Protein labeling approach to improve lysosomal targeting and efficacy of antibody-drug conjugates

Article abstract of DOI:10.1039/d0ob00265h

An anti-EGFR nanobody was labeled at the C-terminus with a lysosome-sorting NPGY (Asn-Pro-Gly-Tyr) motifviasortase-mediated ligation to enhance the engagement of the clathrin-mediated endocytosis. The synergistic effects of NPGY motif and nona-arginine peptide were found to induce robust internalization and lysosomal trafficking, which in turn improved anti-tumor activity of an antibody-drug conjugate.

Full text of DOI:10.1039/d0ob00265h

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I . J. Dmochowski et al.
Oligonucleotide modifi cations enhance probe stability for
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Protein labeling approach to improve lysosomal targeting and
efficacy of antibody-drug conjugates
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
b
a
a
a
a
c
Ying Han,† Yifan Da,† Mingjia Yu, Yaping Cheng, Xin Wang, Jiale Xiong, Guoying Guo, Yan Li,
a
a
Xianxing Jiang, and Xiaoqing Cai*
DOI: 10.1039/x0xx00000x
An anti-EGFR nanobody was labeled at the C-terminus with a pathway, in which proteins are taken up in a non-selective manner.
lysosome-sorting NPGY (Asn-Pro-Gly-Tyr) motif via sortase- Especially, macropinosomes formed during macropinocytosis
mediated ligation to enhance the engagement of the clathrin- appear independent of the canonical endocytic pathways and tend
6
mediated endocytosis. The synergistic effects of NPGY motif and to undergo a unique efflux step in higher eukaryotes. Therefore,
nona-arginine peptide were found to induce robust internalization approaches that increase clathrin-mediated endocytosis of ADCs
and lysosomal trafficking, which in turn improved anti-tumor are highly desired. Currently, the strategy to improve the
activity of an antibody-drug conjugate.
internalization properties of ADCs is by constructing new antibody
structures, such as bispecific antibodies, in which one arm binds to
tumor-associated antigen and the second arm is designed to
Antibody-drug conjugates (ADCs) are emerging as a promising class
of anticancer therapeutics that combine the specificity of antibodies
and the cytotoxicity of chemotherapeutic drugs. The mechanism of
7
facilitate internalization and lysosomal trafficking . Despite some
1
encouraging advances, it remains a significant challenge to
manipulate the endocytic pathways as a mean to enhance the
lysosomal targeting of ADCs.
action of an ADC involves antibody binding to a cell surface antigen,
followed by receptor-mediated endocytosis. After internalization,
an ideal ADC would go through the endo/lysosomal pathway to
release the cytotoxic payload, which ultimately triggers tumor cell
Many transmembrane proteins are marked for clathrin-
mediated endocytosis by a tyrosine-containing cytoplasmic motif
2
death. The release of the payload can be achieved by degradation
8
referred as tyrosine-based sorting signals. NPXY (Asn-Pro-X-Tyr, X
of the cleavable linkage (ADCs with cleavable linkers) or catabolism
of the entire antibody (ADCs with noncleavable linkers), either of
which requires efficient proteolysis by hydrolases within lysosomes.
Therefore, efficient internalization and trafficking to lysosome is
essential for the antitumor efficacy of ADCs. Furthermore, recent
studies indicated that diminution in accumulation or proteolysis of
ADCs in lysosomes could cause drug resistance to ADC-based
stands for any amino acid) is one of the most common tyrosine-
based signals found in many integral membrane proteins, such as
low-density lipoprotein (LDL) receptors, β3-integrin and β-amyloid
9
precursor proteins. Proteins containing NPXY signal were observed
9a
to be internalized via clathrin-coated pits.
crystallographic studies have demonstrated that NPXY-type motifs
NPXY or FXNPXY) adopt a type-I β-turn conformation and bind to
Previous
(
3
therapy.
the phosphotyrosine-binding (PTB) domain of cell surface clathrin-
Due to their size and polarity, proteins, including antibodies,
cannot directly pass through cell membranes, and can only be
internalized into eukaryotic cells via three distinct routes: clathrin-
mediated endocytosis, caveolae-mediated endocytosis, and
10
associated proteins, including Dab2 and ARH. Although NPXY has
been recognized as a critical signal for sorting of membrane
proteins to lysosomes, alone it may not be sufficient for
internalization. It has been shown that fusion of only NPXY motif
2
a
macropinocytosis. Among these pathways, only clathrin-mediated
endocytosis is processed through the classic endo/lysosomal
11
into the transferrin receptor failed to direct rapid endocytosis. To
date, it remains unexplored how to incorporate a minimum NPXY
motif into protein structure to ensure the specific and directed
transport of extracellular proteins to lysosomes.
4
system. Proteins internalized through the caveolae-mediated
endocytosis have been shown to mainly accumulate in the
endoplasmic reticulum or Golgi, two non-proteolytic compartments
In this study, we sought to investigate whether we could
construct an NPXY-labeled antibody that would effectively engage
the clathrin-mediated endocytosis (Fig. 1). We hypothesized that
the complete exposure of the NPXY motif to its recognition proteins
located in the cytoplasm would promote the assembly of clathrin
machinery. Accordingly, three peptides were designed as protein
fusion partners based on the following: (1) NPGY was chosen as the
signal peptide in the hope that a simple glycine residue could avoid
any steric hindrance. (2) We expected that a flexible linker should
facilitate the exposure and proper function of the NPXY motif. Thus,
5
of cells. Macropinocytosis is a receptor-independent endocytosis
a. School of Pharmaceutical Sciences, Sun Yat-sen University, 132 East Outer Ring
Road, Guangzhou, 510006, China. E-mail: caixq7@mail.sysu.edu.cn
Beijing Advanced Innovation Center for Soft Matter Science and Engineering，
b.
Beijing University of Chemical Technology, Beijing, 100029, China.
School of Engineering, Sun Yat-sen University, 132 East Outer Ring Road,
Guangzhou, 510006, China.
c.
†
These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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provides an alternative to avoid the issues involved in recombinant
View Article Online
DOI: 10.1039/D0OB00265H
protein expression.
We began the sortase-mediated ligation through engineering
the mutated sdAb (7D12-S85C) with a C-terminal LPETG sequence
followed by a His tag. The mutation site Ser85, which is distant
6
from the antigen-binding domain, was selected for small molecule
conjugation. In parallel, we synthesized the linker-payload construct
(
mcMMAF) that contains a maleimidocaproyl (mc) spacer and an
antitumor agent, monomethyl auristatin F (MMAF) (Fig. 1b).
Another linker-payload construct (maleimide-PEG -TAMRA) was
obtained from a commercial source to visualize protein endocytic
trafficking. Briefly, 7D12-S85C-LPETG-His was expressed in the E.
coli, followed by the conjugation of linker-payload mcMMAF or
maleimide-PEG -TAMRA to afford conjugates 7D12-MMAF-LPETG-
or 7D12-TAMRA-LPETG-His . The designed peptides containing
3
6
3
Fig. 1 Labeling of antibody-drug conjugates. (a) Computationally His
docked models showing the binding of GGGNPGY motif (highlighted
in green) to the PTB domain of Dab2 (PDBID: 1M7E). α helix and β
sheet of PTB domain are shown in ice blue and yellow. (b)
Structures of maleimidocaproyl-MMAF (1) and maleimidopropionyl-
6
6
a tri-glycine sequence (GGGNPGY, GGGRRRRRRRRRNPGY and
GGGRRRRRRRRR) were prepared using standard solid-phase
1
5a
peptide synthesis. Lastly, srtA∆N59 was prepared and used for the
ligation between 7D12-MMAF-LPETG-His or 7D12-TAMRA-LPETG-
with tri-glycine-containing peptides (Fig. 1c). The obtained
PEG
carboxytetramethylrhodamine. (c) Scheme for the synthesis of His
3
-TAMRA (2). MMAF: monomethyl auristatin F; TAMRA: 5-
6
6
peptide-labeled conjugates of a single domain antibody (7D12).
Conditions: i) 5 mM TCEP, rt, 30 min; ii) 5 equiv of 1 or 2, 4 °C, 2 h;
iii) 2 equiv of SrtAΔN59, 10 equiv of peptides, 150 mM NaCl, 50 mM
7
D12-MMAF-peptide (7D12-MMAF-P
1
, 7D12-MMAF-P
2
and 7D12-
7D12-
) were further characterized by ESI
and 7D12-
were ligated with a simple triglycine peptide to
MMAF-P
TAMRA-P
3
)
and 7D12-TAMRA-peptide (7D12-TAMRA-P
1
,
2
and 7D12-TAMRA-P
3
Tris·HCl pH 7.5 and 5 mM CaCl
2
, rt, 15 h.
mass spectrometry. In addition, 7D12-MMAF-LPETG-His
TAMRA-LPETG-His
remove the LPETG-His
6
6
a short peptide sequence GGGNPGY (P
1
) was designed as the first
6
tag and afforded 7D12-MMAF and 7D12-
fusion partner. (3) To maximize the accessibility of the NPGY motif
to its cytosolic recognition proteins, RRRRRRRRRNPGY sequence
TAMRA.
Confocal imaging study was carried out to visualize the antibody
internalization and lysosomal trafficking in EGFR-positive A431 cells
and EGFR-negative 293T cells. LysoTracker® Green was selected as a
lysosome marker. As shown in Fig. 2, we observed significant
fluorescent signals for all 7D12-conjugates after 4-h incubation in
A431 cells, but not 293T cells (Fig. S19). In addition, we analyzed
and quantified the colocalization of these conjugates with
lysosomes using the Pearson’s correlation coefficient (Fig. S20 &
Table S4). 7D12-TAMRA was used as a control with a correlation
2
(P ) was designed as a second fusion partner, in which a nona-
arginine peptide with excellent cell-membrane penetration ability
was inserted before the NPGY motif. (4) The nona-arginine peptide
1
2
3
(P ) was chosen to probe the importance of the poly-arginine
peptide itself. Furthermore, the interaction between the designed
peptides and the PTB domains of Dab2 was predicted by protein-
1
3
peptide docking method. Fig. 1a shows an energetically favorable
docked structure, in which the NPGY motif adopts an active β-turn
structure accommodated between the secondary structure of helix

3 and strand 5 of DAB homology domain, and introduction of a
flexible linker is likely to enhance the fully binding of NPGY residues.
1
4
An anti-EGFR single domain antibody (sdAb-7D12) was
selected as the model antibody for our studies. Since the antigen-
binding variable region is located at the N-terminal domain, fusion
can only be constructed by adding the designed peptide to the C-
terminus of 7D12. Initially, we applied recombinant DNA technology
for fusion protein expression (see supporting information for
details). Unexpectedly, the proteins of interest could only be
prepared at a low level (Fig. S2). Presumably, the presence of a
lysosome sorting signal together with a polycationic peptide
sequence in protein structure might hamper the protein expression.
To improve the preparation of 7D12 fusions, we exploited sortase-
mediated ligation for protein labeling. Sortases are a class of
bacterial tranpeptidase that catalyze the cleavage of a sortase-
recognition motif (LPXTG) followed by the formation of a covalent
linkage between the LPXTG-containing protein and an oligoglycine
Fig. 2 Internalization and lysosomal trafficking studies of 7D12-
conjugates. A431 cells were treated with TAMRA-labeled 7D12-
conjugates followed by the treatment of LysoTracker® Green for 30
min. The top row shows the lysosome signals (green), the middle
row indicates the 7D12 conjugate signals (red), and the bottom row
shows the merged images (yellow).
1
5
peptide/protein. By directly using two independently active
protein/peptides as substrates, sortase-mediated protein ligation
2
| J. Name., 2012, 00, 1-3
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coefficient of 0.4157. To our delight, NPGY-containing peptides (P
and P ) caused a substantial increase in colocalization (Pearson’s
correlation coefficient values for 7D12-TAMRA-P : 0.7236; for 7D12-
TAMRA-P : 0.7504). Also, nona-arginine was found to increase the
lysosomal trafficking with a PCC value of 0.6508.
Next, we performed time-course internalization study using
flow cytometry. As shown in Fig. 3a, 7D12-TAMRA-P and 7D12-
TAMRA-P exhibited a significantly faster and higher level of
internalization than 7D12-TAMRA and 7D12-TAMRA-P . To obtain a
1
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DOI: 10.1039/D0OB00265H
2
1
2
2
3
1
more accurate account of the internalization event, we conducted a
fluorescence assay to quantify the internalization of the conjugates.
As seen in Fig. 3b, R
D12-TAMRA-P ) displayed the highest level of internalization,
followed by 7D12-TAMRA-P and then 7D12-TAMRA. Notably, the
total cellular uptake of 7D12-TAMRA-P
9 2
-containing conjugates (7D12-TAMRA-P and
7
3
A431 cells. Data are expressed as the mean ± SEM (n=3). ns: not
significant. **P < 0.01 (Student’s t test).
1
2
over the first 6-h was Fig. 4 Antitumor activity of 7D12-conjugates. (a) A431 cells were
calculated to be approximately 1 million molecules per cell (19.4 treated with MMAF, 7D12 or the indicated 7D12-MMAF conjugates
7
of varying concentrations for 96 h. EC50 values are expressed as
mean ± SEM (n=3). (b) Confocal images of A431 tumor spheroids
pmol per 10 cells), which was about 2-fold more than that of 7D12-
TAMRA. Together, the above results indicated that nona-arginine
played a key role in promoting internalization of 7D12-conjugates
while NPGY was more responsible for lysosomal targeting.
2
incubated with TAMRA-labeled 7D12 and 7D12-P for 6 h followed
by the treatment of LysoTracker® Green. Nuclei were stained with
Hoechst 33342 (blue). (c) Representative images of A431 tumor
Encouraged by the internalization data, we studied the in vitro spheroids after exposure to the tested 7D12-conjugates over time.
cytotoxic activity of MMAF-conjugated antibodies against A431 cells
and 293T cells. The free MMAF is reported to have low cell limited internalization. Next, we carried out the viability assay with
permeability, which provides the rationale for the construction of this 3D model. Fig. 4c shows the A431 spheroid morphology with
1
6
the treatment of 2.5 μM conjugates over nine days. A clear
antibody-MMAF-conjugates to deliver MMAF into the target cell.
As shown in Fig. 4a, chimeric 7D12-MMAF conjugates exhibited diminishing was observed on day 3 for 7D12-MMAF-P while no
2
nanomolar-level cell killing activity in EGFR-positive A431 cells, but significant change occurred for 7D12-MMAF over a 9-day period.
no cytotoxicity in EGFR-negative 293T cells (Fig. S23). Compared to Overall, the data obtained from the 3D tumor model are entirely
MMAF and 7D12-MMAF, chimeric 7D12-MMAF showed at least 10- consistent with the enhanced cell-killing potency anticipated from
fold more potency, with 7D12-MMAF-P
potency, followed by 7D12-MMAF-P and then 7D12-MMAF-P
general, the cytotoxicity of the tested conjugates was correlated to the tested antibodies. First, to test whether the internalization was
2
exhibiting the highest efficient internalization and lysosomal trafficking.
. In
Finally, we investigated the possible endocytosis mechanism of
3
1
their internalization and lysosome-targeting ability.
energy-dependent, A431 cells were incubated with the tested
In order to mimic the in vivo tumor development and behavior,
we used a three-dimensional (3D) tumor spheroid model to assess
the anti-tumor activity of 7D12-MMAF-P in comparison with 7D12-
2
MMAF. Accordingly, A431 cells were placed into soft agarose, and
cultured over an approximately 2-day period to obtain spheroids
with a diameter around 100 μm. Following the treatment of the
tested conjugates, morphologic analysis of the spheroids was
performed. First, using this 3D tumor model we visualized the
internalization events of 7D12-TAMRA and 7D12-TAMRA-P
2
(Fig.
4
b). Our data further supported that P could promote a robust
2
cellular uptake and lysosomal trafficking while 7D12-TAMRA only
induced a
Fig. 5 Endocytosis mechanism study. Shown are merged images of
representative cells. (a) Effect of temperature on the internalization
of 7D12-TAMRA. (b) Effect of different inhibitors on the
internalization of TAMRA-labeled 7D12-conjugates. A431 cells
o
Fig. 3 Internalization properties of TAMRA-labeled 7D12-conjugates. incubated with the tested TAMRA-labeled 7D12-conjugates at 37 C
(
a) The time course of antibody internalization into A431 cells via without the pretreatment of any endocytosis inhibitors were taken
flow cytometry. (b) Quantification of antibody internalized into as control samples. Lysosomes were stained by LysoTracker® Green.
(c) Colocalization of TAMRA-labeled 7D12-conjugates with anti-
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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caveolin-1 antibody (Green) or anti-clathrin heavy chain antibody
Notes and references
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DOI: 10.1039/D0OB00265H
(Green). Nuclei of A431 cells were stained in blue by Hoechst 33342.
o
o
1
2
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D12-conjugates at two different temperatures 4 C or 37 C). As
shown in Fig. 5a & S27, the uptake of all tested 7D12-conjugates
o
was significantly inhibited at 4 C, suggesting 7D12-conjugates were
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o
incubation with the tested conjugates at 37 C, i.e., chlorpromazine
2
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clathrin-mediated endocytosis, caveolae-mediated endocytosis and
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1
7
conjugates was conducted as previously reported, and the results
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different extent. Notably, the clathrin-mediated endocytosis
inhibitor CPZ was found to significantly reduce the cellular uptake
4
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1
1
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was largely involved. To further explore the endocytosis
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7
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further confirmed that 7D12-P
1 2
and 7D12-P were internalized
predominantly through clathrin-mediated pathway while 7D12-P
3
appeared to be internalized by both caveolin- and clathrin-
mediated pathways equally.
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clathrin-mediated endocytosis of antibody conjugates. An anti-EGFR
nanobody (7D12) was labeled with lysosome-sorting NPGY signals
at the C-terminus via sortase-mediated ligation. Labeling with a
GGGNPGY or RRRRRRRRRNPGY motif resulted in two novel protein
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1
2
and 7D12-P ) with significantly improved
lysosome-targeting properties. In addition, the synergistic effects of
NPGY motif and nona-arginine peptide were found to induce robust
internalization and lysosomal trafficking. When conjugated with a
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2
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proteins. Overall, this present work provides a versatile and
promising design strategy to direct an ADC to lysosomes for payload
release, and may represent a unique and powerful approach for
developing lysosome-targeting protein therapeutics.
This work was funded by the National Natural Science
Foundation of China (No. 21708051 to X.C. and No. 91853106 to
X.J.) and the Program for Guangdong Introducing Innovative and
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Conflicts of interest
There are no conflicts to declare.
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Organic & Biomolecular Chemistry
Page 6 of 6
View Article Online
DOI: 10.1039/D0OB00265H
Graphic abstract:
Protein labeling approach to improve lysosomal targeting and
efficacy of antibody-drug conjugates
Ying Han,†a Yifan Da,†a Mingjia Yu, Yaping Cheng, Xin Wang, Jiale Xiong,
b
a
a
a
a
c
a
a
Guoying Guo, Yan Li, Xianxing Jiang, and Xiaoqing Cai*
An anti-EGFR nanobody was labeled at the C-terminus with a lysosome-sorting
NPGY (Asn-Pro-Gly-Tyr) motif via sortase-mediated ligation to enhance the
clathrin-mediated endocytosis.