = 0.0054), with a 93.6% tumor inhibition rate.
1200
A
vehicle
1000
mil40: 5mpk
ADC: 2.5mpk
800
ADC: 5mpk
600
400
200
0
0
10
20
30
t (day)
28
26
24
22
20
vehicle
B
mil40: 5mpk
ADC: 2.5mpk
ADC: 5mpk
0
10
20
30
t (day)
Figure 8. Xenograft studies of the ADC (mil40-6). Nude mice were implanted subcutaneously with
NCI-N87 cells. When the tumors reached ~ 120 mm3, the animals were given vehicle, mil40, and ADC
on: Days 0, 7, 14, and 21. Results are shown as mean
the test animals during the treatment and observation period; (
±
SD, n = 6/group. (
A) The tumor volume of
B
) changes in body weight of the mice
during the observation period. To compare the difference in activity between antibody and ADC,
unpaired two-tailed t tests for multiple comparisons were used, and statistical analyses were performed
using Prism5 for Windows.

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During the treatment period, there was no weight loss observed in all of the treatment groups.
Body weight of the test mice was not significantly different between experimental and control groups,
indicating that the ADC and naked antibody were all preliminarily well-tolerated at the therapeutic
dose (Figure 8B).
2.10. Hematology Safety Study
To further verify the safety of the silyl ether-based acid-cleaving ADC (mil40-6), changes in
hematology between vehicle and treatment groups (ADC at doses of 5 mg/kg) were compared at the
end of administration (one week after the end of administration; day 28).
The unpaired two-tailed t tests for the hematological parameters of the blood samples showed
that there was no significant difference (p < 0.05) between the ADC treatment group (5 mg/kg) and the
vehicle (white blood cells: p = 0.6083; red blood cells: p = 0.1655; hemoglobin: p = 0.8078; platelets:
p = 0.5633; neutrophils: p = 0.8072; lymphocytes: p = 0.5030; monocyte: p = 0.9230; eosinophils:
p = 0.2161; large unstained cell: p = 0.4772), with the hematological parameters of the test animals shown
in Figure 9. The above results demonstrated that the ADC did not produce significant hematological
toxicity at therapeutic doses.
WBC
RBC
Neut
Eos
HGB
15
10
5
13
12
11
10
200
190
180
170
160
150
0
vehicle
ADC: 5mpk
vehicle
vehicle
vehicle
ADC: 5mpk
ADC: 5mpk
ADC: 5mpk
vehicle
ADC: 5mpk
PLT
Lymph
1400
1300
1200
1100
1000
900
3
2
1
0
8
6
4
2
0
800
vehicle
ADC: 5mpk
vehicle
ADC: 5mpk
Momo
LUC
0.25
0.20
0.15
0.10
0.05
0.00
0.6
0.4
0.2
0.0
0.25
0.20
0.15
0.10
0.05
0.00
vehicle
ADC: 5mpk
vehicle
ADC: 5mpk
Figure 9. Hematological analysis of acid-sensitive ADC (mil40-6). WBC: white blood cells; RBC:
red blood cells; HGB: hemoglobin; PLT: platelets; Neuts: neutrophils; Lymph: lymphocytes; Mono:
monocyte; Eos: eosinophils; LUC: large unstained cell. Results are shown as mean
±
SD, n = 6/group.
3. Discussion
The design of the linker is one of the core elements for constructing ADCs, as it has a direct
and significant impact on stability, toxicity and pharmacokinetics [47 48]. Linkers with different
,
drug release mechanisms currently need to constantly balance the relationship between efficacy and
toxicity of ADCs to obtain better clinical benefits, making the developing of diverse types of linkers a
historically attractive proposal.

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The MMAE (IC50 ~ 10−11 mol/L) developed by Seattle Genetics (Bothell, WA, USA) is the most
widely used cytotoxin in current ADCs [18 49]. However, the conjugating mode of MMAE-based ADCs
,
is still relatively simple, as the high toxicity of MMAE generally requires the linker to have sufficient
plasma stability to reduce off-target toxicity. Currently, MMAE mostly uses a Val-Cit (VC)-based
cathepsin B-cleavable linker to achieve conjugation with antibodies; this enzymatic linker system
has good stability, but its structural complexity may lead to additional tolerance risks. It is generally
believed that MMAE is not suitable for existing acid-cleavable linkers that are based on hydrazone,
carbonate acid, or others due to its high toxicity; these types of linkers are currently only used for
conjugating cytotoxins with relatively lower activities, such as doxorubicin, calicheamicin, and SN-38.
These acid-cleavable ADCs usually require increased doses, frequency of dosing, or DAR to ensure
efficacy, which may result in dose-limiting toxicity and immunogenic risk [50].
In this study, we generated a novel MMAE-based acid-cleavable ADC with relatively high plasma
stability. It has been reported that silyl ether groups are sensitive to acidity and their cleavage can
be precisely regulated by adjusting the size of the alkyl substituents in their structure [33–35,51].
However, these groups have only been used in the design of prodrugs for hydroxyl-containing drugs
due to the limitation of the silyl ether structure. By introducing PHB, the ADC skillfully achieves
an efficient combination of amino-conjugated MMAE with acid-triggered silyl ether groups. In
addition, the further introduction of an antibody commendably overcomes the drawbacks of prodrug
targeting. This conjugating model may provide a new general strategy for ADC design that is based
on amino-containing cytotoxins.
The produced ADC showed significant stability improvement, which was a basic requirement of
the linkers for MMAE-based ADCs. The plasma half-life of the prepared diisopropyl-substituted silyl
ether-based linker was longer than 7 days, which was greatly improved compared with the half-life of
the current hydrazone and carbonate type linkers (t1/2 ~ 2–3 days or 1 day, respectively) [2,17,52–54].
We speculated that the ideal steric hindrance of the diisopropyl-substituted silyl ether structure better
guarantees the stability of the ADC in neutral environments. Further studies showed that this ADC
can quickly and effectively release the payload in weakly acidic buffer, in which the rate and extent of
drug release show dependence on acidity. The above features of this linker system fully meet the two
basic requirements of the MMAE-based ADCs for linkers.
Further in vitro cell viability studies demonstrated that the ADC showed potent efficacy and
significant antigen selectivity. Endocytic studies also showed that the ADC can be effectively
internalized into target cells and eventually enter the lysosomes, where the weakly acidic environment
(pH ~ 4.5) will ultimately ensure the effective drug releases of the ADC. In addition, the microtubule
polymerization inhibition test showed that the ADC’s potential metabolite, Cys-6, can also produce
microtubule polymerization inhibition that is similar to MMAE, meaning that even if free MMAE is
not completely released, the metabolites can also work synergistically. This may be another reason for
the increased cytotoxicity of this ADC. Furthermore, cell cycle arrest assay indicated that the ADC can
suppress the cell cycle to the G2/M phase, which is most likely caused by inhibition of microtubule
polymerization, further elaborates the anti-tumor mechanism. The properties exhibited by this silyl
ether-based acid-cleavable ADC provide important support and inspiration for our future research.
In the pharmacodynamic study of xenograft models in vivo, this ADC not only exhibited superior
tumor inhibition than naked antibody mil40 (p = 0.0054), but also showed significant dose-effect
dependencies. Compared to the usual administration frequency of the acid-cleavable ADCs of about
2 times per week [17,52,55], the tumor growth inhibition rate can be as high as 93.6% for this ADC
with weekly dosing treatments (5 mg/kg). Moreover, there was no obvious adverse reaction during the
treatment of animals, as the body weight of all of the tested mice increased steadily. In addition, there
were no significant differences in hematological parameters between ADC-treated mice compared with
the control group (p > 0.05), further indicating a lower therapeutic toxicity. In vivo studies showed
that this novel acid-cleavable ADC with MMAE as a potent payload initially maintains a balance

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between efficacy and toxicity, which was expected to make it a potential leader for the development of
acid-cleavable MMAE-based ADCs.
4. Materials and Methods
4.1. Chemistry
Unless stated otherwise, all of the anhydrous solvents were commercially obtained and stored
in sure-seal bottles under dry nitrogen. All of the other reagents and solvents were purchased at the
highest grade available and used as received. Anti-HER2 antibody mil40 (a biosimilar of Herceptin®)
was purchased from Hisun Pharmaceutical CO., Ltd. (Taizhou, Zhejiang, China). Monomethyl
auristatin E (MMAE) was purchased from Concortis Biosystems (San Diego, CA, USA). Zenon
pHrodo iFL red human IgG labeling reagent (Z25612), NucBlue Live ReadyProbes reagent
Lysosomes-GFP, BacMam 2.0 (C10507) were purchased from Invitrogen
™
™
™
™
(R37605), and CellLight
(Carlsbad, CA, USA).
™
Thin layer chromatography was performed using pre-coated silica gel plates (Yantai Dexin
Bio-Technology Co., Ltd., Yantai, China). Column chromatography was performed using silica gel
(200–300 mesh; Yantai Chemical Industry Research Institute, Yantai, China). Proton nuclear magnetic
resonance (1H-NMR) spectra were recorded on a JNM-ECA-400 400 MHz spectrometer (JEOL Ltd.,
Tokyo, Japan) using CDCl3 as solvent. Chemical shifts (
δ) are expressed in ppm, with tetramethylsilane
(TMS) functioning as the internal reference, where ( ) TMS = 0.00 ppm. The mass spectrometry (MS)
δ
systems used were the API 3000 triple-quadrupole mass spectrometer equipped with a Turbo Ion Spray
electrospray ionization (ESI) source (AB Sciex, Concord, ON, Canada) for liquid chromatography-mass
spectrometry (LC/MS) analysis and the Agilent G6230A mass spectrometer for accurate mass detection
(Agilent, Santa Clara, CA, USA). Both of them were equipped with a standard ESI source.
4.2. Synthesis of the Silyl Ether-Based Linker-MMAE Conjugate
The synthetic route for the linker-MMAE conjugate (compound 6) was shown in Figure 2, with
the original Mass and NMR spectrogram details of the above synthesized compounds provided in the
Supporting Information, Figures S2–S13.
4.2.1. Synthesis of 1-(2-(2-hydroxyethoxy)ethyl)-1H-pyrrole-2,5-dione (2)
Compound 2 was synthesized using previously reported experimental protocols [56]. Maleimide
(4.0 g, 41.2 mmol) was dissolved in ethyl acetate (160 mL), then cooled to 0 ◦C in ice water.
N-methylmorpholine (3.7 g, 41 mmol) was then added slowly in a dropwise fashion after stirring for
10 min. A solution of methyl chloroformate (4.92 g, 40 mmol) in ethyl acetate (10 mL) was slowly
added dropwise and the reaction continued for 1 h at room temperature. After the completion of the
reaction, the insoluble salt was removed by filtration. The filtrate was concentrated to give a crude
product methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (1), which was obtained as a red oily
solid (6.55 g, 99% yield) after storage in low temperature. The product was unstable which was directly
subjected to the next reaction without further purification.
2-(2-Aminoethoxy)ethan-1-ol (4.0 g, 41.2 mmol) was dissolved in a saturated sodium bicarbonate
solution (120 mL) and cooled to 0 ◦C. After the desired temperature was reached, the above solution
was transferred to compound 1 (6.55 g, crude product) and the reaction was continued at 0 ◦C for
30 min. The sample was slowly warmed to room temperature and continued to react for 1 h. After the
completion of the reaction, the mixture was extracted with chloroform. The crude product was purified
by column chromatography to obtain compound 2 as a white solid (4.37 g, 57% yield of two-step
1
reaction). H-NMR (400 MHz, CDCl3):
δ 6.73 (s, 2H), 3.75 (t, J = 5.1 Hz, 2H), 3.69 (t, J = 5.1 Hz, 2H),
3.65 (t, J = 5.3 Hz, 2H), 3.57 (t, J = 5.3 Hz, 2H), 2.27 (br, 1H). ESI m/z (M + H)+ calculated for C8H12NO4
186.07; found 186.07. ESI m/z (M + Na)+ calculated for C8H11NNaO4 208.06; found 208.05.

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4.2.2. Synthesis of 4-(((2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)diisopropylsilyl)-oxy)
benzaldehyde (3)
Compound 2 (0.5 g, 2.7 mmol) and triethylamine (0.55 g, 5.4 mmol) were dissolved in
dichloromethane (20 mL) and the mixture was cooled to 0 ◦C. A solution of dichlorodiisopropylsilane
(0.6 g, 2.7 mmol) in DCM (10 mL) was slowly added dropwise. After 40 min, p-hydroxybenzaldehyde
(0.33 g, 2.7 mmol) in tetrahydrofuran (5 mL) was slowly added dropwise for an additional 1 h of
reaction. After completion of the reaction, the solvent was concentrated to give a crude material, which
was purified by column chromatography to obtain compound 3 as a white solid (0.86 g, 76% yield).
1H-NMR (400 MHz, CDCl3):
3.92 (t, J = 5.1 Hz, 2H), 3.72 (t, J = 5.1 Hz, 2H), 3.64 (t, J = 5.3 Hz, 2H), 3.58 (t, J = 5.3 Hz, 2H), 1.16 (m,
δ 9.90 (s, 1H), 7.80 (dt, J = 8.4 Hz, 2H), 7.08 (dt, J = 8.4 Hz, 2H), 6.69 (s, 2H),
2H), 1.05 (m, 12H). 13C-NMR (100 MHz, CDCl3):
δ 191.10, 170.73, 161.05, 134.23, 132.07, 130.58, 120.26,
71.84, 67.98, 62.99, 37.31, 17.20, 12.39. HRMS (ESI) m/z (M + H)+ calculated for C21H30NO6Si 420.1842;
found 420.1838. HRMS (ESI) m/z (M + Na)+ calculated for C21H29NNaO6Si 442.1662; found 442.1655.
4.2.3. Synthesis of 1-(2-(2-(((4-(hydroxymethyl)phenoxy)diisopropylsilyl)oxy)ethoxy)ethyl)-1H-
pyrrole-2,5-dione (4)
Compound 3 (0.5 g, 1.19 mmol) was dissolved in dry THF (15 mL) and the mixture was cooled to
-5 ◦C after dissolution. Sodium borohydride (18 mg, 0.48 mmol) was added to the reaction mixture
and the mixture was reacted for 2 h and then filtered to remove the insoluble salt; the filtrate was
concentrated under reduced pressure and evaporated to dryness to give a crude product, which
was purified by column chromatography to obtain compound 4 as a colorless oil (0.1 g, 23% yield).
1H-NMR (400 MHz, CDCl3):
δ 7.25 (t, 2H), 6.93 (m, 2H), 6.67 (s, 2H), 4.61 (s, 2H), 3.90 (t, J = 5.2 Hz,
2H), 3.69 (t, J = 5.0 Hz, 2H), 3.61 (t, J = 5.3 Hz, 2H), 3.56 (t, J = 5.2 Hz, 2H), 1.73 (br, 1H), 1.13 (m, 2H),
1.05 (m, 12H). ESI m/z (M + Na)+ calculated for C21H31NNaO6Si 444.5; found 444.4.
4.2.4. Synthesis of 4-(((2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)diisopropylsilyl)-oxy)
benzyl(4-nitrophenyl) carbonate (5)
Compound 4 (253 mg, 0.6 mmol) was dissolved in dry DMF (15 mL) and bis(p-nitrophenyl)
carbonate (360 mg, 1.2 mmol) and DIPEA (104 mg, 0.9 mmol) were added; the reaction was stirred at
room temperature for 12 h. After the completion of the reaction, the solvent was concentrated under
reduced pressure to remove the solvent. The obtained residue was subjected to diethyl ether dispersion
and then filtered to obtain crude product, which was further purified by column chromatography to
1
obtain compound 5 as a white solid powder (253 mg, 72% yield). H-NMR (400 MHz, CDCl3):
δ 8.28
(dt, J = 9.0 Hz, 2H), 7.38 (dt, J = 9.2 Hz, 2H), 7.33 (dt, J = 8.4 Hz, 2H), 6.98 (dt, J = 8.7 Hz, 2H), 6.68 (s,
2H), 5.23 (s, 2H), 3.91 (t, J = 5.2 Hz, 2H), 3.72 (t, J = 5.6 Hz, 2H), 3.65 (t, J = 5.6 Hz, 2H), 3.58 (t, J = 5.2
Hz, 2H), 1.14 (m, 2H), 1.06 (dd, 12H). ESI m/z (M + NH4)+ calculated for C28H38N3O10Si 604.3; found
604.6. ESI m/z (M + Na)+ calculated for C28H34N2NaO10Si 609.2; found 609.5.
4.2.5. Synthesis of the Linker-MMAE Conjugate (Compound 6)
Compound 5 (35.2 mg, 0.06 mmol) was dissolved in dry DMF (3 mL), then HOBt (8.28 mg, 0.06
mmol), MMAE (40 mg, 0.06 mmol), and DIPEA (10.68 µL) were added; the reaction was stirred at
room temperature for 18 h. After the reaction, the solvent was concentrated under reduced pressure to
remove the solvent. The obtained residue was further purified by column chromatography to afford
1
compound 6 as a white solid powder (43.4 mg, 62% yield). H-NMR (400 MHz, CDCl3):
δ 7.38-7.31 (m,
6H), 7.25-7.20 (t, J = 8.12 Hz, 2H), 7.13 (d, J = 8.12 Hz, 0.5H), 7.02 (d, J = 9.28 Hz, 0.5H), 6.90 (d, J = 8.44
Hz, 2H), 6.68 (s, 2H), 6.55 (m, 1H), 5.15-5.03 (m, 2H), 4.95 (d, J = 2.8 Hz, 1H), 4.69 (m, 2H), 4.28-4.05 (m,
5H), 3.91 (t, J = 5.05 Hz, 2H), 3.72 (t, J = 5.50 Hz, 2H), 3.64 (t, J = 5.50 Hz, 2H), 3.57 (t, J = 5.05 Hz, 2H),
3.52-3.50 (m, 2H), 3.40 (m, 4H), 3.30 (s, 3H), 3.11 (m, 1H), 3.01 (m, 3H), 2.87 (m, 3H), 2.49-2.32 (m, 3H),
2.21 (m, 1H), 2.04 (m, 3H), 1.85 (m, 2H), 1.33-1.24 (m, 5H), 1.04 (m, 19H), 0.98-0.77 (m, 26H). 13C-NMR
(100 MHz, CDCl3):
δ 174.78, 170.77, 170.54, 169.98, 169.70, 157.58, 155.18, 141.21, 134.21, 130.07, 129.55,

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128.05, 127.39, 126.44, 119.65, 82.04, 78.61, 75.84, 71.94, 67.96, 67.50, 64.95, 62.80, 61.08, 60.19, 58.10,
57.25, 54.21, 51.64, 47.96, 45.10, 37.73, 37.36, 33.33, 32.03, 31.00, 29.81, 26.21, 25.81, 25.10, 25.04, 19.41,
18.61, 17.29, 16.04, 14.54, 14.00, 12.38, 10.98. HRMS (ESI) m/z (M + H)+ calculated for C61H97N6O14Si
1165.6832; found 1165.6828. HRMS (ESI) m/z (M + Na)+ calculated for C61H96N6NaO14Si 1187.6651;
found 1187.6645.
4.3. Preparation of Reagents and Antibody-Drug Conjugates
Reagents and antibody-drug conjugates were prepared with methods as described [38]. Briefly,
humanized anti-HER2 antibodies mil40 (10 mg/mL) in L-histidine buffer (20 mM, pH ~ 7.5), were
treated with TCEP (3.0 equivalents) at 25 ◦C for 90 min. To the reduced mAb was added the maleimide
drug derivative 6 (2 equivalents/SH group) in dimethylacetamide (DMAC) (8% v/v). After 120 min,
the reactions were quenched with excess NAC. The mixture was adjusted to weak acidity (pH ~ 5.5)
with diluted AcOH (0.3 mol/L) and placed on ice for 30 min before buffer exchange by elution through
Sephadex G25, and concentrated by centrifugal ultrafiltration to obtain target ADC (mil40-6). The
conjugates’ concentrations were determined by UV absorption at 280 nm, and were filtered through a
0.2-µm filter under sterile conditions and stored at −80 ◦C before use for analysis and testing.
4.4. The Stability Assays of Linker and ADC in Plasma
Human plasma (LOT#: BRH1343165) was purchased from BioreclamationIVT (Westbury, NY,
USA). NAC (900 µL, 0.41 mg/mL in PBS, pH 7.4) was added to linker-MMAE 6 (100 µL, 70.9 µg/mL in
DMSO) and incubated in a water bath at 25 ◦C for 10 min until complete conversion to NAC-6 [38].
The reaction mixture was mixed into an equal volume of human plasma and incubated in a sterile
incubator at 37 ◦C. Aliquots (50
96, 120, 144, and 168 h) and quenched with cold acetonitrile (150 L) before frozen at
µ
L) were collected at subsequent time points (0, 3, 6, 12, 24, 36, 48, 72,
µ
−
80 ◦C. After
sampling was completed, all of the samples were melted at room temperature, centrifuged to remove
protein, and analyzed by LC/MS. Results were based on the area under curve (AUC) of the off-target
MMAE at each time point, with the total amount of MMAE contained in the substrate defined as
100%. Analogously, ADC (mil40-6) in PBS (1.8 mg/mL, pH 7.4) was mixed into an equal volume of
human plasma and incubated in a sterile incubator at 37 ◦C. Similarly, aliquots (50
subsequent time points (0, 3, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h) and quenched with acetonitrile
(150 L) before being frozen at
80 ◦C. After sampling was completed, the off-target MMAE were
µL) were collected at
µ
−
detected using the methods described above.
4.5. Effect of Acidity Difference on the Drug Release Process
The drug release process was characterized using DAR values over time. ADC (mil40-6) in PBS
(2.0 mg/mL, 9 mL, pH 7.4) was divided into 3 parts; then, diluted acetic acid solution (0.3 mmol/L) was
added to two of them to adjust the pH value to 4.5 and 5.5. The resulting three solutions were placed
in an incubator at 37 ◦C for incubation; aliquots (100
3 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, and 7 d) and frozen at
of each sample was determined using hydrophobic interaction chromatography high-performance
liquid chromatography.
µ
L) were collected at subsequent time points (0,
−
80 ◦C. After sampling was completed, the DAR
4.6. Evaluation of ADC for Cancer Cell Killing in Vitro
The human HER2-positive cell lines (BT-474, NCI-N87, and MDA-MB-453), the HER2-normal level
cell line (MCF-7), and the HER2-negative cell line (MDA-MB-231) were all purchased from American
Type Culture Collection (ATCC, Manassas, VA, USA). All of the cell lines were cultured in medium
supplemented with 10% fetal bovine serum (FBS) at 37 ◦C in a humidified atmosphere containing 5%
CO2 in air; cells were subcultured three times per week at a ratio of 1:4 in medium with 10% FBS and
1% penicillin/streptomycin solution. Cells (3.3
×
104 cells/mL) were added to each well of a 384-well
L compound aliquots were added to the assay
plate and incubated at 37 ◦C overnight, after which 10
µ

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plate. The plate was incubated for 3 days at 37 ◦C, 5% CO2, and 95% humidity. Then, the plates were
incubated at room temperature for about 10 min; 40
L Cell Titer Glo® (CTG) reagent was added to
µ
each well and the plates were incubated for 30 min at room temperature. Luminescence was detected
using the EnSpire Plate Reader (PerkinElmer, Waltham, MA, USA). All of the experiments were
conducted independently and in triplicate. The effects of each agent on the proliferation of cancer cell
lines were expressed as the % cell growth inhibition using the following formula: Remaining activity
(%) = (S–M)/(V–M)
×
100%, in which S is the readout of the test sample, V is the readout of the vehicle
sample, and M is the readout of the well with compounds treatment. The 50% inhibiting concentration
(IC50) was calculated by Prism5 for Windows (Graphpad Software Inc., La Jolla, CA, USA).
4.7. Endocytosis and Transport for the Silyl Ether-Based ADC
The lysosomal trafficking of the resulting ADC was examined with reference to a published
method [46]. BT-474 cells (6
×
105/well) were seeded in 24-well plates containing glass covers and lips
and was cultured with Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS)
at 37 ◦C overnight. Cells were further incubated with CellLight Lysosomes-green fluorescent protein
(GFP, C10507, Invitrogen, Carlsbad, CA, USA) for 30 min on ice or 16 h at 37 ◦C; then, the lysosomal
membrane protein Lamp-1 was labeled with GFP. The antibody was labeled as red fluorescence
(Invitrogen, Z25612; Zenon® Labeling Technology, Carlsbad, CA, USA) with a final concentration
of 6 µ
g/mL and was incubated with the labeled BT-474 cells for 16 h at 37 ◦C. Nucblue (Invitrogen,
R37605) was added to the medium and incubated for another 15–30 min at 37 ◦ C; then, the cells were
washed with cold PBS twice. Fluorescence images were taken using a Nikon A1 confocal microscope
(Nikon Corp., Tokyo, Japan).
4.8. Microtubule Polymerization Assay
The Tubulin Polymerization Assay Kit (Cat. # BK011P, Cytoskeleton, Inc., Denver, CO, USA) was
used according to the manufacturer’s instructions for the fluorescence-based test. Before this test, the
silyl ether-based linker-MMAE conjugate (compound 6, 0.3 mmol/L in DMSO, 100
to the L-cysteine solution (0.3 mmol/L in H2O, 900 L). The reaction mixture was incubated at room
temperature for 10 min, after which HPLC analysis revealed complete conversion to Cys-linker-MMAE
µL) was added
µ
(Cys-6), as previously reported [57]. MMAE and the Cys-linker-MMAE conjugate were used at 3.0 µM
and 0.3 µM, respectively.
4.9. Cell Cycle Arrest in HER2-Positive Cancer Cells
Effects of the novel silyl ether-based ADC (10 ng/mL, 50 ng/mL, and 100 ng/mL; the concentration
of the ADC was calculated as the concentration of the antibody) on the cell cycle were examined using
the HER2-positive breast cancer cell line BT474, and using vehicle as a comparator. Cells were cultured
with RPMI 1640 medium containing 10% FBS in a humidified atmosphere, each plate was seeded with
2
×
105 cells for each group, and then normal cultured overnight at 37 ◦C, 5% CO2, and 95% humidity
before adding the drug. The ADC was diluted with medium and added into the corresponding
wells, and the medium was renewed every 3 days during the 6 days’ treatments. The Cell Cycle and
Apoptosis Analysis Kit (C1052, Beyotime, Shanghai, China) was applied for PI labeling. The entire
procedure was performed as outlined in the manufacture’s of manual. Results were analyzed with
NovoCyte (ACEA Biosciences, San Diego, CA, USA).
4.10. In Vivo Antitumor Activity in Human Gastric Xenograft Tumors
Female BALB/c nude mice (6–8 weeks old) were purchased from Beijing Ankaiyibo Biological
Technology Co. Ltd. (Beijing, China) and were acclimated for 1 week before the experiment. All of the
animal experiments were performed by following the protocol approved by the Institutional Animal
Care and Use Committee at Pharmaron Co., Ltd. (ON-CELL-XEM-06012017, 1 June 2017 of approval)
To develop the human tumor xenografts, female nude mice, with an average weight of approximately

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22 g, were inoculated subcutaneously with 5
×
106 NCI-N87 gastric cancer cells in 0.2 mL of serum-free
RPMI1640 medium for tumor development, one tumor per mouse. When tumors reached ~ 120 mm3,
mice were randomly divided into treatments and vehicle control groups (n = 6/group). The mice were
given ADC (mil40-6), antibody (mil40), and vehicle (physiological saline) on days 0, 7, 14, and 21. For
this subcutaneous xenograft model, tumor volume and body weight were monitored 2–3 times per
week from day 0 by electronic calipers during the treatment period, which was calculated using the
formula: TV = a
×
b2/2, where “a” and “b” are long and short diameters of a tumor, respectively. When
the subcutaneous xenografts were over 2000 mm3, or at the end of the experiments, animals were
sacrificed according to institutional guidelines.
4.11. Hematology Analysis during Treatment
One week after the end of administration of the nude mice NCI-N87 xenograft model (day 28), the
animals were subjected to blood collection (100–180 µL) for whole blood analysis. For some smaller
blood samples, the volume was diluted twice and the whole blood count was performed, with the
number of cells counted and analyzed based on the corrected dilution factor. The measured blood
cell parameters mainly include white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB),
platelets (PLT), neutrophil (Neut), lymphocytes (Lymph), monocytes (Momo), eosinophils (Eos), and
large unstained cells (LUC). In this experiment, the vehicle group (saline) was selected as the control
group. The final results were expressed as mean
±
SD. The data was analyzed based on the value with
a corrected dilution factor. For comparison of hematological indicators, unpaired two-tailed t tests for
multiple comparisons were used. The level of significance was set at p < 0.05. Statistical analyses were
performed using Prism5 for Windows.
5. Conclusions
Acid-cleavable linkers were first used in clinical ADCs, but their structural category is still limited,
and its stability is usually insufficient. The application of novel acid-cleavable linker technology to
produce stable ADCs allowed us to assess the critical impact of drug-linker design upon stability
and in vivo efficacy. In this study, we generated a novel, silyl ether-based acid-cleavable ADC with
MMAE as the potent payload. By introducing PHB, the ADC skillfully achieves efficient combination
of amino-conjugated MMAE with acid-triggered silyl ether groups. The resulting ADC exhibits
improved stability, effective release of payload, appropriate efficacy in vitro and in vivo, and controlled
therapeutic toxicity. This strategy may deepen the cognition of acid-cleavable linker and provide a new
conjugating model for MMAE. As an addition to the existing acid-cleavable linkers, this linker system
is expected to provide more options for the development of ADCs that load high-efficiency cytotoxins.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/11/7/957/s1,
Figure S1: Synthesis and screening process of key intermediates of the designed silyl ether-based acid-cleavable
1
linker, Figure S2: H-NMR spectrometry of compound 2, Figure S3: Mass spectrometry of compound 2, Figure
1
S4: H-NMR spectrometry of compound 3, Figure S5: 13C-NMR spectrometry of compound 3, Figure S6: High
resolution mass spectrometer of compound 3, Figure S7: 1H-NMR spectrometry of compound 4, Figure S8: Mass
spectrometry of compound 4, Figure S9: 1H-NMR spectrometry of compound 5, Figure S10: Mass spectrometry
of compound 5, Figure S11: 1H-NMR spectrometry of compound 6, Figure S12: 13C-NMR spectrometry of
compound 6, Figure S13: High resolution mass spectrometer of compound 6, Figure S14: Hydrophobic interaction
chromatography analysis of the ADC mil40-6 with an average DAR of about 5.5.
Author Contributions: Conceptualization, W.Z. and X.Z.; Data curation, Y.W.; Formal analysis, F.X.; Funding
acquisition, W.Z.; Investigation, D.X.; Methodology, Y.W. and X.Z.; Project administration, S.F.; Resources, S.F.;
Supervision, W.Z.; Validation, W.L.; Writing—original draft, Y.W.; Writing—review & editing, X.Z.
Funding: This research was funded by the National Science and Technology Major Project for Major New
Drugs Innovation and Development under grant no. 2018ZX09711003-009 and Chinese National Natural Science
Foundation under grant no. 81872736.
Acknowledgments: The authors gratefully acknowledge research support from Dan Jiang laboratory for the
structural confirmation. Authors would also like to thank Fanhua Meng for the helpful assistance when needed
while executing certain experiments.

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Conflicts of Interest: The authors declare no conflict of interest.
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