receptor ligands
Weterings, J.J.
Citation
Weterings, J. J. (2008, November 27). Synthesis and evaluation of peptide and nucleic acid based Toll-like receptor ligands. Bio-organic Synthesis, Leiden Institute of Chemistry, Faculty of Science, Leiden University. Retrieved from https://hdl.handle.net/1887/13284
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Chapter 4
Toll-like Receptor Ligand-Peptide conjugates to study antigen processing in Dendritic Cells 1
Introduction
Toll-like receptors2 (TLR) are receptors expressed mainly on cells of the innate immune system, such as granulocytes, macrophages and dendritic cells (DCs). Toll like receptors are important in sensing infectious agents through recognition of pathogen-associated molecules, and act as a communicator between innate and adaptive immune responses. The receptors are expressed either on the cell surface or in the endosomal organelles. This compartimentalization of the TLRs correlates with the type of ligands with which they interact. The TLRs expressed on the cell surface bind to extracellular components of the microorganisms, for instance bacterial lipopolysaccharide3 binds to TLR4 and bacterial lipopeptide4 binds to TLR2. In contrast, the TLRs found in the endosomes bind to ligands derived from intracellular molecules of the pathogen, such as unmethylated CpG DNA sequences to TLR95, and ssRNA to TLR7.6 Studies have shown that ligands interacting with the latter type of TLRs are internalised independently of the TLRs7. Upon binding of the ligand to its receptor, a cascade of intracellular signalling events is initiated, which involves docking of different adaptor molecules such as MyD88, and TRAM to the TLR receptors and recruitment of proteins belonging to the IRAK-family, that ultimately culminate in the activation of the NF-B transcription factor and gene transcription leading to production of proinflammatory cytokines2.
Dendritic cells are both initiators and regulators of T cell responses.8 Dendritic cells constantly screen the environment for potential foreign antigens by a variety of mechanisms such as phagocytosis, macropinocytosis, caveolin-mediated or clathrin-dependent endocytosis. The manner of uptake depends on the size and nature of material to be internalized.9,10 As specialized antigen presenting cells, DCs have the capacity to efficiently process exogenous proteins and present the peptides in major histocompatibility complex (MHC) class I molecules, a process known as cross-presentation. In this scenario, exogenously derived antigens are internalised and translocated from the endosomal route into the cytosol, where the proteasome complex processes the antigens. The generated peptides are transported from the cytosol into the endoplasmic reticulum via the peptide transporter TAP11, after which the peptides undergo further trimming and are finally loaded onto MHC class I molecules, which translocate to the cell surface, where the peptide is presented to CD8+ T- cells. The ability of DCs to cross-present peptides on MHC class I to CD8+ T-cells together with the capacity of TLR ligands to deliver maturation signals, have inspired efforts to explore the use of DCs as vaccine vehicle in the fight against infectious diseases and cancer.12,13 It has also been shown that covalent linkage of immunogenic peptides to the TLR9 ligand, CpG DNA or TLR2 ligands, such as Pam3CysSS and Pam3CysSK4 induces a more prominent T-cell response than administration of free TLR2-L or TLR9-L mixed with protein.7, 14-20
H
N DE VSGLE QLESIINFEKL-O H N
O O
O
H
N DE VSGLE QLESIINFEKLA AAAAK -NH2
H2N DEVSGLE QLESIINFEKL- OH
H2N DE VSGLE QLESIINFEKLA AAAAK -NH2
H2N SK KKKDEVS GLEQL ESIINFE KL-OH
H2N SK KKKCDEVSG LEQLE SIINFEK L-OH
H2N SK KKKDEV SGLEQL ESIINFE KLAAA AAK-NH2
HN DEV SGLEQ LESIINFEKL-OH O
1
2
3
4
5
6
7
8 N
O O
O
Figure 4.1. Structures of compounds 1-29
CCATGACGTTCCTGACGT 5'
O 3'
P O S
O-
O N NH
O
O O
P O
S O- O
HO N
NH O
O
O P S -O
O
S H
CCATGACGTTCCTGACGT 5'
O 3'
P O S
O-
O N NH
O
O O
P O
S O- O
HO N
NH O
O
HO
CCATGAGG CTTCTTGAT 5'
O 3'
P O S
O-
O N
O P O
S O- O
HO N
NH O
O
O P S -O
O
SH
CCATGAGG CTTCTTGAT 5'
O 3'
P O S
O-
O N
O P O
S O- O
HO N
NH O
O
HO O
O O
N NH
O
O
P S
O-
T
O O O
N N
NH2
O
P S
O-
C
O O O
N
P S
O-
A
O O O
N
P S
O-
G
N
N N
NH2
N
N NH
O
NH2
9 10
11 12
N N
N NH
N NH
O
O
NH2 NH2
O O O O
CCATGACGTTCCTGACGT 5'
O 3'
P O S
O-
O N NH
O
O O
P O
S O- O
HO N
NH O
O
O P S -O
O
CCATGAG GCTTCTTGAT 5'
O 3'
P O S
O-
O N
O P O
S O- O
HO N
NH O
O
O P S -O
O
13
15
N
N NH
O
NH2
H
N DEVSG LEQLE SIINFEK L-OH S
N O
O CCATGACGTTCCTGACGT
5'
O 3'
P O S
O-
O N NH
O
O O
P O
S O- O
HO N
NH O
O
O P S -O
O 14
H
N DEVSG LEQLE SIINFEK LAAAA AK-NH2 S
N O
O
S
N O
O
DEV SGLEQ LESIINFEKL-OH O
O
H N O
N
O
O NH
O N N F B
F -+
O
O
NH2+ SO3- S O3- H2N
O N
O O
16
17
HN DEV SGLEQ LESIINFE O
O HN
NH
NH O
OH O O
O
NH2+ SO3- SO3- H2N
20 HN DEV SGLEQ LESIINFEKL
O NH
S
O
S KKKK
N
O
O NH
O N N F B
F -
+
H2N
19 O
O
NH2+ SO3- S O3- H2N
18
COO- COO H
COO- 2Li+
NH
N O
O
Na+
A -10254 OH
6
5 5
6
5 6
CCATGACGTTCCTGACGT 5'
O 3'
P O S
O-
O N NH
O
O O
P O
S O- O
HO
N NH
O
O
O P S -O
O
CCATGAGG CTTCTTGAT 5'
O 3'
P O S
O-
O N
O P O
S O- O
HO
N NH
O
O
O P S -O
O 21
22
N
N NH
O
NH2 H
N DEVSGL EQLESIINFE S
N O
O
S N O
O
DEVS GLEQL ESIINFE O HN
NH
H N O
OH O O
O
NH2+ SO3- SO3-
H2N
O NH
HN O
O H O O
O
NH2+ S O3- SO3-
H2N
HN
COOH
CO OH
HN O
O
6
5
6
5
Figure 4.1. continued
O C15H31
O O C15H31
O S
N H
OH
O
*
C15H31 O
O C15H31
O O C15H31
O S
N H
O
*
C15H31 O
HN SKKK KDEVSGL EQLESIINFEKL -OH
O C15H31
O O C15H31
O S
N H
O
*
C15H31 O
HN SKKK KDEVSGL EQLESIINFEKLA AAAA K-NH2
O C15H31
O O C15H31
O S
N H
O
*
C15H31 O
HN SKKK K O C15H31
O O C15H31
O S
N H
O
*
C15H31 O
HN SKKK KCDEV SGLEQ LESIINFEKL-O H
HN
O O
HN DEVSG LEQLE SIINFEK L-OH S
N
O
O NH
O N N F B
F -
+ 23
26
27 25
29 O C15H31
O O C15H31
O S
N H
O
*
C15H31 O
HN SKKK K-NH2
24
O
O
NH2+ SO3- S O3- H2N
COO H
NH
N O
O O C15H31
O O C15H31
O S
NH O
*
C15H31 O
H N SKKK K
HN
O O H
N DEVSG LEQLE SIINFEK L-OH S
28
6
5
Figure 4.1. continued
To explore the mode of action of TLR-L antigen conjugates, a set of well-defined conjugates was designed, composed of peptides containing the model antigen ovalbumin CD8+ cytotoxic T-lymphocyte (CTL) epitope (SIINFEKL) chemically linked to either the TLR9- ligand CpG DNA or the TLR2 ligand Pam3CysSK4. Both constructs were either fluorescently labeled or unmodified (Figure 4.1, compounds 13-15, 21-22, 25-29). The suitability of the conjugates to study the uptake, intracellular routing and processing of the peptide antigen derived from the synthetic covalent vaccines was evaluated in vitro and in vivo.
Results and Discussion
CCA TGA CG TTCCTGA CGT 5'
O 3'
P O S
O-
O N NH
O
O O
P O
S O- O
OH N
NH O
O
O P S -O
O 31
S S
OH
DMTO S S O
O O
NH
CCA TGA CG TTCCTGA CGT 5'
O 3'
P O S
O-
O N NH
O
O O
P O
S O- O
OH N
NH O
O
O P S O-
O 9
SH 30
S S
O O O
B eaucage reagent
1. TCA 2. repeating cycle a) 5'-DMT-T-amidite, DCI b) Tac2O , 1-methylimidazole
THF/Pyrid ine c) Beaucage reage nt d) TCA
3. A q. NH3 4. Ion excha nge
1. DTT, ace tate b uffer 2. PD- 10 column
CCATGAGG CTTCTTGAT 5'
O 3'
P O S
O-
O N
O P O
S O- O
HO N
NH O
O
O P S -O
O 32
N
N NH
O
NH2
1. DTT, ace tate b uffer 2. PD- 10 column 1. TCA
2. repeating cycle
a) 5'-DMT-G(Tac)-amidite, DCI b) Tac2O , 1-methylimidazole
THF/Pyrid ine c) Beaucage reage nt d) TCA
3. A q. NH3 4. Ion excha nge 30
10 S
S HO
Scheme 4.1 Solid phase synthesis of 3’-thiolated phosphorothioate DNA fragments 9 and 10.
The synthesis of the TLR9-L peptide conjugates (13-15, 21 and 22, Figure 1) is depicted in Scheme 4.3 and entails the chemoselective Michael-type reaction21 of thiolated DNA phosphorothioate (9, 10) with peptide (7, 8) provided with a maleimide moiety (Michael acceptor). This approach requires the availability of N-maleimidated peptides 7 and 8, on one hand, and 3’-thiolated phosphorothioate deoxyoligonucleotides 9 and 10, on the other hand.
Oligonucleotide 9 was prepared according to Scheme 4.1 starting from commercially available CPG support with the 3’-thiol modifier22 30. Elongation was performed using 3’- phosphoramidite derivatives of DMT protected nucleosides 5’-DMT-A(TAC)-OH, 5’-DMT- C(TAC)-OH, 5’-DMT-G(TAC)-OH and 5’-DMT-T-OH under the agency of dicyanoimidazole. After each coupling free 5’-hydroxyls were capped (Tac2O/1- methylimidazole23 in THF/pyridine), followed by sulfurization of the phosphate linkage to the phosphorothioate using Beaucage reagent.24 The 5’-DMT was removed with TCA after which elongation continued. After final DMT removal, cleavage from solid support was achieved by treatment with aqueous ammonia after which disulfide 31 was purified by means of ion
tubing. Concentration and lyophilisation yielded 31 as a white solid in 20% yield. Reactive thiol 9 was obtained by disulfide reduction of 31 using a buffered DTT solution.25,26 DTT was removed using a PD-10 column.27 Oligonucleotide 10 was prepared identical to 9 except that the DNA sequence was different.
O
O O
NH
1. Automated oligonu cleo tide synthesis 2. Aq. NH3 3. ion excha nge
CCATGACGTTCCTGACGT 5'
O 3'
P O S
O-
O N NH
O
O O
P O
S O- O
HO
N NH
O
O
HO 11
33 O
N HN
DMTO O
O 3'
O
O O
NH
1. Automated oligonu cleo tide synthesis 2. Aq. NH3 3. ion excha nge 34
O N
DMTO 3'
CCATGA GGCTTCTTGAT 5'
O 3'
P O S
O-
O N
O P O
S O- O
HO
N NH
O
O
HO 12
N
N NH
O
NH2 N
HN N
O
NH(Tac)
Scheme 4.2. Synthesis of TLR9-L CpG DNA 11 and GpC 12.
CpG DNA 11 was prepared as shown in Scheme 4.2. Oligonucleotide 11 was assembled on commercially available preloaded 5’-DMT-dT-3’-Control Pore Glass 33 by oligonucleotide automated synthesis. Cleavage from solid support by aqueous ammonia, followed by ion exchange yielded fractions containing 11. Collection, concentration, dialysis (1-kDa cut-off), concentration and lyophilisation resulted in purified 11 as a white solid (overall yield 43%).
GpC DNA 12 was prepared identically to CpG DNA but with a different DNA sequence starting from preloaded 5’-dG(Tac)-3’-CPG resin 34.
Next, the synthesis of the target deoxyoligonucleotide-peptide conjugates 13-15 and 21-22 was undertaken (Scheme 4.3). The purified N-maleimidopeptides 7 was coupled to 3’- thiolated CpG-phosphorothioates 9 prepared via automated synthesis (Scheme 4.1). Coupling was performed in a 50 mM phosphate buffer. After shaking for 2 days conjugate 13 was purified by gel filtration (0.15 M TEAA) giving a good overall yield (58%).
CCATGACGTTCCTGA CG T 5 '
O 3 '
P O S
O-
O N NH
O
O O
P O
S O- O
HO
N NH
O
O
O P S -O
O 9
SH H
N DEV SGLEQ LESIINFEKL-OH +
CCA TGA CG TTCCTGA CGT 5'
O 3'
P O S
O-
O N NH
O
O O
P O
S O- O
HO
N NH
O
O
O P S -O
O 1 3
N
H DEVS GLEQL ESIINFE KL-OH S
N O
O
CCATGACGTTCCTGACGT 5'
O 3'
P O S
O-
O N NH
O
O O
P O
S O- O
HO
N NH
O
O
O P S -O
O 21
H
N DEVSG LEQLES IINFE S
N O
O
O HN
NH
H N O
OH O O
O
NH2+ SO3- SO3- H2N
O
O
NH2+ SO3- S O3- H2N
O N
O
O 19
+
1. 300 mM Ca rbonate buffer , pH 8 ACN/H2O 2. RP -HP LC 1. 50 mM Phosphate bu ffe r
pH 7, EDTA He lium Atm.
2. gel filtra tion 7
COO H
COOH 58%
60%
O N
O
O
O
O
6
5
6
5
Scheme 4.3. Synthesis of TLR9-L conjugates 13 and 21.
Conjugates 21 and 22 were prepared by specific labeling of lysine28 (Scheme 4.3) present in the peptide as a part of CTL epitope. Conjugate 13 was dissolved together with an excess of Alexa FL 488 carboxylic acid succinimidyl ester 19 in a 300 mM NaHCO3 buffer (300 mM in 30% ACN/water, pH 8). Alexa FL 488 was chosen, based on the assumption that hydrophilic conjugate 13 combines best with a hydrophilic fluorescent label, resulting in a fluorescent conjugate without internally contradicting properties. The green solution was shaken overnight after which purification of conjugate 21 was performed on RP-HPLC yielding an orange solid in 60% (21) yield. Compounds 14 and 15 were prepared similar as 13, except that 14 is a combination of 9 and 8 (36%), and 15 is obtained after ligation of 10 and 7 (63%).
Fluorescent conjugate 22 was obtained by a nearly identical protocol used for the preparation of 21 combining 15 and 19 (15%).
Fmoc HN
O O
DEVSGL EQLESIINFEKL H
N Fmoc
O
O
DEVSGL EQLESIINFEKL H
N
O
OH O
DEVS GLEQL ESIINFE KL H
N
O
O H O
35
36
1. 20% pip/NMP
2. HCTU, maleimid epropionic acid, DiPEA 3. TFA/TiS/H2O, RP-HP LC
1. 20% pip/NMP 2. Ac2O, DiPEA 3. TFA/TiS /H2O, RP-HPLC
6 7
1. TFA/TiS/H2O
DEVSG LEQLE SIINFEK L H2N
O
1 O H 2. RP-HPLC
HN DEVSGLE QLESIINFE O HN
NH
H N O
O H O O
O
NH2+ S O3- SO3- H2N
O O
O
NH2+ SO3- SO3- H2N
O N O
O 19
20 Fmoc SPPS
Fmoc-Leu -S-PHB resin
CO OH
COOH
1. 300 mM NaHCO3 30% ACN/H2O, 20 2. RP-HPLC FmocHN
Fmoc-Tentagel S RAM
37
N O
O
6
5
6
5
Scheme 4.4. Synthesis of peptides 1-8, 17 and 20.
The peptides 7 and 8 containing the requisite maleimide residue at the N-terminus were prepared as shown in Scheme 4.4. The immobilized, side-chain protected peptide fragment 36 was obtained by automated Fmoc-based solid phase peptide synthesis29 starting from 35.
After Fmoc deprotection using 20% piperidine in NMP the N-terminal free amine was treated with 3-maleimidopropionic acid in the presence of HCTU and DIPEA. The resulting peptides containing the N-terminal maleimide residue were cleaved from the resin with TFA/TIS/H2O, precipitated with diethyl ether and purified by RP-HPLC to give the pure building block 7 in good overall yield (47%). Maleimidated peptide 8 was prepared according to a similar procedure, but starting from Tentagel S RAM resin 37 (18%). Peptides 1-5 were prepared by Fmoc SPPS starting from either Fmoc-Leu S PHB 35 or Tentagel S RAM 37. Labeled construct 20 was obtained by Fmoc deprotection of 36 and subsequent N-terminal acetylation.
After cleavage and purification 6 was dissolved in a 300 mM carbonate buffer and Alexa Fluor carboxylic acid succinimidyl ester 19 was added. Overnight shaking followed by RP- HPLC yielded 20 as an orange solid (22%).
S KKKK CDEVS GLEQL ESIINFE KL HN
Fmoc
38
O C15H31
O O C15H31
S O
NH O H
O
*
C15H31
O 23
O C15H31
O O C15H31
O S
NH O
*
C15H31 O
H
N S KKKK CDEV SGLEQL ESIINFE KL 39
O O O
O
O C15H31
O O C15H31
O S
N
H O
*
C15H31 O
HN S KKKK CDEV SGLEQL ESIINFE KL
25
O H O
O C15H31
O O C15H31
S O
NH O
*
C15H31 O
HN S KKKK HN
O O
HN DEVSGL EQLES IINFEKL -OH S
N
O
O NH
O
N N F B
F -
+
29 N
O
O HN
O
N B N F F
1.TFA/TiS/H2O 2. RP-HPLC
16
PyBOP, DIPEA
1. 50 mM Phospha te buffer , 16 2. RP-HPLC - +
1. 20% pip eridine -NMP
2.
1. 20% Pi p-NMP 2. TFA /TiS/H2O 3. RP-HPLC 4. 50 mM Phospha te
buffer , 16 5. RP-HPLC
H2N S KKKK HN
O O
HN DEVSGL EQLES IINFEKL -OH S
N
O
O NH
O
N N F B
F -
+
17
Tenta gel S PHB
Scheme 4.5: Synthesis of Pam3Cys-conjugate 29 and peptide 17.
The synthesis of unlabeled Pam3Cys containing lipopeptides 24-29 was performed as outlined in Scheme 4.5. Resin bound peptide 38 was synthesized via automated solid phase Fmoc- based peptide synthesis. Pam3Cys-OH 23 was coupled to the N-terminus under the agency of PyBOP and DiPEA30 after N-Fmoc removal using piperidine in NMP. Using TFA/TIS/H2O/m-cresol/EDT as cleavage buffer crude peptide 25 was obtained uneventfully.
RP-HPLC purification over a semi-prep CN-phase HPLC column yielded pure hydrophobic Pam3Cys-peptide conjugate 25. Reaction of the thiol function in 25 with Bodipy fluor-N-(2- aminoethyl)maleimide 16 (50 mM phosphate buffer, 1mM EDTA, 2/1/1 H2O/MeOH/ACN) gave fluorescent conjugate 29 as an orange solid in 9% yield after RP-HPLC. Lipopeptides 25 and 26 were prepared similar as 25 but with a different peptide sequence. Peptides 24 and 27 were synthesized with a similar protocol but the initial resin used is Tentagel S RAM. Labeled peptide 17 was obtained by Fmoc deprotection of 38, cleavage, purification and subsequent labeling (Bodipy FL maleimide 16, 50 mM phosphate buffer, 3/2 H2O/ACN) of the peptide, followed by RP-HPLC. Fluorescent lipopeptide 28 was prepared as described for 29 (Scheme 4.5) using Alexa Fluor 488 C5 maleimide 18 as the reactive dye (8%).
Induction of CD8+ T Cell response by the peptide-TLR-L conjugates
The ability of TLR-L-peptide conjugates 14 and 27 to induce an endogenous T-cell response was investigated by subcutaneous injection of TLRL-conjugated peptides 27 and 14 or free peptide 4 into naïve C57BL/6 mice. After 10 days, the induction of SIINFEKL-specific CD8+ T-cells was analysed. As is shown in Figure 4.2A, the magnitude of the SIINFEKL -specific T-cell response induction by either 27 or 14 was significantly higher than that in mice injected with non-conjugated peptide 4 mixed together with either free CpG 11 or free Pam3CysSK4
24. To address whether the induction of specific T-cells depended on activation of the DCs, conjugate 15, which contains a non-stimulatory oligonucleotide (GpC-DNA 12, Figure 4.1) as shown by its lack of capacity to induce IL-12 production by DCs was injected (Figure 4.2C). Injection of conjugate 15 into naïve mice led to a significantly lower induction of specific CD8+ T-cells than of CpG-DNA conjugated peptide 14, but was still as high as the response obtained after mixing of peptide with the CpG-DNA 11 (Figure 4.2A). Importantly, only when a stimulatory TLRL-conjugate was given the majority of CD8+ T-cells were able to produce interferon- (Figure 4.2B), indicating that signalling via the TLR is essential for the generation of large numbers of functional T-cells in vivo. These results suggest that the enhanced induction of specific T-cells is primarily the result of efficient delivery of the TLR- L-conjugated peptide into the antigen-presenting cell.
Figure 4.2. Robust induction of naïve CD8+-specific T-cells mediated by the TLR-L conjugates 14, 16 and 27. A: Antigen presentation; B: Interferon production; C: Interferon production.
TLR-conjugates activate dendritic cells
Next the ability of the different conjugates to induce maturation of DCs was analysed. As is evident from Table 4.1, increased surface marker expression of CD40, CD86, MHC I, and MHC II was observed in dendritic cells after treatment with the different TLR-L-conjugates to a similar extent as the free TLR-Ls. To confirm the involvement of the TLRs in DC activation, bone-marrow derived dendritic cells (BMDCs) from WT mice, TLR2-deficient mice and TLR9-deficient mice were isolated and stimulated with the different conjugates, followed by phenotypic characterization by staining for different surface markers associated with DC maturation.31 No up-regulation of the cell surface markers CD86 and MHC class II was detected when BMDCs from TLR2-deficient mice or TLR9-deficient mice were stimulated with TLR-L-peptide conjugates 14 (TLR9-L) and 27 (TLR2-L) (Figure 4.3). This impaired up-regulation was not due to a general defect in the maturation signalling pathway, as stimulation with LPS could induce up-regulation of CD86 and MHC II in BMDCs derived from both TLR2-and TLR9-deficient mice to a similar extent as observed for BMDCs derived from wild type mice. Taken together, these results demonstrate that the conjugates are as effective as free TLR ligand in activating the DCs, and show that the expression of the cognate TLR is required for activation of DCs by the TLR-L-peptide conjugates.
Mean Fluorescence Treatment Compound
Nr. CD40 CD86 MHC I MHC II
Untreated Peptide CpG
CpG/peptide mix CpG-conjugate Pam3CysSK4
Pam3CysSK4/peptide mix Pam3Cys-conjugate LPS
* 1 11 11/1
13 24 24/1
26
**
8 8 59 48 71 16 18 14 46
21 22 136 134 132 38 40 35 137
923 ND 3421
ND 3229
ND ND 1348 3572
277 271 512 903 725 327 346 329 576
Table 4.1. Cell surface marker expression after TLR-L-conjugate-induced maturation of dendritic cells. * vehicle, **control E. coli LPS a TLR4 ligand, N.D. = Not Determined.
Figure 4.3. TLR dependent DC activation. A: BMDC from either WT or TLR9 deficient mice were treated with 13 or LPS for 48h. B: BMDC from either WT or TLR2 deficient mice were treated with 26 or LPS for 48h.
Efficient uptake of CpG – and Pam-conjugated antigen peptides by dendritic cells
Having demonstrated that TLR signalling was important for DC activation and priming of T- cells, down-stream cellular mechanisms used by the two types of TLR-L’s with respect to uptake, routing, and cellular processing were compared. Therefore the efficiency of antigen uptake of conjugated versus non-conjugated peptide by DCs was determined. DCs were incubated with either fluorescently labeled Pam3Cys-conjugated peptide 29, or free peptide 17. Introduction of the fluorophore (either Alexa 488 or BODIPY-FL) into the conjugates did
not alter the ability of the conjugates to activate DCs as comparable levels of IL-12 was produced by the fluorescent conjugates and the dark conjugates.
Figure 4.4. Efficient antigen uptake mediated by the TLR-L conjugates. A, confocal images of the dendritic cell line D1 incubated for 15 min with either 29 or 17. B, quantification of MFI of fluorescence inside numbered cells selected from A. C, DCs were incubated with either 20, 21 or 28. D, DCs were incubated with either 21 or 22. E, mice were
As indicated by the increased intensity of fluorescence inside the cells, the Pam3Cys- conjugated peptide 29 was taken up far more efficient than the non-conjugated peptide 17 (Figure 4.4 A). Interestingly, Pam-conjugated peptide 29 was found to accumulate in hot spots whereas a more diffuse pattern was observed in DCs incubated with the peptide alone.
Quantification of the mean fluorescence intensity (MFI) revealed a more than 4-fold higher fluorescence in DCs incubated with the Pam-conjugate 29 compared to DCs incubated with the peptide 17 (Figure 4.4 B). Similarly, CpG-conjugated peptide 21 was internalised more efficiently than the unconjugated peptide 20 by DCs (Figure 4.4 C). In addition, the non- stimulatory GpC-conjugate 15 (MFI 63±7.3) was internalized to similar extent as the stimulatory CpG-conjugate 13 (MFI 72±9.1) (Figure 4.4 D). These comparisons indicate that the fluorescent TLR-L conjugates are taken up much more efficiently by DCs than unconjugated peptides in vitro. To examine the uptake efficiency in vivo, mice were injected with either Alexa 488 Fluor labelled CpG-conjugate 21 or peptide 20 labelled with Alexa 488 Fluor mixed with CpG 11. Three days later draining lymph node cells were stained for the DCs surface marker CD11c. In line with the in vitro results, a significantly higher proportion of CD11c+ cells had taken up CpG-conjugated peptide 21 (2.5%), when compared to the population of DCs that ingested unconjugated peptide 20 (0.3%), or non-injected mice (0.1%;
Figure 4.4 E). A similar tendency was observed when comparing BOPIPY-FL labelled Pam- conjugate 29 with peptide labelled with BODIPY-FL 17 mixed with dark Pam 24 (Figure 4.4 F). Collectively, these results indicate that it is the covalent linking of the peptide to the TLR- L that is responsible for the enhanced uptake by the DCs.
Conjugation of peptide leads to pronounced enhancement of antigen presentation in vitro Having established that the conjugates were taken up much more efficiently than the free peptide harboring the OVA CTL epitope SIINFEKL, the effect ofconjugation of peptide to TLR-L on antigen presentation was addressed next (Figure 4.5).
Figure 4.5. TLR-L conjugates strongly enhance antigen presentation.
DCswere loaded with either the CpG-conjugated peptide 13, Pam-conjugatedpeptide 26, the CpG 11 or Pam3CysSK4 24 mixed with peptide 1, or peptide 1alone (Figure 4.5 A and 4.5 B) before incubation with the peptide-specificT-cell hybridoma B3Z cells that recognize the H- 2Kb, SIINFEKL CTL epitope32. As the concentration of the compounds decreased, antigen recognition was rapidly lost when DCs were incubatedeither with the peptide 1 alone or with the peptide 1 mixed withCpG 11 but not when the CpG-conjugated peptide 13 was used. This indicatesthat the conjugation of peptides to TLR-L enhances antigen presentation(Figure 4.5 A). Likewise, an increased antigen presentation was observedfor the Pam-conjugated peptide 27 (Figure 4.5 B). In this case the differencein antigen presentation between conjugate and non-conjugate was even more prominent. Incubation with a mixture of free TLR-L (Pam3CysSK4 24 or CpG 11) and the peptides 1 resulted in a decreased antigenpresentation by DCs when compared to loading with peptide 1 alone or with conjugated peptide. This might be related to decreased uptake, since it has previously been reported that the endocytotic capacity of DCs declines upon encountering maturation signals.33, 34 To gain insight in the kinetics of antigen presentation, DCswere incubated for various time periods with either TLR-L-conjugatedpeptides 14 and 27, peptide 4 mixed with TLR-L 11 or 24, or peptide 4 alone. As shown in Figure 4.5 C and Figure 4.5 D, it required 24–48 h of
to reach the level of antigen presentation acquired already after 2 h of incubation with the conjugated peptide, as measured by an equal ability to stimulate the peptide-specific B3Z hybridoma T-cells. Thus, conjugation greatly improves the swiftness of presentation of antigen by DCs for stimulation of T-cells. The confocal microscopy results indicated that uptake of theconjugates occurred independently from the expression of therespective TLRs.
Therefore, the impact ofTLR expression upon antigen presentation was evaluated. To this end BMDCsfrom WT, TLR2-/-, and TLR9-/- mice loaded with the conjugates,were incubated in vitro together and subsequently incubatedwith the peptide-specific T-cell hybridoma B3Z cell line. In line with the confocal uptake studies, BMDCs derived from WT or the TLR2- or TLR9-deficient mice strains were recognizedto the same extent (Figure 4.6 A and 4.6 B).
Figure 4.6. Antigen presentation induced by TLR-L conjugates does not require TLR expression but is dependent on receptor-mediated endocytosis. A,B).
Discussion
In this chapter the cellular uptake and trafficking of a set of distinct TLR ligand-antigen conjugates 14 and 27 that ultimatelylead to the induction of an efficient CTL response was evaluated. Strikingly,one single subcutaneous immunization with conjugate in saline induced an impressive systemic expansion of antigen-specific CD8 T-cells (Figure 2). Thus, conjugates consisting of both antigenic peptide and TLR-L resulted in a stronger systemic response than what was observed for mixtures of the individual components. The immunofluorescence analysis revealed that conjugates of both types of TLR ligands were taken up very efficiently comparedwith unconjugated peptides (Figure 3). It should be noted that the fluorescent conjugates 21 and 28 may have slightly differentproperties compared to the unmodified conjugates, which could influence the uptake and function. However, the fluorescent conjugates induced DC maturation to a similar extent as the unmodified conjugates.9 In line with these findings, CpG linked to fluorescein isothiocyanate-labeled ovalbumin protein was recently shown to translocate to LAMP-1-positive endosomal- lysosomal compartments9. Further support of enhanced uptake mediatedby the conjugates was provided from the here described in vivo uptake analysis, which revealed a 6–8-fold increase in uptake of the CpG-conjugatedpeptide by CD11c+ cells and a 2-fold increase in uptake of the Pam-conjugated peptide 28 by CD11c+ cells compared with uptake of non- conjugated peptide 17 (Figure 4B and 4F). The here presented results are in accordance with those of Wagner and co-workers7 who showed that cross-presentation of OVA-linked CpG occurred independently from TLR9 expression but that TLR9 expression nevertheless was essential for activation of theDCs. Both for the Pam3Cys and the CpG constructs it can be concluded that conjugation to the antigenic peptide leads to enhanced MHC-I presentation, when compared to the separate peptide and the TLR-L entities.
Experimental Section
General methods: All chemicals and solvents used in the solid phase peptide synthesis, except of HCTU, Pam3Cys-OH, dithiothreitol and PYBOP were from Biosolve (The Netherlands) and used as received. HCTU was purchased from IRIS Biotech GmbH (Germany) and Pam3Cys-OH bought at Bachem Distribution GmbH (Germany). PYBOP was purchased at Multisynthech GmbH. Fluorescent labels BODIPY-FL N-(2- aminoethyl)maleimide, Alexa Fluor 488 carboxylic acid succinimidyl ester and Alexa Fluor 488 C5 maleimide were purchased at Invitrogen/Molecular Probes. Fmoc-amino acids were from SENN Chemicals or from MultiSynTech GmbH (Germany). Tentagel based resins were bought at Rapp Polymere GmbH (Germany). Dithiothreitol was received from Acros. All chemicals and solvents used in the solid phase DNA synthesis except Beaucage reagent and Control Pore Glass solid support resins were from Biosolve and were used as received. The 3’-Thiol modifier C3 S-S Control Pore Glass used to introduce 3’-thiol modification and Beaucage reagent were purchased at Glen Research. Preloaded 5’-DMT-dT-3’-CPG resin(loading 34 μmol/g) and 5’-DMT-dG(Tac)-3’-CPG resin(loading 0.25 μmol/g) were received from Proligo. All chemicals were used as received. Desalt tubing with a 1kD cut-off for ODN purification was bought at Spectrum. Mass spectra were recorded on a PE/SCIEX API 165 (Perkin-Elmer). Analytical LC/MS was conducted on a JASCO system using an Alltima C18 analytical column (5μm particle size, flow: 1.0 ml/min). Absorbance was measured at 214 and 254 nm. Solvent system: A: 100% water, B: 100% Acetonitrile, C: 1%
TFA. Gradients of B in 10% C were applied over 13.5 or 26 minutes unless stated otherwise.
Purifications were conducted on a BioCAD “Vision” automated HPLC system (PerSeptive Biosystems, inc.), supplied with a semipreparative Alltima C18 column (5μ particle size, running at 4 mL/min). Solvent system: A: 100% water, B: 100% Acetonitrile, C: 1% TFA.
Gradients of B in 10% C were applied over 3 CV unless stated otherwise. The UV absorption was measured with a Varian DMS 200 UV visible spectrophotometer. MALDI-TOF spectra were recorded on a Voyager-DE PRO mass spectrometer (Perseptive Biosystems, inc.).
The solid-phase peptide synthesis was performed on a CS Bio 336 automated instrument (CS Bio, California, USA) applying Fmoc based protocol starting from either preloaded Fmoc- Leu-PHB-Tentagel resin or from Tentagel –RAM resin. The synthesis was performed on a 50 or 250 μmol scale according to established methods29. The consecutive steps performed in each cycle were:
1) Deprotection of the Fmoc-group with 20% piperidine in NMP for 15 min; 2) DMF wash;
3) Coupling of the appropriate amino acid applying a five-fold excess. Generally, the Fmoc
amino acid (0.25 mmol) was dissolved in 0.25 M HCTU in NMP (1 mL), the resulting solution was transferred to the reaction vessel followed by 0.5 ml of 1 M DIPEA in NMP to initiate the coupling. The reaction vessel was then shaken for 45 min. 4) DMF wash; 5) Capping with 0.5 M NMP solution of acetic anhydride in presence of 0.5 mmol DIPEA 6) DMF wash. The Fmoc amino acids applied in the syntheses were: Fmoc-Ala-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu), Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Phe-OH, Fmoc-Ser-OH and Fmoc-Val-OH.
Oligonucleotide synthesis
DMT-based solid-phase phosphorothioate oligonucleotide (ODN)synthesis was performed on an Expedite automated instrument(Perseptive Biosystems) starting from Control Pore Glass support with the 3'-thiol modifier. The syntheses were performed on a 10-μmol scale according to established methods.23 Elongation was performed using 3'-phosphoramidite derivatives of DMT-protected nucleosides 5'-DMT-A(TAC)-OH, 5'-DMT-C(TAC)-OH, 5'- DMT-G(TAC)-OH, and 5'-DMT-T-OH under the agency of dicyanoimidazole. After each coupling, remaining free 5'-hydroxyls were blocked using a capping solution (t- butylphenoxyacetic anhydride (Tac2O)/1-methylimidazole in tetrahydrofuran/pyridine) followed by sulfurization of thephosphite linkage to the phosphorothioate linkage using the Beaucage reagent. Next, the 5'-DMT protecting group was removedby trichloroacetic acid, after which elongation was continued. After final DMT removal the DNA oligomer was cleaved from theresin by 25% ammonium hydroxide solution to give a 3'-disulfidemodified ODN (ODN-SS-propyl-OH). ODNs were purified on a Q-Sepharosecolumn pre-equilibrated with 50 mM NaOAc applying a gradientof 2M NaCl in 50 mMNaOAc. Fractions containing the pure productwere combined and dialyzed three times with Millipore waterusing dialysis tubing with 1-kDa cut-off (Spectrum). Quantification was performed by UV absorbance at 260 nm. Sequences of the ODN prepared in this chapter were CpG; 5’- TCCATGACGTTCCTGACGTT-3'-OH;GpC; 5'-TCCATGAGCTTCCTGATG-3'-OH.
Asp-Glu-Val-Ser-Gly-Leu-Glu-Gln-Leu-Glu-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-OH (1) Peptide synthesis was carried out on a 50 μmol scale using a CS Bio 336 automated instrument applying Fmoc based protocol starting from preloaded Fmoc-Leu-PHB-Tentagel resin 33 (loading 0.25 mmol/g). The resin, after final Fmoc deprotection, was washed with DMF, DCM and dried. A small aliquot of the resin was transferred to a glass tube and treated for 2 hours with a cleavage cocktail TFA/TIS/H2O (95/2.5/2.5). The solution was filtered into cold diethylether and the resin was washed with neat TFA. The diethylether was centrifuged, removed and the precipitate was washed with diethylether. After centrifugation, the ether was removed and the precipitate was air dried and dissolved in 20% HFiP/H2O. LCMS: 10-90%
B, Rt = 6.14 min; ESI-MS: [M+H]+: 2064.4 (calculated 2064.4), [M+H]2+: 1032.6 (calculated 1032.7). MALDI-TOF [M+H]+:2064.1 (calculated 2064.4).
Ser-Lys-Lys-Lys-Lys-Asp-Glu-Val-Ser-Gly-Leu-Glu-Gln-Leu-Glu-Ser-Ile-Ile-Asn-Phe- Glu-Lys-Leu-OH (2) Fmoc-Ser(OtBu)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Asp(OtBu)- Glu(OtBu)-Val-Ser(tBu)-Gly-Leu-Glu(OtBu)-Gln(Trt)-Leu-Glu(OtBu)-Ser(tBu)-Ile-Ile-
Asn(Trt)-Phe-Glu(OtBu)-Lys(Boc)-Leu-Tentagel-S-PHB (34) was prepared using a CS Bio 336 automated instrument applying Fmoc based protocol. A small aliquot of the resin was transferred to a glass tube and treated for 2 hours with a cleavage cocktail TFA/TIS/H2O (95/2.5/2.5). The solution was filtered into cold diethylether and the resin was washed with neat TFA. The diethylether was centrifuged, removed and the precipitate was washed with diethylether. After centrifugation, the ether was removed and the precipitate was air dried and dissolved in tBuOH/ACN/H2O, 3/1/1. LCMS: 10-70% B, Rt = 8.27 min; ESI-MS: [M+H]+: 2664.0 (calculated. 2664.0), [M+H]2+: 1332.4 (calculated 1332.5), [M+H]3+: 888.6 (calculated 888.7).
Ser-Lys-Lys-Lys-Lys-Cys-Asp-Glu-Val-Ser-Gly-Leu-Glu-Gln-Leu-Glu-Ser-Ile-Ile-Asn- Phe-Glu-Lys-Leu-OH (3) Fmoc-Ser(OtBu)-Lys(Boc)-Lys(Boc)-Lys(Boc)-Lys(Boc)- Cys(Trt)-Asp(OtBu)-Glu(OtBu)-Val-Ser(tBu)-Gly-Leu-Glu(OtBu)-Gln(Trt)-Leu-Glu(OtBu)- Ser(tBu)-Ile-Ile-Asn(Trt)-Phe-Glu(OtBu)-Lys(Boc)-Leu-Tentagel-S-PHB was prepared on a CS Bio 336 automated instrument applying fmoc based protocol. After final Fmoc deprotection the resin (25 μmol) was transferred to a glass tube and treated for 2 hours with 4 ml cleavage cocktail TFA/TIS/H2O/EDT (3760/100/40/100). The solution was filtered into cold diethylether and the resin was washed with neat TFA. The diethylether containing the crude product was centrifuged, removed and the precipitate was washed with diethylether.
After centrifugation, the ether was removed and the precipitate was air dried and dissolved in
5 ml tBuOH/H2O/H2O, 3/1/1 (3) Purification by RP-HPLC (Gradient: 10-45% B in C) yielded 1.71 mg (0.62 μmol, 2.5%) of white solid. LCMS: 10-90% B, Rt = 4.72 min; ESI-MS:
[M+H]+: 2767.4 (calculated 2766.5), [M+H]2+: 1384.4 (calculated 1383.8), [M+H]2+: 923.0 (calculated 922.8). MALDI-TOF: [M+H]+:2766.2 (calculated 2766.5). MALDI-TOF:
[M+H]+:2766.2 (calculated 2766.5).
Asp-Glu-Val-Ser-Gly-Leu-Glu-Gln-Leu-Glu-Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu-Ala-Ala- Ala-Ala-Ala-Lys-NH2 (4) Peptide synthesis was carried out on a 50 μmol scale using a CS Bio 336 automated instrument applying fmoc based protocol starting from Tentagel RAM resin 35 (loading 0.25 mmol/g). The resin, after final Fmoc deprotection, was washed with DMF, DCM and dried. A small aliquot of the resin was transferred to a glass tube and treated for 2 hours with a cleavage cocktail TFA/TIS/H2O (95/2.5/2.5). The solution was filtered into cold diethylether and the resin was washed with neat TFA. The diethylether was centrifuged, removed and the precipitate was washed with diethylether. After centrifugation, the ether was removed and the precipitate was air dried and dissolved in tBuOH/ACN/H2O, 3/1/1. LCMS:
10-90% B, Rt = 5.09 min; ESI-MS: [M+H]+: 2546.8 (calculated 2546.8), [M+H]2+: 1274.4 (calculated 1273.9).
Ser-Lys-Lys-Lys-Lys-Asp-Glu-Val-Ser-Gly-Leu-Glu-Gln-Leu-Glu-Ser-Ile-Ile-Asn-Phe- Glu-Lys-Leu-Ala-Ala-Ala-Ala-Ala-Lys-NH2 (5) 350 mg of N-terminal Fmoc deprotected (20% piperidine in NMP) H-Asp(OtBu)-Glu(OtBu)-Val-Ser(tBu)-Gly-Leu-Glu(OtBu)- Gln(Trt)-Leu-Glu(OtBu)-Ser(tBu)-Ile-Ile-Asn(Trt)-Phe-Glu(OtBu)-Lys(Boc)-Leu-Ala-Ala- Ala-Ala-Ala-Lys(Boc)-Tentagel S RAM was elongated with Ser(OtBu)-Lys(Boc)-Lys(Boc)- Lys(Boc)-Lys(Boc) using a CS Bio 336 automated instrument applying the Fmoc based protocol. A small aliquot of the resin was transferred to a glass tube and treated for 2 hours with a cleavage cocktail TFA/TIS/H2O (95/2.5/2.5). The solution was filtered into cold diethylether and the resin was washed with neat TFA. The diethylether was centrifuged, removed and the precipitate was washed with diethylether. After centrifugation, the ether was removed and the precipitate was air dried and dissolved in tBuOH/ACN/H2O, 3/1/1. LCMS:
10-70% B, Rt = 7.10 min; ESI-MS: [M+H]2+: 1573.8 (calculated 1573.4), [M+H]3+: 1049.6, [M+H]4+: 787.2 (calculated 787.2).
