Neutrophil Elastase Promotes Linker Cleavage and Paclitaxel Release from an Integrin-Targeted Conjugate

Article abstract of DOI:10.1002/chem.201805447

This work takes advantage of one of the hallmarks of cancer, that is, the presence of tumor infiltrating cells of the immune system and leukocyte-secreted enzymes, to promote the activation of an anticancer drug at the tumor site. The peptidomimetic integrin ligand cyclo(DKP-RGD) was found to accumulate on the surface of α_vβ₃ integrin-expressing human renal cell carcinoma 786-O cells. The ligand was conjugated to the anticancer drug paclitaxel through a Asn-Pro-Val (NPV) tripeptide linker, which is a substrate of neutrophil-secreted elastase. In vitro linker cleavage assays and cell antiproliferative experiments demonstrate the efficacy of this tumor-targeting conjugate, opening the way to potential therapeutic applications.

Full text of DOI:10.1002/chem.201805447

A Journal of
Accepted Article
Title: Neutrophil Elastase Promotes Linker Cleavage and Paclitaxel
Release from an Integrin-Targeted Conjugate
Authors: André Raposo Moreira Dias, Arianna Pina, Amelia Dean,
Hans-Georg Lerchen, Michele Caruso, Fabio Gasparri, Ivan
Fraietta, Sonia Troiani, Daniela Arosio, Laura Belvisi, Luca
Pignataro, Alberto Dal Corso, and Cesare Gennari
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201805447
Link to VoR: http://dx.doi.org/10.1002/chem.201805447
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Key Topic: Tumor Targeting
COMMUNICATION
Exploiting inflammation in cancer:
A RGD-drug conjugate accumulates
on the surface of integrin-expressing
human renal cell carcinoma cells and
releases paclitaxel upon linker
cleavage mediated by neutrophil-
secreted elastase.
A. Raposo Moreira Dias, A. Pina, A.
Dean, H.-G. Lerchen, M. Caruso, F.
Gasparri, I. Fraietta, S. Troiani, D.
Arosio, L. Belvisi, L. Pignataro, A. Dal
Corso and C. Gennari*
Page No. – Page No.
Neutrophil Elastase Promotes Linker
Cleavage and Paclitaxel Release from
an Integrin-Targeted Conjugate
.
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Neutrophil Elastase Promotes Linker Cleavage and Paclitaxel
Release from an Integrin-Targeted Conjugate
André Raposo Moreira Dias,[a] Arianna Pina,[a] Amelia Dean,[a] Hans-Georg Lerchen,[b] Michele
Caruso,[c] Fabio Gasparri,[c] Ivan Fraietta,[c] Sonia Troiani,[c] Daniela Arosio,[d] Laura Belvisi,[a,d] Luca
Pignataro,[a] Alberto Dal Corso,[a] and Cesare Gennari*[a,d]
in intracellular compartments (e.g. lysosomes), upon receptor-
mediated internalization and selective cleavage of a linker (e.g.
a short peptide sequence or a reducible disulfide bond)
connecting the targeting unit to the payload.[9]
Abstract: We took advantage of one of the hallmarks of cancers, i.e.
the presence of tumor infiltrating cells of the immune system and
leukocyte-secreted enzymes, to promote the activation of an
anticancer drug at the tumor site. The peptidomimetic integrin ligand
cyclo(DKP-RGD) was found to accumulate on the surface of αvβ3
integrin-expressing human renal cell carcinoma 786-O cells. The
ligand was conjugated to the anticancer drug paclitaxel through a
Among the protein antigens that have been explored for tumor
targeting applications, integrin αvβ3 is
a
heterodimeric
transmembrane receptor overexpressed in a variety of cancer
types (such as melanoma, glioblastoma, renal cell carcinoma,
and tumors of lung, ovary, breast, prostate and colon), where it
is involved in disease progression.[10] Upon the observation that
cyclic peptides bearing the Arg-Gly-Asp (RGD) sequence are
potential αvβ3 integrin ligands, a large number of RGD-bearing
peptides and peptidomimetics have been explored for SMDC
development.[11]
Asn-Pro-Val (NPV) tripeptide linker, which is
a substrate of
neutrophil-secreted elastase. In vitro linker cleavage assays and cell
antiproliferative experiments demonstrate the efficacy of this tumor-
targeting conjugate, opening the way to potential therapeutic
applications.
The conjugation of anticancer drugs to specific ligands, capable
of selective binding to tumor-associated receptors, represents a
widely explored strategy to improve the accumulation of
cytotoxic agents at the tumor site, sparing healthy tissues and
resulting in better therapeutic outcomes. Antibody-Drug
Conjugates (ADCs) represent the first-in-class in this oncology
area.[ 1 ] To date, four ADC products have gained marketing
authorization
(Adcetris™,
Kadcyla™,
Mylotarg™
and
Besponsa™), while more than 50 are presently under clinical
investigation.[ 2 ] Similarly, anticancer agents have also been
coupled to small ligands (e.g. peptides/peptidomimetics,[ 3 ]
vitamins,[4] steroids[5] and enzyme inhibitors[6]), targeting specific
receptors overexpressed by cancer cells. An increasing body of
evidences suggests that these so-called Small Molecule-Drug
Conjugates (SMDCs)[ 7 ] may accumulate in the tumor mass
homogeneously and with high tumor/organ and tumor/blood
ratios, potentially showing better anticancer efficacy than
conventional cytotoxic agents and ADC products.[8] In general,
these conjugates have been designed to bind the target receptor
on the surface of cancer cells and to release the cytotoxic cargo
[a] A. Raposo Moreira Dias, A. Pina, A. Dean, Prof. Dr. L. Belvisi,
Dr. L. Pignataro, Dr. A. Dal Corso, Prof. Dr. C. Gennari
Università degli Studi di Milano, Dipartimento di Chimica
Via C. Golgi, 19, I-20133, Milan (Italy)
E-mail: cesare.gennari@unimi.it
Figure 1. Molecular structure of the αvβ3 integrin ligand cyclo(DKP-RGD) (1)
and its analog cyclo(DKP-RβAD) (3), alongside with their relative sulfo-
cyanine5 (sCy5) conjugates cyclo(DKP-RGD)-sCy5 (2) and cyclo(DKP-
RβAD)-sCy5 (4). Data of inhibition of biotinylated vitronectin binding to the αvβ3
receptor are shown.
http://sites.unimi.it/gennarigroup/
[b] Dr. H.-G. Lerchen
Medicinal Chemistry Wuppertal, BayerAG,
42113 Wuppertal (Germany)
[c] Dr. M. Caruso, Dr. F. Gasparri, Dr. I. Fraietta, Dr. S. Troiani
Nerviano Medical Sciences,
Viale Pasteur, 10, I-20014, Nerviano (Italy)
[d] Dr. D. Arosio
Within this frame, our group synthesized a cyclic peptidomimetic
compound bearing the RGD integrin recognition motif and a
diketopiperazine (DKP) scaffold, as a low nanomolar αvβ3
integrin ligand (compound 1, Figure 1).[ 12 ] The ligand was
successfully linked to different cytotoxic payloads (i.e.
paclitaxel,[ 13 ] camptothecin[ 14 ] and α-amanitin[ 15 ]) and the
resulting conjugates maintained high affinity for the integrin
receptor. In line with literature data reporting evidences of
internalization of integrin ligands equipped with fluorescent
CNR, Istituto di Scienze e Tecnologie Molecolari (ISTM)
Via C. Golgi, 19, I-20133, Milan (Italy)
Supporting information, including the synthetic procedures, along
with the 1H- and 13C-NMR spectra, HPLC and MS data, all
procedures for biological and biochemical assays, and the ORCID
identification number(s) for the author(s) of this article can be found
under: https://doi.org/10.1002/chem.xxxxxxxx
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dyes,[ 16 ] our cyclo(DKP-RGD)-drug conjugates were endowed
with suitable linkers for selective payload release in intracellular
compartments of the cancer cells. The ability of these SMDCs to
selectively hit αvβ3-positive cells was quantified through in vitro
cell antiproliferative assays performed using cancer cell lines
expressing the integrin receptor at different levels (i.e.
expressing vs. non-expressing cells). To our delight, some of
these SMDCs proved to be highly selective for the αvβ3-
expressing cells,[17] indicating that 1 effectively recognizes the
integrin receptor on the cancer cell membrane. However, the
potency of all our conjugates against αvβ3-positive cells proved
to be significantly lower (typically one order of magnitude) than
that of the free drug. This observation is in contrast with
literature data reporting the high potency of internalizing ADCs
and SMDCs,[18,19] and it may indicate a suboptimal endocytosis
of our ligand-drug conjugates. In order to confirm this hypothesis
and evaluate the internalization properties of the cyclo(DKP-
RGD) integrin ligand, we labeled it with the near-infrared (NIR)
dye sulfo-cyanine5 (sCy5, Figure 1). The ability of the resulting
conjugate cyclo(DKP-RGD)-sCy5 (2) to compete with
biotinylated vitronectin for the binding to the isolated αvβ3
receptor was measured. The low-nanomolar IC50 value obtained
confirmed that integrin recognition was not affected by the
conjugation with the probe (2, Figure 1). Moreover, we devised
the preparation of
a negative control, possessing almost
identical physical-chemical properties, while showing negligible
affinity for the receptor. Inspired by the literature,[20] the Arg-Gly-
Asp tripeptide was mutated into a Arg-[βAla]-Asp sequence,
giving rise to the cyclo(DKP-RβAD) ligand 3 (Figure 1). In
comparison with the parent ligand 1, the new compound showed
a dramatically lower integrin binding affinity (micromolar IC50
value) and a similar result was obtained for the fluorescent
conjugate cyclo(DKP-RβAD)-sCy5 4 (Figure 1).
Figure 2. Confocal microscopy images of live αvβ3-expressing human renal cell carcinoma 786-O cells after exposure to conjugates 2 (A), 4 (B) and free sCy5 (C)
(1 µmol/l, exposure time = 2 h). Accumulation at the cell membrane was detected only upon exposure to conjugate 2. No fluorescent signal was detected in the
intracellular compartments for all compounds tested. Blue: Hoechst; Red: NIR dye.
Figure 3. Molecular structures of the αvβ3 integrin-targeted conjugate cyclo(DKP-RGD)-NPV-PTX (5), of its diastereoisomer cyclo(DKP-RGD)-NPv-PTX (6),
whose linker bears D-valine (v), and of the uncleavable conjugate cyclo(DKP-RGD)-unc-PTX (7). Mechanism of PTX release upon linker cleavage is depicted,
consisting in the fast 1,6-elimination of the p-aminobenzyl carbamate 8, followed by amine cyclization in compound 10, which results in the formation of cyclic
urea 11 and free Paclitaxel (12).
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Confocal microscopy experiments were carried out with live
human cancer cells 786-O (renal cell carcinoma), expressing
αvβ3 integrin (as detected by Western blot analysis, see Figure
S1 in the Supporting Information), in the presence of conjugates
2 and 4. The uncoupled sulfo-cyanine5 probe was also included
in the experiment as additional control. As shown in Figure 2, a
fair accumulation of the cyclo(DKP-RGD)-sCy5 conjugate (2) on
the membrane of cancer cells was detected, whereas no
significant intracellular uptake was observed. On the other hand,
no accumulation of control compounds cyclo(DKP-RβAD)-sCy5
(4) and free sulfo-cyanine5 was detected neither in intracellular
compartments, nor on the cell surface.
Table 1. Inhibition of biotinylated vitronectin binding to the αvβ3 receptor.
Conjugate
Structure
αvβ3 IC50 [nM][a]
12.9 ± 1.4
5
6
7
cyclo(DKP-RGD)-NPV-PTX
cyclo(DKP-RGD)-NPv-PTX
cyclo(DKP-RGD)-unc-PTX
24.9 ± 2.1
5.8 ± 1.2
[a] IC50 values were determined as the concentration of compound required for
50% inhibition of biotinylated vitronectin binding as estimated by GraphPad
Prism software. All values are the arithmetic mean ± the standard deviation
(SD) of duplicate determinations.
Overall, this analysis confirmed that the integrin ligand
cyclo(DKP-RGD) 1 accumulates on the cell membrane of αvβ3
integrin-expressing cancer cells, while is poorly internalized by
receptor-mediated endocytosis. This finding may be related to
the antagonist behavior of 1, which has been found to display
inhibitory effects on the FAK/Akt integrin activated transduction
pathway and on integrin-mediated cell infiltration processes.[21]
The elucidation of the link between the agonist/antagonist
behavior of integrin ligands and the receptor-mediated
internalization is currently a hot topic in this field.[22]
Table 2. Antiproliferative activity of conjugates 5-7 and free PTX in αvβ3-
expressing human renal cell carcinoma 786-O cells after 96 h treatment, in the
presence (or absence) of elastase from human leukocytes.
IC50 [nM][a]
Added elastase
Structure
NO
YES
Interestingly, the importance of conjugate internalization has
been recently challenged.[23] Indeed, ADCs and SMDCs specific
to non-internalizing receptors (e.g. collagen IV,[ 24 ] carbonic
anhydrase IX,[25] fibrin[26] and splice variants of fibronectin[27] and
tenascin-C[28]) were found to elicit strong antitumor responses in
vivo, while proving significantly less potent than the parent drug
in in vitro cell antiproliferative assays.[29] For these reasons, we
envisioned the preparation of a non-internalizing, αvβ3 integrin-
targeted conjugate, featuring a specific linker for the extracellular
release of the payload.
Paclitaxel (PTX) (12)
35.8 ± 16.7
> 5000
29.5 ± 7.6
19.6 ± 4.1
> 5000
cyclo(DKP-RGD)-NPV-PTX (5)
cyclo(DKP-RGD)-NPv-PTX (6)
cyclo(DKP-RGD)-unc-PTX (7)
> 5000
> 5000
> 5000
[a] IC50 values were determined as the concentration of compound required for
50% inhibition of cell viability in presence or absence of 50 nM elastase
(ELANE, 324681, Millipore). Samples were measured in triplicate.
In 2002, researchers at Bayer developed a new SMDC, whose
linker consisted in the tripeptide Asn-Pro-Val (NPV), a specific
substrate of neutrophil elastase.[ 30 ] The latter is a serine
protease stored in azurophilic granules of neutrophils and
released into the extracellular space upon infections or
inflammation stimuli. High levels of elastase have been reported
in primary tumors and metastasis, where it promotes oncogenic
signaling and inhibits tumor suppressors. As a result, elevated
neutrophil elastase levels correlate with poor prognosis in
different types of solid tumors.[31] To the best of our knowledge,
no further investigations of this promising NPV peptide linker
have been reported in the literature. For these reasons, we
designed an extracellularly-cleavable conjugate (5, Figure 3) in
which the αvβ3 integrin ligand cyclo(DKP-RGD) is connected to
paclitaxel (PTX) via a self-immolative spacer, the NPV linker and
a hydrophilic PEG4 spacer. Two additional conjugates were
prepared as negative controls: the cyclo(DKP-RGD)-NPv-PTX
conjugate (6), featuring the non-proteinogenic amino acid [D]-
valine, and the cyclo(DKP-RGD)-unc-PTX conjugate (7), in
which the tripeptide moiety is replaced by a proteolytically stable
(or “uncleavable”) tertiary amide bond. This panel of compounds
was designed to gain insights into the specificity of elastase
cleavage and the stability of the whole linker system. All
synthetic details and analytics are included in the Supporting
Information.
To evaluate the cleavage of the tripeptide linker and the
subsequent paclitaxel release in the presence of neutrophil
elastase, conjugate 5 was treated with the enzyme in PBS
solution at 37 ºC, and metabolites were detected by HPLC-MS
analysis. The enzymatic cleavage of the tripeptide linker was
observed over a 2 h period, resulting in the complete formation
of metabolite 10[32] (Figure 3), whereas the use of inactivated
enzyme did not lead to metabolite formation (Figure S2 in the
Supporting Information). The selectivity of linker cleavage was
analyzed treating 5 with rat liver-derived lysosome extract,
composed by a mixture of proteolytic enzymes. The broad-
scope cysteine proteases inhibitor E-64 was used in this
experiment to gain insight into the effector enzymes involved in
the cleavage. Upon 2 hours exposure to lysosome extract,
conjugate 5 was digested only partially (Figure S4 in the
Supporting Information), possibly indicating the presence of
elastase in the lysosome extract.[ 33 ] This hypothesis was
supported by the partial formation of metabolite 10 also in the
presence of the E-64 inhibitor, (Figure S4 in the Supporting
Information) indicating that cysteine proteases are not
responsible for the cleavage of the NPV linker.
Additionally, conjugate 5 was treated with mouse plasma at 37
ºC, showing an excellent stability under these conditions (t1/2
=
35.3 h, Figure S5 in the Supporting Information).
Following the protocol reported by Bayer,[30] the in vitro cell
antiproliferative activity of conjugates 5-7 was tested against
786-O cancer cells, in the presence or absence of elastase.
Importantly, Western blot analysis showed no significant
elastase expression in these cancer cells (see Figure S1 in the
Supporting Information). This model assay aimed at evaluating
The affinity of compounds 5-7 for αvβ3 integrin was estimated as
described previously and low-nanomolar values were obtained
throughout the series (Table 1).
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the extracellular cleavage of the NPV linker upon activation of
neutrophils and release of elastase in the extracellular tumor
environment, followed by the PTX internalization into cancer
cells by passive diffusion through the cell membrane. In the
absence of elastase, conjugates 5-7 did not exhibit a significant
cytotoxic activity (IC50 > 5 µM, Table 2), whereas free PTX
Colombo (Nerviano Medical Sciences) for the plasma stability
experiments.
Keywords: Drug delivery, Elastase, Antitumor agents,
Inflammation, Neutrophils.
inhibited cell proliferation at nanomolar concentrations (IC50
=
35.8 ± 16.7 nM). Interestingly, conjugate 5 displayed a > 250-fold
increased activity (IC50 = 19.6 ± 4.1 nM) upon addition of
elastase (50 nM), and the observed potency was comparable to
References
the one exhibited by free PTX under the same conditions (IC50
=
29.5 ± 7.6 nM). On the other hand, the presence of elastase did
not modify the original cell antiproliferative activity of conjugates
6 and 7. This result indicates not only that the PTX payload is
inactive when it is not released from the targeting vehicle, but
also that the use of the natural amino acid [L]-Val at the linker C-
terminus is crucial for the recognition of the tripeptide sequence
by elastase.
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In conclusion, integrin ligands represent promising vehicles for
the selective release of anticancer drugs at the tumor site.
However, it is still not clear whether different RGD-based ligands
may have different effects on the receptor internalization and
recycling in cancer cells. Upon the observation that the integrin
ligand cyclo(DKP-RGD) is not efficiently internalized by αvβ3-
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the free payload would diffuse in the tumor mass, and act
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