Molecular Pharmaceutics
Article
target side effects. The concept of AgDCs is similar to
antibody-drug conjugates (ADCs); however, AgDCs likely
achieve high affinity specificity by targeting endogenous
autoantibodies or cognate B cell receptors, thus essentially
flipping the mechanism of ADCs.
other chemicals and reagents were of analytical grade and were
used as received without further purification.
The peptides PLP139−151-Alk (N-terminal 4-pentynoic acid
PLP139−151) and PLP139−151-N3 (N-terminal 3-(2-(2-(2-
azidoethoxy)ethoxy)ethoxy)-propanoic acid PLP139−151) have
been synthesized in our laboratory via solid- phase peptide
synthesis on a Wang resin, but larger quantities of each peptide
were obtained from Biomatik Corporation (Wilmington, DE).
In each case, the linker 3-(2-(2-(2-azidoethoxy)ethoxy)-
ethoxy)propanoic acid was purchased from PurePEG, LLC
(San Diego, CA), and 4-pentynoic acid was purchased from
Sigma-Aldrich (St. Louis, MO).
The design of AgDCs requires the selection of an
appropriate antigen or epitope, an immune modulator, and a
linking scheme. With this in mind, a murine experimental
autoimmune encephalomyelitis (EAE) model exhibiting
clinical and histopathological similarities to relapsing−remit-
2
ting multiple sclerosis (RRMS) in humans was selected to
probe the proposed AgDC concept. The model is induced by
administering an adjuvanted “vaccine” containing the antigenic
Synthesis of Azide-Functionalized DEX (DEX-N ). DEX
3
+
epitope PLP139−151. This particular EAE model is the CD4 T
(842 μmol) was added to a flame-dried 250 mL round bottom
flask with a stir bar and septa. Anhydrous MeCN (100 mL)
was added under nitrogen then DIPEA (919 μmol, 1.09 eq.)
via glass syringe. The flask was stirred for 10 min before
azidoacetic acid NHS ester (908 μmol, 1.08 eq.) was added as
a powder. The reaction mixture was stirred overnight at room
temperature before being analyzed by HPLC. Additional
equimolar aliquots of azidoacetic acid NHS ester were added,
followed by stirring for 2 h at room temperature and analyzing
by HPLC, until no additional benefit was observed. The crude
reaction mixture was evaporated under a reduced pressure,
cell-mediated disease with B cell involvement, leading to
primary demyelination of the axonal tracks in the CNS and
10,11
subsequent progressive paralysis of the hind limbs.
Most
importantly, this particular EAE model provided a simplified
system for testing the efficacy of AgDCs wherein the disease
may be induced with a specific peptide, PLP139−151, and
subsequently treated with an AgDC utilizing the same
PLP139−151 epitope.
Dexamethasone (DEX) was selected as the immune
modulator for testing the AgDC concept. Our laboratory
previously screened multiple immune modulators in combina-
then dissolved in 4:6 MeCN/H O, and purified by preparative
2
HPLC. The resulting column fractions were evaporated under
tion with PLP
by using splenocytes derived from EAE
1
39−151
1
2
a reduced pressure to yield the final product as a white powder.
mice. DEX emerged as one of the most potent suppressors of
proinflammatory cytokines when rechallenging EAE spleno-
cytes with PLP139−151 and also showed evidence of inducing
markers of immune tolerance (e.g., upregulation of IL-10).
DEX possesses the necessary potency required (pmol−nmol
1
H NMR (500 MHz, DMSO-d , δ): 7.30 (d, J = 10.2 Hz, 1H),
6
6.23 (dd, J = 10.1, 1.9 Hz, 1H), 6.01 (t, J = 1.7 Hz, 1H), 5.45
(dd, J = 5.0, 1.4 Hz, 1H), 5.23 (s, 1H), 5.17 (d, J = 17.5 Hz,
1H), 4.90 (d, J = 17.6 Hz, 1H), 4.32−4.19 (m, 2H), 4.19−4.11
(m, 1H), 2.88 (dqd, J = 11.5, 7.2, 4.1 Hz, 1H), 2.62 (tdd, J =
13.6, 6.0, 1.7 Hz, 1H), 2.44−2.32 (m, 1H), 2.35−2.28 (m,
1
3,14
range)
because the dose typically used for the antigen-
specific immunotherapy is on the order of milligrams of
15
1
1
3
4
H), 2.22−2.05 (m, 3H), 1.77 (dt, J = 11.2, 5.2 Hz, 1H),
.70−1.58 (m, 1H), 1.56 (dd, J = 13.8, 2.0 Hz, 1H), 1.49 (s,
H), 1.35 (qd, J = 12.9, 5.0 Hz, 1H), 1.08 (ddd, J = 12.1, 8.2,
.1 Hz, 1H), 0.89 (s, 3H), 0.80 (d, J = 7.2 Hz, 3H). C NMR
126 MHz, DMSO, δ): 204.36, 185.30, 168.28, 167.10, 152.77,
antigen per injection. Finally, we rationalized that DEX must
be released in order to escape the typical binding and
internalization pathway associated with antigen recognition
and processing. Thus, we designed an ester linker capable of
releasing DEX from PLP139−151 via hydrolysis with accelerated
release under acidic conditions.
1
3
(
1
4
3
29.03, 124.12, 102.00, 100.61, 90.52, 70.63, 70.34, 69.00,
9.34, 48.09, 48.05, 47.87, 43.33, 35.69, 35.53, 33.67, 33.51,
1.92, 30.28, 27.32, 23.03, 22.98, 16.31, 15.15, 1.19. Expected
In this article, we outline the synthetic strategy, character-
ization, and biological screening of an AgDC containing
PLP139−151 and DEX. These two components were conjugated
utilizing copper-catalyzed azide−alkyne cycloaddition
+
+
[M + H] = 476.2191 Da, observed [M + H] = 476.2067 Da.
Synthesis of PLP139−151-DEX. To a solution of PLP139−151
-
Alk (93.6 μmol) in 120 mL of deionized H O was added DEX-
(
CuAAC) chemistry, and this linkage was characterized by
2
N (189.4 μmol, 2.02 eq.) in 12 mL of EtOH. A premixed
HPLC and mass spectrometry. Additionally, AgDC linker
stability was monitored over a short time frame to confirm the
desired release of DEX. Finally, the efficacy of this AgDC was
demonstrated first in vitro through the use of EAE splenocytes
induced against PLP139−151 and subsequently in vivo through
subcutaneous administration to EAE mice.
3
solution (5.7 mL) of CuSO ·5H O (38.1 μmol) and THPTA
4
2
(
189.8 μmol) in deionized H O was added to the reaction
2
mixture, followed by 7.2 mL of NaAsc (726.9 μmol) in
deionized H O. The reaction was allowed to stir at room
2
temperature for 1 h before an aliquot was removed for
analytical HPLC to monitor the reaction progress. After 3 h,
the reaction mixture was concentrated under a reduced
pressure and purified by preparative HPLC on a Waters
XBridge BEH C , 5 μm, 130 Å stationary phase (19 × 250
MATERIALS AND METHODS
■
DEX, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA),
and sodium ascorbate (NaAsc) were purchased from Sigma-
Aldrich (St. Louis, MO). Copper(II) sulfate pentahydrate
1
8
mm) column using a gradient of MeCN in H O (constant
2
0.05% TFA). The isolated fractions were evaporated under a
reduced pressure to remove residual MeCN, then frozen at
−20 °C, and lyophilized to yield a white powder. Expected [M
(
CuSO ·5H O) was purchased from Acros Organics (Geel,
4 2
Belgium). 2,5-Dioxopyrrolidin-1-yl 2-azidoacetate, DBCO-
PEG -maleimide, DBCO-NH , and MMAE-DBCO were
2
+
3+
+ 2H] = 1039.5224 Da, [M + 3H] = 693.3507 Da;
4
2
2
+
3+
purchased from Click Chemistry Tools, LLC (Scottsdale,
AZ). Doxorubicin hydrochloride was purchased from LC
Laboratories (Woburn, MA). Mertansine (DM1) was
purchased from Carbosynth Limited (Berkshire, UK). All
observed [M + 2H] = 1039.5145 Da, [M + 3H]
693.3478 Da.
=
Analytical Characterization. All HPLC chromatographic
analyses were conducted on a Waters Alliance HPLC system
B
Mol. Pharmaceutics XXXX, XXX, XXX−XXX