Re-engineering the Immune Response to Metastatic Cancer: Antibody-Recruiting Small Molecules Targeting the Urokinase Receptor

Article abstract of DOI:10.1002/anie.201510866

Developing selective strategies to treat metastatic cancers remains a significant challenge. Herein, we report the first antibody-recruiting small molecule (ARM) that is capable of recognizing the urokinase-type plasminogen activator receptor (uPAR), a uniquely overexpressed cancer cell-surface marker, and facilitating the immune-mediated destruction of cancer cells. A co-crystal structure of the ARM-U2/uPAR complex was obtained, representing the first crystal structure of uPAR complexed with a non-peptide ligand. Finally, we demonstrated that ARM-U2 substantially suppresses tumor growth in vivo with no evidence of weight loss, unlike the standard-of-care agent doxorubicin. This work underscores the promise of antibody-recruiting molecules as immunotherapeutics for treating cancer.

Full text of DOI:10.1002/anie.201510866

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International Edition: DOI: 10.1002/anie.201510866
German Edition: DOI: 10.1002/ange.201510866
Antitumor Agents
Re-engineering the Immune Response to Metastatic Cancer:
Antibody-Recruiting Small Molecules Targeting the Urokinase
Receptor
Anthony F. Rullo, Kelly J. Fitzgerald, Viswanathan Muthusamy, Min Liu, Cai Yuan,
Mingdong Huang, Minsup Kim, Art E. Cho, and David A. Spiegel*
Abstract: Developing selective strategies to treat metastatic
cancers remains a significant challenge. Herein, we report the
first antibody-recruiting small molecule (ARM) that is capable
of recognizing the urokinase-type plasminogen activator
receptor (uPAR), a uniquely overexpressed cancer cell-surface
marker, and facilitating the immune-mediated destruction of
cancer cells. A co-crystal structure of the ARM-U2/uPAR
complex was obtained, representing the first crystal structure of
uPAR complexed with a non-peptide ligand. Finally, we
demonstrated that ARM-U2 substantially suppresses tumor
growth in vivo with no evidence of weight loss, unlike the
standard-of-care agent doxorubicin. This work underscores the
promise of antibody-recruiting molecules as immunotherapeu-
tics for treating cancer.
Antibody-recruiting molecules (ARMs) are bifunctional
molecules capable of delivering endogenous antibodies to
disease-causing entities, leading to their destruction and/or
clearance by the immune system. Our group and others have
previously developed ARMs that target various cancers and
infectious agents. These novel immunotherapies have the
potential both to complement protein-based agents while
overcoming their challenges, such as poor oral bioavailability,
high molecular weights, and immunogenicity.[5]
Our group previously reported an ARM capable of
targeting uPAR-expressing cancer cells for immune-mediated
cell death.[5] Although effective in vitro, the reported con-
struct (termed ARM-U1) contained the uPA protein at its
target binding terminus (TBT), imparting many of the
limitations of biologics. We therefore hypothesized that the
therapeutic potential of this agent could be significantly
improved upon by replacing the uPA protein with a high-
affinity uPAR-binding small molecule (Figure 1A).
Herein, we report the design, synthesis, and in vitro and
in vivo evaluation of a second-generation, low-molecular-
weight (< 1000 amu) ARM derivative termed ARM-U2. In
place of uPA, ARM-U2 incorporates a restructured analogue
of IPR-803, a uPAR inhibitor identified previously using
a virtual screen, into its TBT.[6] ARM-U2 targets the uPA
binding site on uPAR with low nanomolar affinity, induces
immune-mediated phagocytosis and cytotoxicity of uPAR-
expressing cells in culture, and also inhibits tumor progression
in vivo, possessing comparable efficacy to the standard-of-
care agent doxorubicin but without the substantial weight loss
associated with doxorubicin treatment. We also report a co-
crystal structure of the ARM-U2/uPAR complex, which to
our knowledge represents the first uPAR/small-molecule
inhibitor complex to appear in the literature. This work
underscores the promise of small-molecule immunothera-
peutics for treating patients with uPAR-expressing metastatic
cancers.
T
umor metastasis involves the invasion of cancer cells into
surrounding tissues, a process often accelerated by cell-
surface proteases. One such protease, the urokinase-type
plasminogen activator (uPA), breaks down extracellular
matrix proteins and activates migration-inducing signal
cascades upon binding to the urokinase-type plasminogen
activator receptor (uPAR).[1] Both uPA and uPAR expression
are substantially upregulated in invasive cancer cells com-
pared to healthy cells or benign tumors.[2] In clinical settings,
uPAR levels have been shown to correlate with metastatic
potential and poor clinical outcomes.[1a] As such, uPAR has
gained recognition as a promising target for treating meta-
static cancers from diverse tissues of origin, including breast,
colon, stomach, and bladder.[3,4]
[*] Dr. A. F. Rullo, Dr. V. Muthusamy, Prof. Dr. D. A. Spiegel
Department of Chemistry, Yale University
225 Prospect Street, New Haven, CT 06511 (USA)
E-mail: david.spiegel@yale.edu
K. J. Fitzgerald, Prof. Dr. D. A. Spiegel
Department of Pharmacology, Yale School of Medicine
333 Cedar Street, New Haven, CT 06520 (USA)
In designing ARM-U2, we first sought to identify a site in
the IPR-803 scaffold at which to affix a linker connecting the
TBT to a 2,4-dinitrophenyl (DNP) antibody-binding terminus
(ABT). To this end, we performed computationally assisted
docking experiments,[7] which suggested that functionaliza-
tion of the IPR-803 core at position 2 of ring C with
triethylene glycol derived PEG-3 linker 9 would be sufficient
to drive solvent exposure of the ABT (1; Figure 1B). Also,
computational studies performed previously and in our
laboratory suggested that electrostatic interactions between
R53 and the carboxylate of IPR-803 are critical for driving
M. Liu, Dr. C. Yuan, Prof. Dr. M. Huang
State Key Laboratory of Structural Chemistry
Fujian Institute of Research on the Structure of Matter
Chinese Academy of Sciences
155 Yang Qiao West Road, Fuzhou, Fujian 350002 (China)
M. Kim, Prof. Dr. A. E. Cho
Department of Bioinformatics, Korea University
2511 Sejong-ro, Sejong, 339-700 (Korea)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201510866.
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Figure 1. A) The reported antibody-recruiting small molecule (ARM) approach to targeting metastatic cancer cells that over-express uPAR. An
ARM binds uPAR-expressing cancer cells, followed by the recruitment of anti-DNP antibodies to the cell surface, which in turn mediate a range of
effector cell functions, including antibody-dependent cellular phagocytosis (ADCP) and cytotoxicity (ADCC). The bifunctional ARM-U2 is
composed of an antibody-binding terminus (ABT), a linker region, and a uPAR-target-binding terminus (TBT). B) The docking of IPR-803-
substituted derivative 1 to the crystal structure of uPAR. The ABT region remains solvent-exposed (black arrow) while the small molecule TBT is
engaged in binding uPAR. Proposed interactions with residues R53 and K50 are indicated. C) The chemical structure of derivative 1 and derivative
2 (ARM-U2); the latter was predicted to display high-affinity uPAR binding.
ligand affinity.[8] We therefore hypothesized that repositioning
this carboxylate function closer to R53, and/or incorporating
additional acidic sites into the ligand, would improve its
overall binding affinity. To test this, we performed further
docking studies with model derivatives differing in the
number or position of negatively charged substituents
within ring D. We explored biscarboxylate 15, extended
monocarboxylate 16, and the mono- and bissulfonate deriv-
atives 17 and 2, respectively (Figure 2). Compound 15 was
predicted to bind uPAR with comparable affinity to 1,
whereas the other derivatives were calculated to have
significantly stronger docking scores, likely both because of
stronger electrostatic contacts and additional bonding inter-
actions with the hotspot residues R53 and K50 (Table 1,
Figure S1).
U2 derivatives for further evaluation (Figure 2). These were
synthesized in a divergent manner starting from isoxazole 3
(Figure 2, Scheme S2).[8b] Thus, exposure of 3 to a diverse set
of commercially available functionalized anilines under Lewis
acid catalysis at ambient temperature led to the chemo-
selective displacement of the 4-bromo substituent to afford
variously substituted intermediates 4–8 in high yields. Treat-
ment of 4–8 with amine base and (S)-3-piperidinecarboxylate
derivatives 9–12, containing linkers of various lengths and
antibody-binding motifs, led to a second displacement process
of the bromine atom in the 2 position. Ester deprotection
where appropriate yielded products 1, 2, and 13–20 in good to
excellent yields.[8b]
We then evaluated the binding of compounds 1, 2, and 13–
20 to uPAR using a competition ELISA protocol (Table 1).
We first observed that the KI and Kd values determined for
1 were in close agreement with the reported 200 nm affinity of
Informed both by these computational results and pub-
lished SAR studies,[8] we prepared several candidate ARM-
Figure 2. Chemical synthesis of the ARM-U candidate derivatives employed in this study.
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Table 1: Docking scores and in vitro binding affinities calculated using a compet-
itive ELISA binding assay for selected ARM-U candidate derivatives.
ARM-U2 was also capable of eliciting antibody-
dependent cellular cytotoxicity (ADCC), which was
suppressed in the presence of competitors (Fig-
ure S4). Taken together, these results support that
ARM-U2 is capable of inducing both ADCP and
ADCC in a manner dependent on binding anti-DNP
antibodies and cell-surface uPAR.
To further assay the effector cell activation by
ARM-U2, we investigated the release of pro-inflam-
matory cytokines by U937 monocytes in the context
of ADCP assays. Thus, cellular supernatants were
isolated from ADCP assay conditions and evaluated
using a multi-inflammatory cytokine ELISA proto-
col.
Higher levels of IL-8 secretion were observed in
assays containing 50 nm or 1 mm ARM-U2 compared
to control samples lacking ARM-U2 (Figure 3B).
We believe that the slight decrease in IL-8 release at
1 mm versus 50 nm of ARM-U2 is another manifes-
tation of the prozone effect, and coincides well with
Entry Compound R1
R2
R3
R4
H
Docking
KI[a]
[nm]
[kcalmolÀ1
]
1
2
3
4
1
H
H
H
SO3H
CO2H
CO2H
NH-Gly-OH
H
H
H
H
SO3H
À12.175
640Æ180
408Æ22
240Æ39
12Æ1
15
16
2
CO2H À11.124
H
H
À36.809
À64.655
[a] Values calculated from triplicate IC50 measurements using a competitive binding
ELISA as described in the Supporting Information. Inhibitory constants calculated
from the IC50 values generated using the Cheng–Prusoff equation as described in the
Supporting Information.
IPR-803 for uPAR (see also Figure S2).[8a] This result
supports computational predictions that linker ABT installa-
tion at position 2 of ring C would minimally perturb uPAR
binding affinity. ARM-U candidates (1, 2, and 13–20) were
also found to bind uPAR competitively with uPA possessing
affinities ranging from low double- to triple-digit nanomolar
values (Table 1, Table S1, and Figure S2). Although the
absolute magnitudes of the computational docking energies
did not correlate with experimental binding affinities, the
highest ranked compound, bissulfonate 2, was found to
possess the highest uPAR binding affinity with a KI value of
12 nm (Table 1). These binding results validated our hypoth-
esis that repositioning and/or increasing the negative charge
on the D ring of ARM-U2 derivatives could afford significant
increases in uPAR binding affinity.
Figure 3. Demonstration of the in vitro efficacy and potency of ARM-
U2 (2) in immune effector cell assays using A172 glioblastoma cells as
targets. A) Antibody-dependent cellular phagocytosis (ADCP) of A172
glioblastoma cells in the presence of anti-DNP antibodies (133 nm) at
the indicated concentrations of ARM-U2 (2) and competitor. Studies
were conducted at both 378C and 48C and in the presence or absence
of either exogenous uPAR or derivative 20, as indicated. B) ARM-U2
induces the release of the inflammatory cytokine IL-8 from U937 cells
in ADCP assays at concentrations of both 50 nm and 1 mm as shown.
We next evaluated the ability of 1 and 2 (henceforth
termed “ARM-U2”) to induce responses from immune
effector cells. We measured the antibody-dependent cellular
phagocytosis (ADCP) of uPAR-expressing A172 glioblas-
toma cells by IFNg-activated U937 monocytes using a two-
color flow-cytometry procedure. Both 1 and ARM-U2
exhibited concentration-dependent phagocytosis with max-
imum efficacy observed at approximately 1 mm and 100 nm,
respectively (Figure 3A, Figure S4). The bell-shaped, auto-
inhibitory dose-response curve observed at ARM-U2 con-
centrations greater than 100 nm has been termed the “pro-
zone” effect, and is expected for agents that function through
ternary complexes, as has been described in detail previ-
ously.[9] When present at high concentrations, unbound
bifunctional agents compete with both antibody and cell-
surface interactions in the ternary complex, driving the
equilibrium to constituent binary complexes, thus preventing
phagocytosis.
the concentration dependence observed in the ADCP experi-
ments shown in Figure 3A. These results are consistent with
previous reports demonstrating that IL-8 is among the first
cytokines released by monocytes and macrophages during the
innate immune response, and serves as a downstream indica-
tor of antibody-dependent cell-surface clustering and activa-
tion of CD64 (FcgRI).[10] Also, the apparent absence of other
cytokines in these experiments likely results from our
exclusive use of CD64-expressing monocytic effector cells.
To gain insight into the ARM-U2 binding mode, we
obtained a co-crystal structure of ARM-U2 bound to the
uPAR soluble extracellular domain (suPAR).[11] These studies
revealed that ARM-U2 binds in a deep hydrophobic pocket
within the uPA binding site on uPAR, and that the anthra-
quinone core (rings A–C) is engaged in hydrophobic inter-
actions with residues L55, L123, L150, L168, V125, and A255
(Figure S7, Table S2). The two D ring sulfonates in ARM-U2
are positioned within 2.8 and 3.5 Š of R53 and T66,
The dependence of ADCP on the presence of cell-surface
uPAR was next assessed through competition experiments.
Competition with soluble uPAR (suPAR) or 20, an ARM-U
derivative lacking the DNP function, substantially suppressed
ARM-U2-mediated phagocytosis (Figures 3A, S4, and S6).
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respectively, suggesting the presence of close-range electro-
static and perhaps hydrogen-bonding contacts (Figure 4A,B).
These results suggest that enhanced polar contacts between
the sulfonates present in ARM-U2 and hot-spot residues on
uPAR are responsible for high-affinity binding.
Figure 4. A,B) X-ray crystal structure of ARM-U2 bound to the uPA
binding site of suPAR. Key interactions between the TBT sulfonates
and the uPA binding-site residues R53 and T66 (distances of 3.5 and
2.8, respectively) are indicated with dotted red lines. In contrast to
computational predictions, residue K50 is 9.1 Š away from the TBT,
outside the hydrogen-bonding range.
Finally, we examined the efficacy of ARM-U2 in a B16-
uPAR mouse allograft model developed in our laboratory.
Mice were first immunized to produce anti-DNP IgG anti-
bodies (Figure S8), then engrafted with B16-uPAR tumors as
detailed in the Supporting Information. Animals received
daily treatment with the indicated agents by intraperitoneal
(IP) injection and were monitored for tumor growth. Mice
treated with ARM-U2 (20 mpk or 100 mpk) showed signifi-
cant decreases in the rate of tumor growth relative to those
injected with PBS only, corresponding to a calculated tumor
growth inhibition (TGI) of approximately 90% (Figure 5A).
This level of TGI was comparable to mice treated with
doxorubicin (Dox, 1 mpk), an antimitotic chemotherapeutic
agent often used as a standard of care in treating metastatic
malignancies, or a combination of ARM-U2 and Dox (20 mpk
and 1 mpk, respectively). Furthermore, following treatment
cessation, tumor regression persisted in all treatment groups
for significantly longer than in the PBS control group
(Figure 5B). Specifically, median survival durations of
between 27 and 37 days were observed for mice treated
with Dox, ARM-U2, or a combination of these agents,
whereas a median survival of 17.5 days was recorded for
animals in the PBS control group. Remarkably, mice treated
with ARM-U2 at either 20 or 100 mpk doses did not lose
Figure 5. A) Tumor growth inhibition in a B16 mouse melanoma
allograft model expressing human uPAR. Tumor growth is measured
over the indicated timeframe upon daily treatment with PBS, doxor-
ubicin at 1 mpk, combined doxorubicin (1 mpk)/ARM-U2 (20 mpk), or
ARM-U2 at 20 mpk and 100 mpk in mice grafted with uPAR-expressing
B16 melanoma cells. B) Kaplan–Meier curves demonstrating the
prolongation of the survival of mice allografted with human uPAR-
positive tumors upon treatment with ARM-U2 at both 20 mpk and
100 mpk, doxorubicin, or doxorubicin/ARM-U2 compared to mice
treated with PBS. Dox=doxorubicin, mpk=milligrams per kilogram,
MS=median survival (in days). C) Measured weight loss associated
with ARM-U2 or doxorubicin treatment.
weight throughout the course of the experiments, in contrast
to significant levels of weight loss observed among Dox-
treated animals (Figure 5C). Although lack of weight loss is
not a definitive indicator of compound safety, these results
represent a positive indication that ARM-U2 might possess
an improved side-effect profile compared to doxorubicin.
In conclusion, we have developed a promising strategy for
cancer immunotherapy that enables both the disruption of the
oncogenic uPA–uPAR interaction and the ability to target
uPAR-expressing cancers for immune-mediated cell death.
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We would like to thank Dr. Julian Vastl, Dr. Tina Wang, and
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of this manuscript. D.A.S. is a paid consultant for Bristol-
Myers Squibb. This work was supported by funding from
Bristol-Myers Squibb (OCR4997.11 to D.A.S.). We also
acknowledge
grants
from
the
NRF
(2013R1A2A2A01067638 and 2012M3C1A6035362 to
A.E.C.).
Keywords: antitumor agents · cell recognition · drug design ·
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