Impact of Polymer-TLR-7/8 Agonist (Adjuvant) Morphology on the Potency and Mechanism of CD8 T Cell Induction

Article abstract of DOI:10.1021/acs.biomac.8b01473

Small molecule Toll-like receptor-7 and -8 agonists (TLR-7/8a) can be used as vaccine adjuvants to induce CD8 T cell immunity but require formulations that prevent systemic toxicity and focus adjuvant activity in lymphoid tissues. Here, we covalently attached TLR-7/8a to polymers of varying composition, chain architecture and hydrodynamic behavior (～300 nm submicrometer particles, ～10 nm micelles and ～4 nm flexible random coils) and evaluated how these parameters of polymer-TLR-7/8a conjugates impact adjuvant activity in vivo. Attachment of TLR-7/8a to any of the polymer compositions resulted in a nearly 10-fold reduction in systemic cytokines (toxicity). Moreover, both lymph node cytokine production and the magnitude of CD8 T cells induced against protein antigen increased with increasing polymer-TLR-7/8a hydrodynamic radius, with the submicrometer particle inducing the highest magnitude responses. Notably, CD8 T cell responses induced by polymer-TLR-7/8a were dependent on CCR2+ monocytes and IL-12, whereas responses by a small molecule TLR-7/8a that unexpectedly persisted in vaccine-site draining lymph nodes (T1/2 = 15 h) had less dependence on monocytes and IL-12 but required Type I IFNs. This study shows how modular properties of synthetic adjuvants can be chemically programmed to alter immunity in vivo through distinct immunological mechanisms.

Full text of DOI:10.1021/acs.biomac.8b01473

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Article
Impact of polymer-TLR-7/8 agonist (adjuvant) morphology
on the potency and mechanism of CD8 T cell induction
Geoffrey M. Lynn, Petr Chytil, Joseph R. Francica, Anna Lagova, Gray Kueberuwa, Andrew S. Ishizuka,
Neeha Zaidi, Ramiro A. Ramirez-Valdez, Nicolas J. Blobel, Faezzah Baharom, Joseph Leal, Amy Q. Wang,
Michael Y. Gerner, Tomáš Etrych, Karel Ulbrich, Leonard W. Seymour, Robert A. Seder, and Richard Laga
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01473 • Publication Date (Web): 04 Jan 2019
Downloaded from http://pubs.acs.org on January 6, 2019
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Impact of polymer-TLR-7/8 agonist (adjuvant)
morphology on the potency and mechanism of CD8
T cell induction
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Geoffrey M. Lynna, Petr Chytilb, Joseph R. Francicaa, Anna Lagovác, Gray Kueberuwac,
Andrew S. Ishizukaa, Neeha Zaidia, Ramiro A. Ramirez-Valdeza, Nicolas J. Blobela,
Faezzah Baharoma, Joseph Leald, Amy Q. Wange, Michael Y. Gernerd, Tomáš Etrychb,
Karel Ulbrichb, Leonard W. Seymourc, Robert A. Sedera, ‡ and Richard Lagab,c, ‡,*
aVaccine Research Center, National Institute of Allergy and Infectious diseases,
National Institutes of Health, 40 Convent Drive, Bethesda, MD 20892, USA
bInstitute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského
nám. 2, 162 06 Prague 6, Czech Republic
cDepartment of Oncology, University of Oxford, Old Road Campus Research Building,
Oxford, OX3 7DQ, United Kingdom
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dDepartment of Immunology, University of Washington, South Lake Union E-411,
750 Republican St., Seattle, WA 98109
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eTherapeutics for Rare and Neglected Diseases, National Center for Advancing
Translational Sciences, 9800 Medical Center Drive, Rockville, MD 20850.
KEYWORDS
Adjuvants, hydrophilic polymers, Toll-like receptor-7 & -8 agonists, polymer
immunostimulants, cellular immunity, vaccine delivery
ABSTRACT
Small molecule Toll-like receptor-7 and -8 agonists (TLR-7/8a) can be used as vaccine
adjuvants to induce CD8 T cell immunity but require formulations that prevent systemic
toxicity and focus adjuvant activity in lymphoid tissues. Here, we covalently attached
TLR-7/8a to polymers of varying composition, chain architecture and hydrodynamic
behavior (~ 300 nm submicron particles, ~ 10 nm micelles and ~ 4 nm flexible random
coils) and evaluated how these parameters of polymer-TLR-7/8a conjugates impact
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adjuvant activity in vivo. Attachment of TLR-7/8a to any of the polymer compositions
resulted in a nearly 10-fold reduction in systemic cytokines (toxicity). Moreover, both
lymph node cytokine production and the magnitude of CD8 T cells induced against
protein antigen increased with increasing polymer-TLR-7/8a hydrodynamic radius, with
the submicron particle inducing the highest magnitude responses. Notably, CD8 T cell
responses induced by polymer-TLR-7/8a were dependent on CCR2+ monocytes and IL-
12, whereas responses by a small molecule TLR-7/8a that unexpectedly persisted in
vaccine-site draining lymph nodes (T1/2 = 15 hours) had less dependence on
monocytes and IL-12 but required Type I IFNs. This study shows how modular
properties of synthetic adjuvants can be chemically programmed to alter immunity in
vivo through distinct immunological mechanisms.
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INTRODUCTION
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While most currently licensed vaccines mediate protection through antibodies 1,
vaccines that generate CD8 T cell immunity are needed for certain infectious diseases
(e.g., Malaria 2and Ebola 3) as well as cancer 4-6. A promising vaccine approach to elicit
CD8 T cell immunity is to combine protein or peptide antigens with specific adjuvants
that activate antigen-presenting cells (APCs) that promote the priming and expansion of
CD8 T cells 7.
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Some of the most studied adjuvants for inducing CD8 T cell immunity include a
structurally diverse class of synthetic and naturally occurring molecules that bind to
specific Toll-like receptors (TLRs) 8. TLR agonists (TLRa) function as adjuvants by
binding to cognate surface (e.g., TLR-1, 2, 4, 5 and 6) or intracellular (e.g., TLR-3, 7, 8
and 9) receptors on antigen-presenting cells (APCs), such as dendritic cells, leading to
a downstream signaling cascade that results in APCs migrating from the periphery to
draining lymph nodes (DLN); upregulating antigen presentation and co-stimulatory
molecule expression; and, producing specific cytokines required for driving T cell
responses to co-administered antigen 8.
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Particular TLRa can be selected for use in vaccines to induce qualitatively distinct
immune responses. Agonists that bind to endosomally localized TLR-7 and -8 (TLR-
7/8), which naturally recognize single stranded RNA from viruses 9, have garnered
interest for use as vaccine adjuvants based on their ability to induce APCs to produce
specific cytokines (i.e. IL-12 and Type I Interferons (IFNs)) that promote CD8 T cell
immunity 10, 11. While a broad range of synthetic TLR-7/8a structurally related to the
nucleotide bases adenine and guanine have been described 12-15, the imidazoquinoline
class, which includes two compounds, Imiquimod (Aldara) and Resiquimod (R848), that
are approved by the FDA for the treatment of skin pathologies 14, 16, are some of the
most studied and promising for use as adjuvants for inducing CD8 T cell immunity 10.
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Despite having favorable immunological characteristics for use as adjuvants for
inducing CD8 T cell immunity, imidazoquinoline-based TLR-7/8a are currently restricted
to topical uses clinically because these low-molecular-weight, amphiphilic compounds
have suboptimal pharmacokinetic (PK) properties for use in parenterally administered
vaccines. For instance, following common routes of vaccination (e.g., intramuscular or
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subcutaneous), unformulated, small molecule TLR-7/8a rapidly enter the blood and
cause systemic immune activation that results in flu-like symptoms in patients 17-20, with
only a limited quantity of the agonist reaching lymph nodes where it is needed for
priming T cells 21-23. Furthermore, free TLR-7/8a may have lower avidity for binding
cognate receptors or result in suboptimal receptor clustering as compared with natural
agonists, which are presented as repeating macromolecular units on pathogen particles
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24
.
A variety of approaches have been developed to improve the pharmacokinetic
properties of TLR-7/8a, and other adjuvants, for use as adjuvants in vaccines. One
strategy is to physically entrap the TLR-7/8a within nano- and micro-sized particles
based on PLGA 25-27, liposomes 28, 29, polypropylene sulfide-based polymersomes 30,
polymer micelles 31 or cross-linked polysaccharides 32. As an alternative to physically
entrapping the adjuvant within particles, which can be limited by relatively low and
variable adjuvant loading, TLR-7/8a can be covalently linked (“conjugated”) to
macromolecular carriers, including proteins33-35, dendrimers 36 and functionalized
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hydrophilic or amphiphilic polymers 37. Potential advantages of conjugating adjuvants
(and/or antigens) to macromolecular carriers is that loading can be controlled by the
number of functional groups used for adjuvant attachment and that the macromolecular
conjugate is a chemically defined, single molecule that permits facile sterile-filtration but
can be chemically programmed for assuming different hydrodynamic properties
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depending on the environment 21, 38-40
.
Prior studies have established that a critical role of adjuvant delivery systems based on
particle emulsions and macromolecular conjugates is to physically restrict adjuvant
distribution to vaccine-site DLN where T cell priming occurs, while at the same time
preventing systemic distribution associated with adjuvant toxicity 21, 41-43. However, it
remains unclear how various characteristics of the delivery platform, such as size and
morphology, influence the efficiency and mechanism of the adjuvant for eliciting T cell
immunity. Accordingly, while nanoparticles with a size range between ~ 20-200 nm in
diameter have been reported to be optimal for promoting T cell immunity based on their
ability to passively traffic to lymph nodes and promote uptake by lymph node resident
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dendritic cells 24, 44, 45, submicron-sized particles (> 200 nm) that reach lymph nodes
through active trafficking by migratory APCs and some passive drainage 46 also induce
robust T cell immunity 21, 47. Moreover, it has not been thoroughly investigated how
various properties of macromolecular carriers, including architecture (e.g., di-block
versus co-polymer) and hydrodynamic behavior (e.g., random coil, micelle or submicron
particle) impact CD8 T cell immunity.
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To evaluate how characteristics of the adjuvant carrier impacts CD8 T cell immunity, we
synthesized polymer-TLR-7/8a conjugates with different chemical compositions and
chain architectures exhibiting distinct hydrodynamic characteristics (random coil,
polymer micelle and submicron particle) and evaluated how these parameters impact
the efficiency and mechanism of the adjuvant for inducing CD8 T cells in vivo. While we
previously evaluated how the density of an R848 analog linked to water-soluble and
temperature-responsive polymer carriers impacts immunogenicity 21, herein we
expanded on this prior work by using a higher potency TLR-7/8a comprising an
imidazoquinoline with a C2-butyl substituent and an N1-xylylamine substituent, referred
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to herein as 2BXy (also known as IMDQ48) linked to polymers with different chain
architectures and hydrodynamic properties.
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The data presented herein provides insights as to how properties of macromolecular
conjugates of TLR-7/8a, including polymer chain composition and hydrodynamic
behavior, can be chemically tuned to alter the magnitude and quality of innate and
adaptive CD8 T cell immunity. Overall, this work has important implications for the
development of vaccines for inducing CD8 T cells for the treatment of infectious
diseases and cancer.
MATERIALS AND METHODS
Chemicals
(RS)-1-Aminopropan-2-ol, 6-aminohexanoic acid (ε-Ahx), 3-aminopropionic acid (β-Ala),
(+)-sodium L-ascorbate, azobisisobutyronitrile (AIBN), 4,4′-azobis(4-cyanovaleric acid)
(ACVA), N-(tert-butoxycarbonyl)-ethylenediamine (Boc-EDA), copper(II) sulfate
pentahydrate, 2-cyano-2-propyl benzodithioate, 4-cyano-4-thiobenzoylsulfanyl
pentanoic acid (CTP), dicyclohexylcarbodiimide (DCC), N,N′-diisopropylethylamine
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(DIPEA), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 4-
(dimethylamino)pyridine (DMAP), N-ethylmaleimide (EtMI), methacrylic acid,
methacryloyl chloride, poly(propylene glycol) triol (Mr 4800), propargylamine, sodium
ascorbate, 4,5-dihydrothiazole-2-thiol (TT), triethylamine, tris[(1-benzyl-1H-1,2,3-triazol-
4-yl)methyl]amine (TBTA) and 2,4,6-trinitrobenzenesulfonic acid solution (1 M in H2O)
were purchased from Sigma-Aldrich, UK. N-(2-Aminoethyl)methacrylamide
hydrochloride was obtained from Polysciences, PA, USA. 2-[(tert-
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Butoxycarbonyl)amino]-3-cyanopropanoic acid dicyclohexylammonium salt (Boc-
PGGly·DCHA) was purchased from Anaspec, CA, USA. Atto-488 NHS-ester fluorescent
label was purchased from ATTO-TEC, Germany. All solvents used in this work were
HPLC grade and purchased from Fisher Scientific, UK. Imiquimod, R848, HEK-Blue™-
hTLR7 cell line, Blasticidin, Zeocin™, Normocin™ and QUANTI-Blue™ alkaline
phosphatase detection reagent were obtained from InvivoGen, CA, USA. Human IL-6
and IP-10 ELISA kits were purchased from R&D Systems, MN, USA.
Synthesis of monomers
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N-(2-Hydroxypropyl)methacrylamide (HPMA) was synthesized by reacting methacryloyl
chloride with (RS)-1-aminopropan-2-ol in dichloromethane in the presence of sodium
carbonate as described in49.
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N-[2-(tert-Butyloxycarbonyl)aminoethyl]methacrylamide (Boc-AEMA) was synthesized
by the reaction of methacryloyl chloride with N-Boc-ethylenediamine in chloroform in
the presence of triethylamine according to 50.
3-(6-Methacrylamidohexanoyl)thiazolidine-2-thione (Ma-ε-Ahx-TT) was synthesized by
the Schotten–Baumann acylation of ε-Ahx with methacryloyl chloride in an aqueous
alkaline medium followed by the reaction of formed 6-methacrylamidohexanoic acid with
TT in tetrahydrofuran in the presence of DCC and DMAP as described in 51.
N-Propargylmethacrylamide (PGMA) was synthesized by reacting methacrylic acid with
propargylamine in dichloromethane (DCM) in the presence of EDC. A mixture of
methacrylic acid (1.567 g, 18.2 mmol) and EDC (4.525 g, 23.6 mmol) was dissolved in
86 mL of DCM and cooled to -18 °C. Propargylamine (1,0 g, 18,2 mmol) and a few
crystals of DMAP were added to the cooled solution and the reaction mixture was
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stirred 2 h at -18 °C and then overnight at r.t. The DCM solution was washed with brine
(3 × 100 mL) and the organic layer was dried over anhydrous MgSO4. After filtration of
MgSO4, the DCM solution was concentrated under reduced pressure and the product
was isolated by crystallization from DCM/hexane/diethyl ether mixture. The yield was
1.23 g (55 %). The purity of the product was checked by reversed-phase HPLC, which
showed a single peak with a retention time of 4.0 min (UV detection at 230 nm).1H-
NMR (300 MHz, DMSO) δppm: 1.85 (s, 3H, -CH3), 3.05 (s, 1H, ≡CH), 3.88 (d, 2H, -
CH2-), 5.37 and 5.68 (s, 1H, =CH2), 8.37 (s, 1H, -NH-).
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tert-Butyl [1-({2-[(2-methacryloyl)amino]ethyl}amino)-1-oxopent-4-yn-2-yl]carbamate
(Boc-PGGly-AEMA) was prepared by the condensation reaction of 2-[(tert-
butoxycarbonyl)amino]pent-4-ynoic acid (Boc-PGGly) with N-(2-
aminoethyl)methacrylamide hydrochloride (AEMA) in dichloromethane in the presence
of DIPEA, EDC and DMAP. Boc-PGGly·DCHA (0.5 g, 1.3 mmol) was dissolved in 5 mL
of aqueous KHSO4 (1.9 mmol) and washed with ethyl acetate (3 × 5 mL). The combined
organic portions were further washed with distilled water (3 × 5 mL) and dried over
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anhydrous MgSO4. After filtration of MgSO4, ethyl acetate was evaporated off under
reduced pressure yielding 0.287 g of the Boc-PGGly in the carboxylic acid form. A
mixture of AEMA (0.244 mg, 1.5 mmol) and DIPEA (516 μL, 3.0 mmol) was suspended
in 5 mL of DCM and added to a cooled solution (-18 °C) of Boc-PGGly (0.287 g, 1.4
mmol) and EDC (0.284 g, 1.5 mmol) in 5 mL of DCM. The reaction mixture was stirred 2
h at -18 °C and then overnight at r.t. After that, the DCM solution was sequentially
washed with aqueous 1 M KHSO4 (3 × 10 mL), 1 M NaCl (2 × 10 mL) and distilled water
(1 × 10 mL). The organic layer was dried over anhydrous MgSO4, followed by filtration
and solvent evaporation. The product was isolated by crystallization from ethyl
acetate/hexane mixture. The yield was 190.2 mg (43.6 %). The purity of the product
was checked by reversed-phase HPLC, which showed a single peak with a retention
time of 7.3 min (UV detection at 230 nm).1H-NMR (300 MHz, methanol) δppm: 1.44 (s,
9H, -C(CH3)3), 1.92 (s, 3H, -CH3), 2.34 (s, 1H, ≡CH), 2.47 - 2.67 (m, 2H, -CH2-C≡),
3.28 and 3.34 (t, 2H, -CH2-NH-), 4.14 (m, 1H, -CH-), 5.36 and 5.71 (s, 1H, =CH2).
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Synthesis of PPG-based macro chain transfer agent (PPG-(CTP)3)
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A mixture of poly(propylene glycol) triol (0.100 g, 54.2 µmol) and EDC (0.102 g, 0.53
mmol) was dissolved in 10 mL of DCM, cooled to -18 °C and added to a cooled solution
(-18 °C) of 4-cyano-4-thiobenzoylsulfanyl pentanoic acid (0.151 g, 0.54 µmol) in 5 mL of
DCM. After that, a few crystals of DMAP were added to the reaction mixture and the
solution was stirred 2 h at -18 °C and then overnight at r.t. The DCM solution was
washed with brine (3 × 30 mL) and the organic layer was dried over anhydrous MgSO4.
After filtration of MgSO4, the DCM solution was evaporated off under reduced pressure,
the oily residue was dissolved in methanol and the crude product was purified by GPC
using the Sephadex™ LH-20 (GE Healthcare Life Sciences, UK) in methanol. Methanol
was removed under reduced pressure yielding 189.6 mg of the oily liquid. The content
of DTB end groups was 0.51 mmol/g corresponding to ~2.6 DTB groups/PPG chain.
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Synthesis of the Toll-like receptor 7/8 agonists
1-(4-[[(5-Azidopentanoyl)amino]methyl]phenyl))-2-butyl-1H-imidazo[4,5-c]quinolin-4-
amine (2BXy-N3) and 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-
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amine (2BXy-NH2) were prepared by the multistep synthesis starting from quinolone -
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2,4-diol as described elsewhere21, 48
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Synthesis of the mannose-derived targeting unit
2-Imino-2-methoxyethyl-1-thiomannose (Man-IME) was prepared by multistep
modification of mannose as described in 52.
Synthesis of polymer precursors
Statistical ter-polymer P[(HPMA)-co-(PGMA)-co-(AEMA)] (PP1) was synthesized by
reacting HPMA, PGMA and Boc-AEMA in the presence of ACVA as an initiator and
CTP as a RAFT agent (Supplementary Scheme S1). A mixture of ACVA (1.3 mg, 4.7
µmol) and CTP (2.6 mg, 9.3 µmol) was dissolved in 0.65 mL of dioxane and added to a
solution of HPMA (250.0 mg, 1.75 mmol), PGMA (12.0 mg, 97.0 µmol) and Boc-AEMA
(22.1 mg, 97.0 µmol) in 1.29 mL of H2O. The reaction mixture was thoroughly bubbled
with Ar and polymerized at 70 °C for 7 h. The polymer was isolated by precipitation to
acetone followed by re-precipitation from methanol to acetone/diethyl ether mixture
(3:1). The yield was 164.8 mg of pink powder. Crystalline NaBH4 (20.8 mg, 0.55 mmol)
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was added to the solution of the polymer (164.8 mg, 5.5 µmol DTB) in 1.8 mL of
methanol and the reaction mixture was shaken for ~1 h until the pink solution become
decolorized. After that, EtMI (13.7 mg, 0.11 mmol) was added to the solution and the
mixture was shaken for next 2 h. The low-molecular-weight impurities were removed
from the polymer solution by GPC using Sephadex™ LH-20 in methanol. Methanol was
removed under reduced pressure; the residue was dissolved in 2.0 mL of 3M
HCl/methanol mixture and allowed to incubate overnight. The solvent was evaporated to
dryness; the polymer was re-dissolved in methanol and precipitated to diethyl ether
yielding 148.2 mg of white powder. The molecular weight characteristics of the polymer
precursor PP1 are stated in Supplementary Table S1.
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Statistical co-polymer P[(HPMA)-co-(PGGly-AEMA)] (PP2) was synthesized by
copolymerizing HPMA and Boc-PGGly-AEMA in the presence of ACVA as an initiator
and CTP as a RAFT agent (Supplementary Scheme S2). A mixture of ACVA (1.3 mg,
4.7 µmol) and CTP (2.6 mg, 9.3 µmol) was dissolved in 0.61 mL of dioxane and added
to a solution of HPMA (250.0 mg, 1.75 mmol) and Boc-PGGly-AEMA (29.7 mg, 91.9
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µmol) in 1.23 mL of H2O. The reaction mixture was thoroughly bubbled with Ar and
polymerized at 70 °C for 7 h. The polymer was isolated by precipitation to acetone
followed by re-precipitation from methanol to acetone/diethyl ether mixture(3:1). The
yield was 176.9 mg of pink powder. Crystalline NaBH4 (22.3 mg, 0.59 mmol) was added
to the solution of the polymer (176.9 mg, 5.9 µmol DTB) in 1.8 mL of methanol and the
reaction mixture was shaken for ~1 h until the pink solution become decolorized. After
that, EtMI (14.8 mg, 0.12 mmol) was added to the solution and the mixture was shaken
for 2 h. The low-molecular-weight impurities were removed from the polymer solution by
GPC using Sephadex™ LH-20 in methanol. Methanol was removed under reduced
pressure; the residue was dissolved in 2.0 mL of 3M HCl/methanol mixture and allowed
to incubate overnight. The solvent was evaporated to dryness; the polymer was re-
dissolved in methanol and precipitated to diethyl ether yielding 169.1 mg of white
powder. The molecular weight characteristics of the polymer precursor PP2 are stated
in Supplementary Table S1.
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Di-block co-polymer P[(HPMA)-co-(PGMA)]-b-P[(HPMA)-co-(AEMA)] (PP3) was
produced in two synthetic steps. First, the P[(HPMA)-co-(PGMA)] block was
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synthesized by copolymerizing HPMA and PGMA using ACVA as an initiator and CTP
as a RAFT agent. Next, the initial block was used as a macro-RAFT agent and
subjected to a chain extension polymerization with HPMA and Boc-AEMA in the
presence of ACVA (Supplementary Scheme S3). A mixture of ACVA (7.3 mg, 25.9
µmol) and CTP (14.5 mg, 51.7 µmol) was dissolved in 2.59 mL of dioxane and added to
a solution of HPMA (1.0 g, 6.98 mmol) and PGMA (95.6 mg, 0.78 mmol) in 5.17 mL of
H2O. The reaction mixture was thoroughly bubbled with Ar and polymerized at 70 °C for
7 h. The polymer was isolated by precipitation to acetone followed by re-precipitation
from methanol to acetone/diethyl ether mixture (3:1). The yield was 550.0 mg of
P[(HPMA)-co-(PGMA)] in the form of pink powder. The molecular weight characteristics
of the polymer precursor were: Mw = 18 000 g/mol, Mn = 15 200 g/mol and Mw/Mn =
1.19. The content of DTB end groups was 0.65 mmol/g corresponding to ~0.99 DTB
groups/polymer chain. Next, a mixture of HPMA (63.1 mg, 0.44 mmol), Boc-AEMA (11.2
mg, 0.49 µmol), P[(HPMA)-co-(PGMA)] (50.0 mg, 3.26 µmol DTB groups) and ACVA
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(0.18 mg, 0.65 µmol) were dissolved in 0.33 mL of dioxane/H2O mixture (2:1),
thoroughly bubbled with Ar and polymerized at 70 °C for 16 h. The diblock copolymer
was isolated by precipitation to acetone yielding 74.7 mg of pale pink powder.
Crystalline NaBH4 (6.2 mg, 0.17 mmol) was added to the solution of the polymer (74.7
mg, 1.7 µmol DTB groups) in 1.0 mL of methanol and the reaction mixture was shaken
for ~1 h until the pink solution become decolorized. After that, EtMI (4.1 mg, 33.0 µmol)
was added to the solution and the mixture was shaken for next 2 h. The low-molecular-
weight impurities were removed from the polymer by GPC using Sephadex™ LH-20 in
methanol. Methanol was removed under reduced pressure; the residue was dissolved in
1.0 mL of 3M HCl/methanol mixture and allowed to incubate overnight. The solvent was
evaporated to dryness, the polymer was re-dissolved in methanol and precipitated to
diethyl ether yielding 65.7 mg of white powder. The molecular weight characteristics of
the polymer precursor PP3 are stated in Supplementary Table S1.
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Multiblock co-polymer PPG-b-P[(HPMA)-co-(PGMA)-co-(AEMA)] (PP4) was
synthesized by copolymerizing HPMA, PGMA and Boc-AEMA in the presence of
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azobisisobutyronitrile (AIBN) as an initiator and PPG-(CTP)3 (for the synthesis see
above) as a trifunctional macro-RAFT agent. A mixture of HPMA (198.4 mg, 1.39
mmol), PGMA (9.5 mg, 77.0 μmol), Boc-AEMA (17.6 mg, 77.0 µmol), AIBN (0.4 mg,
2.57 µmol) and PPG-(CTP)3 (10.0 mg, 5.13 µmol) were dissolved in 3,08 mL of tert-
butanol, thoroughly bubbled with Ar and allowed to polymerize at 70 °C for 18 h. The
product was isolated by precipitation to diethyl ether and purified by GPC using
Sephadex™ LH-20 in methanol. The purified polymer was precipitated to diethyl ether
yielding 66.1 mg of pink powder. Crystalline NaBH4 (3.8 mg, 101.4 µmol) was added to
the solution of the polymer (66.1 mg, 1.0 µmol DTB) in 661 µL of methanol and the
reaction mixture was shaken for ~1 h until the pink solution become decolorized. After
that, EtMI (2.5 mg, 20.3 µmol) was added to the solution and the mixture was shaken
for 2 h. The low-molecular-weight impurities were removed from the polymer by GPC
using the Sephadex™ LH-20 in methanol. Methanol was removed under reduced
pressure; the residue was dissolved in 1.0 mL of 3M HCl/methanol mixture and allowed
to incubate overnight. The solvent was evaporated to dryness; the polymer was re-
dissolved in methanol and precipitated to diethyl ether yielding 40.0 mg of white powder.
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The molecular weight characteristics of the polymer precursor PP4 are summarized in
Supplementary Table S1.
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Statistical co-polymer p[(HPMA)-co-(Ma-ε-Ahx-TT)] (PP5) was synthesized by
copolymerizing HPMA and Ma-ε-Ahx-TT using the free radical polymerization technique
as described elsewhere 21. Briefly, a mixture of HPMA (9.8 wt%), 2-Methyl-N-[6-oxo-6-
(2-thioxo-thiazolidin-3-yl)-hexyl]-acrylamide (Ma-ε-Ahx-TT) (5.2 wt%) and AIBN (1.5
wt%) were dissolved in DMSO (83.5 wt%) and polymerized at 60°C for 6 hours under
argon atmosphere. The polymer was precipitated from a 1:1 mixture of acetone and
diethyl ether and then dissolved in methanol and precipitated from a 3:1 mixture of
acetone and diethyl ether. The content of TT reactive groups determined by UV-Vis
spectrophotometry was 14.8 mol% (ε305 =10,300 L/mol); the weight- and number-
average molecular weights determined by size exclusion chromatography (SEC) were
Mw = 31,850 g/mol and Mn = 20,330 g/mol, respectively. The molecular weight
characteristics of the polymer precursor PP5 are summarized in Supplementary Table
S1.
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Synthesis of polymer-TLR-7/8a conjugates
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Polymer-TLR-7/8a conjugates, with the exception of conjugate PC13, were synthesized
using azide-alkyne Huisgen cycloaddition to react the polymer-bound propargyl groups
with the azide group-functionalized agonist (2BXy-N3) in the presence of copper catalyst
(for conjugate structures see Figure 1; for reaction schemes see Supplementary
Scheme S1 – S3). 10 mg of polymer precursors PP1 – PP4 were dissolved in the
mixture of TBTA (3.52 µmol; 20 µL of 76.51 mg/mL stock solution in DMF), sodium
ascorbate (3.52 µmol; 20 µL of 68.57 mg/mL stock solution in water) and 2BXy-N3 (0.70
µmol for PC1, PC4, PC7 and PC10, 1.76 µmol for PC2, PC5, PC8 and PC11 and 3.52
µmol for PC3, PC6, PC9 and PC12; corresponding to 8, 20 or 40 µL of 42.69 mg/mL
stock solution in DMF) in a total volume of 100 µL of DMF and 80 µL of water. The
solution was thoroughly bubbled with Ar and mixed with a solution of CuSO4 (3.52 µmol;
20 µL of 28.03 mg/mL stock solution in water bubbled by Ar). The reaction mixture was
briefly bubbled with Ar again and allowed to shake for 24 h at r.t. Afterwards, 1.8 mL of
phosphate saline buffer containing 15 wt% of EDTA was added and the polymer
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product was isolated by gel filtration using Sephadex™ PD-10 columns with water and
as an eluent. The collected polymer fractions were freeze-dried. The molecular weight
characteristics of the polymer-TLR-7/8a conjugates PC1 – PC12 are summarized in
Supplementary Table S1.
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Conjugate PC13 was synthesized by the acylation reaction of the polymer-bound TT
groups (PP5) with amino group-functionalized agonist (2BXy-PEG4-NH2) according to
21
the procedure described in . The molecular weight characteristics of the PC13
conjugate as well as the content of bound 2BXy are summarized in Supplementary
Table S1.
Synthesis of mannose-targeted polymer-TLR-7/8a conjugates
The polymer-TLR-7/8a conjugates with the highest content of TLR-7/8a were also
prepared with a mannose targeting unit to yield targeted polymer conjugates, TPC3,
TPC9 and TPC12, which were produced by the polymer-analogous reaction of the
polymer-bound primary amino groups with an imidoester derivative of mannose (Man-
IME, for conjugate structures see Figure 1; for reaction schemes see Supplementary
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Scheme S1 – S3). 5 mg of polymer conjugates PC3, PC9 and PC12 were dissolved in
100 µL of phosphate saline buffer at pH 8.0 and thoroughly bubbled by Ar. The solution
of Man-IME (4.7 mg, 17.6 µmol - ten molar excess related to amino groups) in 100 µL of
the buffer was added to the polymer solution. The reaction mixture was bubbled by Ar
for 5 min and shaken for 24 h at r.t. Polymer conjugates were purified by gel filtration
using Sephadex™ PD-10 column with water as an eluent and isolated by freeze-drying.
The molecular weight characteristics of the mannose-targeted polymer-TLR-7/8a
conjugates TPC3, TPC9 and TPC12 are summarized in Supplementary Table S1.
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Size-exclusion chromatography (SEC)
The molecular weights and molecular weights distributions of the polymers were
determined by SEC on a HPLC system (Shimadzu VP, Japan) equipped with internal
UV–VIS, and external RI, dual LS and viscometric detectors (Agilent Technologies, CA,
USA). TSK-Gel SuperAW4000 column (6.0 × 150 mm, Tosoh Bioscience, Japan) and
80 % methanol / 20 % sodium acetate buffer (0.3 M, pH 6.5) mixture as a mobile phase
(flow rate 0.6 mL/min) were used in all experiments. A method based on the known total
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injected mass with an assumption of 100 % recovery was used for the calculation of the
refractive index increments (dn/dc) needed for the molecular weights determination from
light scattering data.
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High-performance liquid chromatography (HPLC)
The purity of synthesized monomers and other low-molecular-weight reagents were
determined by liquid chromatography on a HPLC system (Shimadzu Nextera, Japan)
using a reversed-phase column Chromolith RP18-e (4.6 × 100 mm, Merck, Germany),
with a linear gradient (0 − 100 %) of water/acetonitrile mixture containing 0.1% TFA at a
flow rate 1.0 mL/min and a UV-VIS photodiode array detector (Shimadzu, Japan).
Determination of small molecule TLR-7/8a pharmacokinetics using UPLC-MS/MS
UPLC-tandem mass spectrometry (UPLC-MS/MS) methods were developed to
determine 2BXy and R848 concentrations in mouse blood, spleen and lymph node
samples. Mass spectrometric analysis was performed on a Waters Xevo TQ-S triple
quadrupole instrument using electrospray ionization in positive mode with selected
reaction monitoring (SRM). The SRM for 2BXy and R848 was 360.1/241 and 315.2/197,
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respectively. The separation was performed using an Acquity BEH C18 column (50 x
2.1 mm, 1.7 µ) and a Waters Acquity UPLC system with 0.6 mL/min flow rate. The
column temperature was maintained at 60oC. The mobile phase A was 0.1% formic
acid in 10%MeOH/water and the mobile phase B was 0.1% formic acid in acetonitrile.
The calibration standards (0.5 -1000 ng/mL for 2BXy and 0.05-500 ng/mL for R848)
were prepared in the control blood and tissue homogenates from untreated animals. 10
µL blood sample or 40 µL of tissue homogenate was mixed with 200 µL internal
standard in acetonitrile to precipitate proteins in a 96-well plate. 1.0 µL supernatant was
injected for HPLC-MS/MS analysis. Pharmacokinetic parameters were calculated using
WinNonlin V6.4.
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UV-VIS spectrophotometry
The spectrophotometric analyses of the polymers were carried out in quartz glass
cuvettes on a UV-VIS spectrophotometer Shimadzu UV-mini 1240 (Shimadzu, Japan).
The content of dithiobenzoate (DTB) end groups in the polymers were determined at
302 nm in methanol using the molar absorption coefficient ε302 (DTB) = 12,100
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L/mol·cm. The functionality of the polymer (f, the amount of the functional end groups
per one polymer chain) was defined as the ratio between Mn obtained from the SEC
measurement and Mn calculated from the end group analysis. The content of primary
amine groups in the polymers was determined using a standard TNBSA assay. The
determination of the 2BXy ligand content in the polymer-TLR-7/8a conjugates was
performed at 325 nm in methanol using the molar absorption coefficient ε325 (2BXy) =
5,012 L/mol·cm. The content of carbonylthiazolidine-2-thione (TT) reactive groups in the
polymer precursor was determined at 305 nm using the molar absorption coefficient ε305
(TT) = 10,300 L/mol·cm.
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Static light scattering (SLS)
SLS measurements were carried out with an ALV-Goniometer System (Model ALV /
CGS-8F, ALV, Germany) equipped with a 30 mW 632.8 nm He–Ne laser in the angular
range 40–150° in PBS buffer (pH 7.4). The weight-average molecular weight (Mw) and
radius of gyration (Rg) were calculated using the Zimm plot procedure.
Dynamic light scattering (DLS)
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The hydrodynamic radii (RH) of the PPG-based multiblock polymers were measured by
the DLS technique at a scattering angle θ = 173° in PBS buffer (pH 7.4) using a Nano-
ZS instrument (Model ZEN3600, Malvern Instruments, UK) equipped with a 632.8 nm
laser. For the evaluation of the dynamic light scattering data, the DTS(Nano) program
was used. The resulting RH values were arithmetic means of at least three independent
measurements.
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Flow cytometry
Samples were acquired on a modified LSR II flow cytometer (BD). Results were
analyzed using FlowJo version 9.3, Pestle version 1.6.2, and SPICE version 5.22
software (Mario Roederer, Vaccine Research Center, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD).
Animals
C57BL/6 (B6), B6.129S1-Il12btm1Jm/J (IL-12p40 KO), B6.129S2-Ifnar1tm1Agt/Mmjax
(IFNαβR KO) and B6.129S4-Ccr2tm1Ifc/J (CCR2 KO) mice were obtained from The
Jackson Laboratory (Bar Harbor, ME) and maintained at the Vaccine Research Center’s
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Animal Care Facility under pathogen-free conditions. Wild type mice used in this study
were female and between 8 and 12 weeks old at the start of experiments. Both male
and female KO mice were used and were between 6 to 18 weeks old at the start of
experiments.
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Animal protocol
All animal experiments were conducted at the National Institutes of Health (Bethesda,
MD) and were in compliance with the guidelines set by the Association for the
Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and
the Institutional Animal Care and Use Committee (ACUC). All experimental animal
protocols underwent review and were approved by the Vaccine Research Center ACUC
prior to the start of experiments.
Immunizations
Vaccines were prepared in sterile, endotoxin-free (<0.05 EU/mL) PBS (Gibco, Life
Technologies) and administered subcutaneously in a total volume of 50 μL. All
immunogens were certified endotoxin free (<1 EU/mg) by the manufacturer or were
ACS Paragon Plus Environment
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