Synthesis and Biological Evaluation of Hapten-Clicked Analogues of The Antigenic Peptide Melan-A/MART-126(27L)-35

Article abstract of DOI:10.1002/cmdc.202000038

A click-chemistry-based approach was implemented to prepare peptidomimetics designed in silico and made from aromatic azides and a propargylated GIGI-mimicking platform derived from the altered Melan-A/MART-1_26(27L)-35 antigenic peptide ELAGIGILTV. The Cu^I-catalyzed Huisgen cycloaddition was carried out on solid support to generate rapidly a first series of peptidomimetics, which were evaluated for their capacity to dock at the interface between the major histocompatibility complex class-I (MHC-I) human leucocyte antigen (HLA)-A2 and T-cell receptors (TCRs). Despite being a weak HLA-A2 ligand, one of these 11 first synthetic compounds bearing a p-nitrobenzyl-triazole side chain was recognized by the receptor proteins of Melan-A/MART-1-specific T-cells. After modification of the N and C termini of this agonist, which was intended to enhance HLA-A2 binding, one of the resulting seven additional compounds triggered significant T-cell responses. Thus, these results highlight the capacity of naturally circulating human TCRs that are specific for the native Melan-A/MART-1_26-35 peptide to cross-react with peptidomimetics bearing organic motifs structurally different from the native central amino acids.

Full text of DOI:10.1002/cmdc.202000038

Accepted Article
Title: Synthesis and biological evaluation of hapten-clicked analogues
of the antigenic peptide Melan-A/MART-126(27L)-35
Authors: Marion Tarbe, John J. Miles, Emily S. J. Edwards, Andrew K.
Sewell, Brian M. Baker, and Stéphane Quideau
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To be cited as: ChemMedChem 10.1002/cmdc.202000038
Link to VoR: http://dx.doi.org/10.1002/cmdc.202000038
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10.1002/cmdc.202000038
ChemMedChem
FULL PAPER
Synthesis and biological evaluation of hapten-clicked analogues
of the antigenic peptide Melan-A/MART-126(27L)-35
Marion Tarbe,[a] John J. Miles,[b] Emily S. J. Edwards,[c]# Kim M. Miles,[c] Andrew K. Sewell,[c] Brian M.
Baker,[d] and Stéphane Quideau*[a]
[a]
Dr M. Tarbe, Prof. S. Quideau (0000-0002-7079-9757),
Univ. Bordeaux, ISM (CNRS-UMR 5255)
351 cours de la Libération, 33405 Talence Cedex, France
& Institut Universitaire de France
1 rue Descartes, 75231 Paris Cedex 05, France
E-mail: stephane.quideau@u-bordeaux.fr
Prof. J. J. Miles (0000-0001-5506-6304),
Australian Institute of Tropical Health and Medicine
James Cook University
Cairns, QLD 4878, Australia
Dr E. S. J. Edwards (0000-0002-0240-4370), Mrs K. M. Miles, Prof. A. K. Sewell (0000-0003-3194-3135),
Division of Infection and Immunity
Cardiff University School of Medicine
Cardiff, CF14 4XN, UK
Prof. B. M. Baker (0000-0002-0864-0964),
Department of Chemistry & Biochemistry
University of Notre Dame
[b]
[c]
[d]
251 Nieuwland Science Hall, Notre Dame, IN 46556, USA
#
Emily S. J. Edwards is currently at the Department of Immunology and Pathology, Central Clinical School, Monash University, Melbourne, Victoria,
Australia.
Supporting information for this article is given via a link at the end of the document.
Abstract: A click chemistry-based approach was implemented to
prepare peptidomimetics designed in silico and made from aromatic
azides and a propargylated GIGI-mimicking platform derived from
the altered Melan-A/MART-126(27L)-35 antigenic peptide ELAGIGILTV.
The Cu(I)-catalyzed Huisgen cycloaddition was carried out on solid
support to generate rapidly a first series of peptidomimetics, which
were evaluated for their capacity to dock at the interface between
the major histocompatibility complex class-I (MHC-I) human
leucocyte antigen (HLA)-A2 and T-cell receptors (TCRs). Despite
being a weak HLA-A2 ligand, one of those 11 first synthetic
(MART) from melanoma patients has raised new prospects in
the development of immunotherapeutic agents to treat this
cancer. Melan-A/MART-1 (hereafter referred to as Melan-A) is a
tissue-specific differentiation antigen expressed in melanocytes
and melanoma.[1] In the late 1990s, Romero and co-workers
identified the peptide segment Melan-A(26-35) (sequence:
EAAGIGILTV, hereafter referred to as EAA) as being an
immunodominant epitope frequently recognized by CD8+ tumor-
infiltrating T- lymphocytes (TIL).[2,3] Indeed, a recent analysis of
the dominant tumor-reactive clonotypes in the blood of an HLA-
A2+ stage IV melanoma patient successfully treated with TIL
therapy found that three out of the five most expanded
clonotypes, accounting for >44% of the total response to tumor
in blood following complete remission, were specific for the EAA
epitope.[4] Moreover, actual therapeutic strategies aiming at
blocking the down-regulation of CD8+ T-cells boost Melan-A
specific T-cell immunity, which is promising for the development
of melanoma immunotherapy.[5]
The EAA antigenic peptide stems from the degradation of
Melan-A antigen. This agonist is then loaded onto the major
histocompatibility class-I (MHC-I) HLA-A2 molecule in the
endoplasmic reticulum, and finally transported to the cell surface
for inspection by T-cell receptors (TCRs).[6] MHC-I proteins bind
peptides adopting an extended or bulged conformation through
the combination of conserved hydrogen bonds with the peptide
main-chain and allele-specific pockets that accommodate some
of the side-chains of peptide. For the HLA-A2 molecule, the key
binding sites are two anchoring pockets that accommodate
hydrophobic amino acid side-chains at P2 and C-terminal
positions.[7] Because the EAA epitope presents a weak binding
affinity to HLA-A2 due to the lack of an optimal residue at P2,
compounds bearing
a
p-nitrobenzyl-triazole side-chain was
recognized by the receptor proteins of Melan-A/MART-1-specific T-
cells. After modifications of the N- and C-termini of this agonist,
which was intended to enhance HLA-A2 binding, one of the resulting
7 additional compounds triggered significant T-cell responses. Thus,
these results highlight the capacity of naturally circulating human
TCRs that are specific for the native Melan-A/MART-126-35 peptide to
cross-react with peptidomimetics bearing organic motifs structurally
different from the native central amino acids.
Introduction
Melanoma is a malignant tumour arising from melanocytes and
causes the majority (75%) of deaths in patients suffering of skin
cancer. Over the past decade, the percentage of persons who
have developed melanoma has considerably increased.
According to the world health organization (WHO), this cancer
disease currently affects 132,000 individuals, and globally each
year incidence is increasing (AICR). The identification of
melanoma-associated antigens recognized by CD8+ T-cells
1
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10.1002/cmdc.202000038
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the sequence of EAA has been optimized replacing the alanine
residue at P2 by a leucine (sequence: ELAGIGILTV, hereafter
referred to as ELA). The ELA peptide was found to be a high-
affinity ligand capable of triggering CD8+ T cell responses
stronger than those triggered by the EAA peptide.[8] However,
further studies have shown that ELA and EAA prime different
TCRs in vivo when used in clinical trials,[9] even though X-ray
crystal structures of both EAA/HLA-A2 and ELA/HLA-A2
complexes revealed no major differences in the global
architecture of the peptides with conservation of the bulged
conformation.[10]
used a click chemistry-based methodology to embed a selection
of organic haptens in the GIGI-mimicking platform of our ELA-
mimetic core structure (Scheme 1b). The copper(I)-catalyzed
azide-alkyne Huisgen 1,3-dipolar cycloaddition (CuAAC)
reaction was chosen for this task, since it offers a convenient
and selective access to 1,4-disubstituted 1,2,3-triazoles and has
found many useful applications in bioconjugate chemistry and
drug discovery research.[16,17] Our selection of structures was
again guided by a simulation of their docking at the TCR/MHC-I
interface. The best performing ELA-mimetics were further
modified to increase their HLA-A2 binding affinity and their
resistance to proteases. Herein, we describe the identification of
a highly heteroclitic HLA-A2 ligand for Melan-A specific CD8+ T-
cells.
Other structural modifications of ELA were also performed in
the aim of enhancing its stability in biological fluids, while
maintaining high HLA-A2 binding affinity and efficacious
immunogenicity.[11] These modifications consisted of replacing
the N- and/or C-terminal regions, which are known to be more
exposed to the action of proteases, by chemically altered or non-
natural amino acids.[12] A few years ago, we opted for a less
classical peptidomimetic approach for the construction of ELA
analogues. Instead of iteratively modifying the terminal ELA
parts bound to the HLA-A2 protein, we focused our efforts on
alterations of the central TCR-contacting GIGI portion by relying
on non-peptidic units, which were either tethered to this
tetrapeptide or used to replace it.[13] These studies demonstrated
the possibility of TCR recognition of haptens with chemical
features drastically different from those of standard amino acids,
while maintaining significant Melan-A specific T-cell recognition.
In particular, a structure-guided rational design led us to prepare
an ELA-mimetic presenting a central unit equipped with an
indole acetic acid side-chain (see 1 in Scheme 1a), which
triggered potent Melan-A specific T-cell responses.[13b] Our X-ray
crystal structure of this ELA-mimetic 1 bound to the HLA-A2
protein[14] revealed that the central indole moiety of 1 is oriented
toward the 2 helix of the HLA-A2 binding groove instead of
bulging out toward the TCR loops as our in silico simulation of
the A6-TCR/1/HLA-A2 ternary complex predicted.[13b,14]
Nevertheless, since 1 has the ability to stimulate Melan-A
specific T-cells, it likely undergoes some conformational
changes when contacting TCRs, as previously observed for
several HLA-A2-binding antigenic peptides.[15]
Results and Discussion
In silico rational design. CD8+ T-cell-mediated immune
responses are initiated by molecular contacts between the TCR
and peptide/MHC-I complex (pMHC-I). It is now well established
that even though antigen specificity is the hallmark of the CD8+ T
cell activation, a given TCR can exhibit a degree of plasticity,
hence allowing it to interact with different pMHC-I ligands.[18] This
cross-reactivity is, in part, the result of conformational
adjustments at the TCR/pMHC-I interface that enable the
formation of ternary complexes. Numerous structural and
biophysical studies have emphasized the structural adaptability
of TCRs and have demonstrated that significant changes
between their unbound and bound states can occur upon
binding to pMHC-I.[19] In this context, Baker and co-workers
have explored the A6-TCR cross-reactivity by replacing the
tyrosine at P5 in the viral Tax peptide sequence with a lysine,
whose side-chain amino group was coupled to indole-3-butyric
acid (i.e., Tax5K-IBA, depicted in orange in Figure 1).[20] The X-
ray crystal structure of the A6-TCR/Tax5K-IBA/HLA-A2 complex
revealed that binding proceeded through changes in the
conformation of the complementarity determining region
3
(CDR3) loops of the TCR (see Figure 1), although the overall
A6-TCR binding orientation was conserved.[20]
a) ELA-mimetic 1
HN
GIGI-mimicking platform
O
O
O
N
H-bAla-Leu-Ala
N
N
H
N
H
Leu-Thr-Val-OH
H
P1 P2 P3
P8 P9 P10
b) Hapten-clicked ELA-mimetics
R
N
N
N
O
O
N
H-bAla-Leu-Ala
N
N
H
N
H
Leu-Thr-Val-OH
H
Scheme 1. a) ELA-mimetic 1 triggers potent Melan-A specific CD8+ T-cell
responses.[13b] b) Novel ELA-mimetics equipped with various clicked haptens
(1R-1,2,3-triazole).
Figure 1. Pymol superimposition of the clicked ELA-mimetic variant 11 (green)
in the Tax5K-IBA (orange) binding interface of the A6-TCR/Tax5K-IBA/HLA-A2
ternary complex (PDB 2GJ6). The orientation of the anchoring residue side-
chains, as well as the overall conformations, are maintained. The clicked
hapten side-chain of 11 bulges out from the HLA-A2 binding groove and is well
The promising results thus obtained with the ELA-mimetic 1
encouraged us to pursue this peptidomimetic approach. We
2
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10.1002/cmdc.202000038
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FULL PAPER
accommodated by the CDR3 loops of the A6-TCR. In the top right corner
frame is depicted the phthalimide moiety with H-bonding interactions predicted
by LigandFit.
synthesis CuAAC reaction of the full peptoid alkyne resin 5 with
various azides R–N3 (Scheme 2). Inclusion of the alkyne was
thus accomplished during the course of the solid phase
synthesis (SPS) via a nucleophilic substitution reaction between
the -bromoacetamide resin 3 and propargylamine. A reductive
amination in the presence of N-Fmoc-glycinal was then carried
out to complete the synthesis of the GIGI-mimicking moiety and
was followed by the introduction of the remaining amino acids to
furnish the propargylated resin 5. The CuAAC reactions were
next performed using pre-synthesized azides R–N3 (see the
Supporting Information for details), copper(I) iodide as catalyst
and N,N-diisopropylethylamine (DIPEA) in a dimethylformamide
(DMF)/tetrahydrofuran (THF) solvent mixture. No ascorbic acid
was used to maintain the copper in its +1 oxidation state.[22a,25]
The organic azides R–N3 selected through our preliminary virtual
screening were all benzylic-type azides (see Scheme 2). After
classical cleavage from the resin using trifluoroacetic acid (TFA),
the resulting clicked ELA-mimetics 7-17 were purified by semi-
preparative reverse-phase HPLC (see the Supporting
Information).
These observations on the structural adaptability of TCRs lent
support to our intent of equipping ELA-mimetics with sterically
demanding clicked organic haptens. We explored the impact of
elongating the central side-chain with a triazole-based system
on the overall conformation of our ELA-mimetics within the HLA-
A2 binding groove, and which orientation these central side-
chains could adopt within the TCR/HLA-A2 interface. Since no
ternary complex structure involving the ELA peptide or
analogues thereof was reported at the time we initiated this
project, we surmised that the overall conformation of ELA would
be similar to those of any antigenic peptide presented by the
HLA-A2 protein with a quasi-complete superimposition of the
anchoring residue side-chains.[13b] We thus decided to rely on
the A6/Tax5K-IBA/HLA-A2 structural data (PDB 2GJ6) to guide
our docking studies. Nonetheless, Sewell and co-workers then
reported the first X-ray crystal structure of ELA/HLA-A2 in
complex with the MEL5-TCR (PDB 3HG1).[21] Remarkably, the
conformation of the ELA peptide engaged with the MEL5-TCR is
very similar to its conformation when only bound to the HLA-A2
protein with a root mean squared deviation of 0.369 Å.[10,21]
Furthermore, this study emphasized that the TRAV12-2 gene,
which encodes the CDR1 and CDR2 loops of the MEL5 TCR,
is also expressed by the A6 TCR. Hence, the CDR1 and
CDR2 loops of the A6-TCR utilize a binding mode that is
identical to that observed for MEL5/ELA/HLA-A2 complex. In
particular, the CDR1 loop residue Gln31 makes conserved
contacts with the peptide positions P1 and P4 (Glu1 and Gly4 for
ELA; Leu1 and Gly4 for Tax5K-IBA). These structural and
binding mode analogies between the MEL5 and A6 TCRs
validated our choice of relying on the A6/Tax5K-IBA/HLA-A2
structure as a model to guide the docking of our ELA-mimetics.
The shape-based docking engine LigandFit, from the
Accelrys Cerius2 software package, was utilized to realize a
virtual screening of the envisaged peptidomimetics. Hence, our
initial GIGI-mimicking platform was retained (Scheme 1) and the
incorporation of organic azides clicked on an alkyne installed
O
O
O
i, ii )
Fmoc
Br
N
L-T-V
N
L-T-V
H
H
Wang resin
2
3
O
O
iii, iv )
N
N
Fmoc-HN
N
H
L-T-V
L-T-V
4
v )
O
O
O
O
Boc-bA-L-A-HN
N
H
R
5
vi )
N
N
N
N
vii )
Boc-bA-L-A-HN
N
H
L-T-V
7-17
O
6
F
OTBDMS
NO2
NO2
O
R–N3
=
O
N
NO2
CH2N3
CH2N3
CH2N3
CH2N3
CH2N3
CH2N3
CH2N3
9’
10’
7’
8’
11’
12’
13’
N
NBoc
onto its N-(2-aminoethyl)glycine unit was viewed as
a
convenient synthesis approach (Schemes 1 and 2), notably
given the wider commercial availability of organic azides as
compared to that of alkynes. Moreover, the replacement of
glutamic acid at P1 by a -alanine was maintained as this
substitution improves the stability of the resulting analogues in
biological fluids.[11a] Besides applying the same checkpoints as
those used for designing the ELA-mimetic 1,[13b] here we also
checked for the presence of potential contacts between the
CDR1 residue Gln31 and the N-terminal part of our in silico
engineered clicked ELA-mimetic variants, as it has been shown
to play a key role in the recognition of the ELA peptide.[21]
CH2N3
CH2N3
CH2N3
16’
CH2N3
14’
15’
17’
Scheme 2. Synthesis of clicked ELA-mimetics 7-17 via a CuAAc reaction on
solid support: i) piperidine/DMF (1:4); ii) bromoacetic acid (0.4 M), N,N’-
diisopropylcarbodiimide (DIC, 2 M), DMF, 5 min, rt; iii) propargylamine, DMSO;
iv) Fmoc-Gly-H, NaBH3CN, 1% AcOH/DMF, 20 h, rt; v) end of SPPS; vi) R–N3,
DIPEA, CuI, THF/DMF (1:1) (TBDMS = tert-butyldimethylsilyl); vii) release
from resin and final deprotection using TFA/TIS/H2O (95:2.5:2.5) (see the
Experimental Section and the Supporting Information for details).
Immunological Evaluation. Peptide antigenicity is influenced
by the affinity of the peptide to the MHC-I protein, and in some
cases, enhancing peptide binding has been shown to enhance
T-cell recognition and immunogenicity.[26] Our clicked ELA-
mimetics 7-17 were thus first evaluated by a flow cytometry-
based MHC-I stabilization assay using the HLA-A2+ human
mutant T2 cell line. The ELA peptide and its clicked analogues
were used at a concentration of 10 M and HLA-A2 binding was
determined by the ability of those compounds to stabilize the
Clicked ELA-mimetics Synthesis. Diverse organic azides were
prepared in solution and clicked on the alkyne of the GIGI-
mimicking platform, which was covalently bound to a solid
support (Scheme 2). Several examples of CuAAC reactions on
solid phase have been reported either to introduce 1,2,3-triazole
moiety in peptoid backbones,[22] to realize cyclodimerization of
peptides,[23] as well as to display various triazole-linked organic
motifs on oligopeptoids.[24] Our strategy was based on a post-
3
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HLA-A2 heavy chain/2-microglobulin complex at the surface of
T2 cells. Binding was quantified by mean fluorescence
intensities (MFI), the values of which were then normalized
relative to the value measured for the ELA peptide (Figure 2). All
of our synthetically modified ELA-mimetics bound with weaker
affinities to the HLA-A2 molecule as compared to that of the ELA
peptide. Although the primary anchoring residues at P2 and P10
of HLA-A2 are conserved, the triazole-based central side-chains
of 7-17 are both longer and bulkier than that of our original ELA-
mimetic 1 (see Scheme 1). These results could be in compliance
with the observations made on the crystal structure of the
1/HLA-A2 binary complex,[14] showing the central indolic side-
chain of 1 oriented toward the 2 helix of the HLA-A2 protein.
Thus, increasing both the length and steric demand of the
central side-chain of our peptidomimetic variants could indeed
affect their HLA-A2 binding affinity. Even if such a rationale is
admittedly simplistic, it finds support in the fact that the binding
affinity decreases when the size and hydrophobicity of the
hapten units increases, the ELA-mimetics 14 to 17 exhibited the
lowest HLA-A2 binding affinity. Therefore, we decided to pursue
our investigations without these peptidomimetics, whose HLA-
A2 binding affinities were more than 5-fold weaker than that of
ELA (i.e., 14-17 with MFI below 20 in Figure 2).
blood mononuclear cells (PBMC) with the native EAA peptide.
The resulting Melan-A-specific T-cell line was subsequently
stimulated with our ELA-mimetics or the native EAA peptide, and
T-cells examined by flow cytometry for expression of the
lysosomal-associated membrane protein (LAMP) CD107a[27] and
for intracellular expression of the cytokine interferon-γ (IFNγ)
(Figure 3, and see the Experimental Section). Two of these ELA-
mimetic variants bearing central benzyl-type haptens (i.e., 9 and
11) induced significant Melan-A specific T-cell responses, but
not to the extent of the native EAA peptide ligand. Interestingly,
the p-nitrobenzyl-bearing compound 11, which was the weakest
HLA-A2 ligand of this selection of ELA-mimetics (see Figure 2),
was capable of triggering the production of CD107a and IFN at
notable levels (see Figure 3). The ELA-mimetic 9, which is
equipped with a p-fluorobenzyl-type hapten and characterized
by a much higher HLA-A2 binding affinity, was found to be a
weaker agonist. In contrast to 11, the other two nitrobenzylated
peptidomimetics 12 and 13 were not markedly recognized by T-
cells, despite their higher HLA-A2 binding affinities (see Figure
2). These results further demonstrate that the level of binding
affinity of a ligand to MHC-I proteins does not presume of its
immunogenic potency. Nevertheless, it would seem that the 1-
(4-nitrobenzyl)-1,2,3-triazole side-chain of 11 confers some
minimum threshold for engaging TCRs, even though it is not a
good ligand for the HLA-A2 molecule. At this stage of our
investigations, we then wondered whether further modifying the
structure of 11 by altering its N- and C-terminal portions, which
are essential for the loading of any antigenic peptide onto MHC-I
proteins and also highly sensitive to the action of exoproteases,
would improve immunogenicity.[11a]
Figure 3. CD107a and IFNγ expressions as measured by intracellular cellular
staining (ICS) and quantified by flow cytometry. Data are presented by mean
fluorescence intensity (MFI) after 10 μM stimulation with a select set of our
ELA-mimetics. Negative control autofluorescence has been subtracted from all
values. Positive control (native EAA peptide) is shown.
Modification of N- and C-terminal portions. Structural and
biophysical studies have highlighted the crucial role of
conserved amino acids at P2 and C-terminal positions in binding
to the cleft of HLA-A2 proteins.[7] Thus, to improve the HLA-A2
binding capacity of our clicked ELA-mimetic 11, as well as its
resistance against proteolysis, we first envisioned to install a
non-natural amino acid that could penetrate more deeply into the
hydrophobic pocket occupied by the leucine at P2. To this aim,
we selected the non-natural L-cyclohexylalanine (Cha, Figure 4),
which was previously used as a substituent of phenylalanine to
improve hydrophobic interactions.[28] Moreover, our crystal
structure of the ELA-mimetic 1 bound to the HLA-A2 protein[14]
revealed an unpredicted conformation of the N-terminal -
alanine disrupting key hydrogen bonds with Lys66 and Glu63 of
the HLA-A2 1 helix. In order to recover these interactions, the
-alanine could potentially be replaced by a phthalimide moiety.
Molecular modeling indicated that this rigid, flat dioxoisoindolic
Figure 2. HLA-A2 binding affinities of the 18 clicked ELA-mimetics as
measured by flow cytometry mean fluorescence intensity (MFI). Data are
presented as percentage of positive control (ELA). Negative control
autofluorescence has been subtracted from all values. 10 μM of compound
was used for binding analysis. ELA-mimetics with binding affinities below the
threshold value indicated by the dashed red line (MFI = 20) were excluded
from further analyses (i.e., 14-17).
The selected ELA-mimetics 7-13 were then all evaluated for
their capacity to stimulate Melan-A-specific T-cell responses in
vitro. Here, an intracellular cytokine stain (ICS) was performed
and T-cell activation was measured by flow cytometry. Briefly, a
T-cell line was generated by stimulating HLA-A2+ peripheral
4
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system can promote the establishment of intermolecular
contacts with the side-chains of α1 helix Lys66 and Glu63
residues (Figure 1).[29] The restoration of hydrogen bonds with
these residues should stabilize binary complexes between the
HLA-A2 molecule and our ELA-mimetics, and their N-terminal
phthalimide unit should further protect them against the action of
exoproteases.
Finally, crystal structures involving the ELA peptide or our
ELA-mimetic 1 have shown that the carbonyl oxygen of the
leucine at P8 is not engaged with any HLA-A2 residue.[10,14,21]
We thus introduced a reduced peptide bond ((CH2-NH))
between this leucine residue and the adjacent threonine at P9.
Such a peptide bond isostere, which provides an additional
degree of conformational flexibility, was viewed as a means to
favor adequate positioning of the C-terminal extremity of our
peptidomimetics. These three types of modifications were
implemented in an iterative manner in order to facilitate the
determination of their specific contribution to the improvement of
the HLA-A2 binding affinity (Figure 4 and Scheme 2). Seven
additional clicked ELA-mimetics 18-24, all bearing the same
central 1-(4-nitrobenzyl)-1,2,3-triazole side-chain, were thus
prepared by conventional solid support synthesis (see the
Experimental Section and the Supporting Information).
relative to those of 18 and 19 (see Figure 2). Nevertheless,
when this reduced peptide bond was introduced in combination
with the Cha or phthalamide residues or even both, the resulting
mimetics 21, 22 and 24 exhibited very good binding affinities,
given the number of structural modifications they feature relative
to the structure of the native EAA antigenic peptide. All together,
these results corroborate our predictions about how to restore
good HLA-A2 binding affinity. Moreover, a higher protease
resistance of these ELA-mimetics should be observed, since
these last modifications were implemented at scissile peptide
bonds.[11a]
Evaluating the immunogenicity of this second series of
clicked ELA-mimetic variants 18-24 showed that the ELA-
mimetic 20 was the most potent inducer of CD107a upregulation
and IFN- secretion. Only two other peptidomimetics of the
series, 18 and 23, managed to induce Melan-A-specific T-cell
responses, but to a much lesser extent (Figure 3). Surprisingly,
once again, the weakest HLA-A2 binder 20 of the series
exhibited the strongest immunogenicity. One possible
explanation could be that the lowest binding affinity is the
consequence of a higher flexibility of the peptidoid backbone in
the HLA-A2 binding groove, which would in turn enable more
extensive conformational adjustments upon TCR engagement.
The TCR could perhaps even pull the weakly binding
peptidomimetic away from the HLA-A2 binding groove for
establishing better contacts with both the peptidomimetic and
the HLA-A2 surface.[10a,15] The more flexible ELA-mimetic 20
could thus be more capable of fitting its central 1-(4-nitrobenzyl)-
1,2,3-triazole side-chain within the TCR CDR3 loops. Even
though the observed T-cell responses are about one-third to
one-half lower than those triggered by the native EAA antigenic
peptide in terms of IFNγ and CD107a secretion, these results
constitute an additional and sound experimental demonstration
of the possibility of replacing natural amino acid side-chains of
antigenic peptides with non-peptidic motifs still capable of
eliciting T-cell responses.
NO2
11 H-bAla-Leu-Ala
Leu-Thr-Val-OH
Leu-Thr-Val-OH
Leu-Thr-Val-OH
18
19
N
N
H-bAla-Cha-Ala
N
O
O
Pht-Leu-Ala
N
N
H
Leu(YCH2NH)Thr-Val-OH
Leu(YCH2NH)Thr-Val-OH
Leu(YCH2NH)Thr-Val-OH
Leu-Thr-Val-OH
20 H-bAla-Leu-Ala
21 H-bAla-Cha-Ala
O
NH
O
22
23
24
Pht-Leu-Ala
Pht-Cha-Ala
Pht-Cha-Ala
HN
Pht
Cha
O
Leu(YCH2NH)Thr-Val-OH
Figure 4. Sequences of the ELA-mimetics 18-24, analogues of the ELA-
mimetic 11 designed for improving HLA-A2 binding affinity.
Conclusions
Gratifyingly, most of the new clicked ELA-mimetics 18-24
exhibited a two- to three-fold improvement in HLA-A2 binding
affinity relative to that of 11, with MFI values reaching over two-
thirds of that of the ELA peptide, except for 20, being the
weakest HLA-A2 binder of this series (Figure 2). In particular,
the introduction of the non-natural amino acid Cha in the
structure of 18 fulfilled our expectations based on the hypothesis
of better occupying the hydrophobic P2 anchoring pocket. The
introduction of a phthalimide moiety in place of the N-terminal -
Ala appeared to be also a suitable replacement as the
corresponding mimetic 19 bound well to the HLA-A2 molecule.
Of course, without any confirmation by X-ray structural analysis,
it is, as yet, unclear whether this increase in binding affinity is
the consequence of H-bonding interactions between the
carbonyls of the phthalimide unit and the side-chains of the HLA-
A2 Lys66 and Glu63. Nonetheless, a previous study reported
that replacing the N-terminal glutamate by its constrained
pyroglutamate lactamic analogue was deleterious for binding to
HLA-A2,[11a] whereas herein the phthalimide moiety appeared to
be a valuable N-terminal building block. The reduced peptide
bond at P8-P9 also seems to be well tolerated, although the
mimetic 20 exhibited a slightly weaker HLA-A2 binding affinity
A computer-aided design of peptidomimetics was based on the
replacement of the central amino acid residues of the Melan-
A/MART-1 ELA antigenic peptide by
a clickable hapten-
presenting platform. This approach enabled the generation of
short series of ELA-mimetics equipped with a 1,4-disubstituted
1,2,3-triazole motif bearing variously substituted aromatic
moieties. Even though none of our first eleven ELA-mimetics
could reach the level of binding affinity to the MHC-I HLA-A2
molecule observed for the ELA peptide, the p-nitrobenzyl-
bearing ELA-mimetic 11 was found to elicit Melan-A-specific T-
cell responses. Further modifications of its N- and C-extremities
aimed at restoring a higher level of HLA-A2 binding affinity led to
seven additional ELA-mimetics. All seven compounds were
expectedly observed to be much better HLA-A2 binders than 11,
but it is the weakest ligand 20 of this series, simply featuring a
reduced leucine-to-threonine amide bond, that triggers the most
significantly enhanced T-cell stimulation. Thus, this work (i)
further demonstrates that the affinity of an antigenic peptide (or
mimetics thereof) to MHC-I molecules does not necessarily
correlate with immunogenicity, and (ii) clearly highlights the
flexibility of TCRs to cross-react with synthetic MHC-I bound
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000038
ChemMedChem
FULL PAPER
purification of peptides were performed on a Varian PrepStar system with
SD-1 Dynamax® pumps, using a Microsorb C18 column (2.14 mm  250
mm, 100 Å pore size, 5 m). The mobile phase was similar as for the
analytic system, unless otherwise notified. A gradient elution (0-40 min:
90% to 50% A) was applied at a flow rate of 20 mL.min-1. Column effluent
was monitored by UV detection at 214 and 254 nm using a Varian UV-
Vis Prostar 325 diode array detector. Flash column chromatography was
carried out under positive pressure using 40-60 m silica gel (Merck) and
the indicated solvents. Evaporations were conducted under reduced
pressure at temperatures less than 40 °C. Further drying of the residues
was accomplished under high vacuum. NMR spectra of organic
molecules were recorded at 300 MHz on a Bruker DPX spectrometer in
the appropriated solvent. Electrospray ionization mass spectrometric low-
and high-resolution data (ESIMS, HRMS) were obtained from the Mass
Spectrometry Laboratory at the European Institute of Chemistry and
Biology (IECB), Pessac, France.
peptidomimetics equipped with central organic motifs having
structurally little in common with native central amino acids. A
recent study demonstrated that a non-biologic T-cell ligand could
protect humanized (HLA-A2+) mice against a lethal challenge
with influenza virus.[30] This D-peptide had almost no sequence
similarity to the wildtype L-peptide sequence and was effective
even though HLA-A2 binding was far weaker than that of our
agonistic ELA-mimetics.[30] A further study has shown that an
altered peptide ligand could induce an improved response to the
HLA-A2-restricted EAAGIGILTV epitope studied here.[31] In
combination, these new developments suggest that it might be
possible to build non-biologic, biostable ligands that induce
improved Melan-A/MART-1 specific T-cell responses. Our study
here demonstrates that the design of peptidomimetic MHC-I
ligands remains
a valid option for the elaboration and
development of synthetic antigens as immunotherapeutic agents.
Peptidomimetic Synthesis. Peptidomimetics were synthesized by
manual solid phase peptide synthesis (SPPS) using Fmoc chemistry on
Wang resin (0.65 mmol.g–1) following standard SPPS protocols. Briefly,
to a solution of N-Fmoc-Val-OH (10 equiv. relative to resin loading) in dry
CH2Cl2 (a few drops of DMF were required to ensure complete
dissolution) under N2 was added diisopropylcarbodiimide (DIC, 5 equiv.).
The reaction mixture was stirred for 20 min at 0°C. In the meantime, the
resin was suspended in dry CH2Cl2 and allowed to swell for 20 min. After
filtration, the N-Fmoc-Val symmetrical anhydride solution and 4-
dimethylaminopyridine (DMAP, 0.1 equiv. relative to the anhydride) were
added, and the resin was shaken for 4 h. After filtration, the resin was
successively washed twice with DMF, CH3OH, CH2Cl2 and again DMF.
The Fmoc protecting group was removed by using twice a solution of
piperidine in DMF (1:4, v/v) for 3 min, then for 7 min. After filtration, the
resin was successively washed as before. The SPPS was continued
using N-Fmoc amino acids (3 equiv.) in the presence of HBTU (3 equiv.)
and DIPEA (5 equiv.) in DMF. Each coupling reaction was performed for
45 min, after which time the resin was washed as before.
Experimental Section
Docking Methodology. All molecular modeling calculations were
performed on a Silicon Graphics Octane workstation using Cerius2 4.7
molecular modeling softwares. First, the three-dimensional structure of
the A6-TCR/Tax5K-IBA/HLA-A2 complex was taken from the PDB file
2GJ6. The Tax5K-IBA peptide ligand and all water molecules were
removed. Hydrogen atoms were added using the Cerius2 templates for
the protein residues. The 18 ELA-mimetic structures were constructed
using Catalyst. Partial charges were assigned using the Gasteiger
method[32] as implemented in Cerius2. A site model based on the Tax5K-
IBA ligand, docked within the protein complex, was identified by
LigandFit. Then, the docking[33] of the 18 ELA-mimetic ligands employed
the following protocol: (a) a Monte Carlo conformational search for
generating a candidate ligand conformation, (b) selection of a ligand
position and orientation based on comparing the shape of the binding site
model with that of the ligand conformation, and (c) evaluation of the
goodness of docking by computing the dock energies using a grid-based
energy calculation. The dock energy was expressed as the sum of the
ligand internal strain energy and the interaction energy of the ligand with
the protein. The position and the orientation of the ligand were optimized
by minimizing the dock energy in respect to rigid body translations and
rotations of the ligand using a steepest descent method. The docked
conformations found were then clustered, and the first 20 were saved. A
superimposition of the highest docked structure (the lowest in terms of
energy) was obtained for each peptidomimetic with plausible
consequence of their higher structural diversity. The parental peptide
structure from the 2GJ6 PDB file enabled us to gain insight into the
consequences of backbone modifications on the interactions with the
protein complex.
Preparation of Resin 5. To the H-AMBA-LTV peptoid construct on Wang
resin (1 g, 0.45 mmol) pre-swelled in DMF (5 mL) was added a 0.4 M
solution of bromoacetic acid (438 mg, 3.15 mmol) in DMF and a 2 M
solution of DIC (562 L, 3.60 mmol) in DMF (see Scheme 2). After 5 min,
the resin was filtrated and washed as described above. Then, a 1 M
solution of propargylamine (309 L, 4.50 mmol) in DMSO was added to
the resin and the reaction was stirred for 1 h. After washing of the resin
and confirmation of the presence of a secondary amine via the chloranil
test, the resin was swollen in a solution of 1% acetic acid (AcOH) in DMF,
then Fmoc-Gly-H (567 mg, 2.7 mmol) followed by NaBH3CN (424 mg,
6.75 mmol) were added. After 5 h, the resin was filtered off and
successively washed with CH3OH, DMF, CH2Cl2 and again DMF. After
removal of the Fmoc-protecting group, the other amino acids were
coupled by following standard SPPS protocols as briefly described above
to furnish the resin 5 (see Scheme 2).
Material and Methods. All reagents were either purchased from Aldrich,
Acros, or Fluka. Amino acids, Wang resin, and N,N,N’,N’-tetramethyl-O-
(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) were
purchased from Novabiochem (Switzerland). All solution phase reactions
were carried out under nitrogen (N2) atmosphere with magnetic stirring.
Tetrahydrofuran (THF), diethyl ether (Et2O) and dichloromethane
(CH2Cl2) were dried through alumina columns. All other organic solvents
were of analytical quality and Milli-Q (Millipore) water was used for
reverse phase (RP) HPLC analyses and purifications. Peptide and
peptidomimetic syntheses were performed manually in a glass reactor
(vide infra). RP-HPLC analyses were performed on a Thermo system
using a Chromolith performance RP-18e column (4.6 × 100 mm, 5 m)
with P1000 XR pumps. The mobile phase was composed of 0.1% (v/v)
TFA-H2O (Solvent A) and 0.1% TFA-CH3CN (Solvent B). A gradient
elution (0-10 min: 100% to 50% A) was applied at a flow rate of 3
mL.min-1. Column effluent was monitored by UV detection at 214 and 254
nm using a Thermo UV 6000 LP diode array detector. Semi-preparative
Preparation of Resins 6 by Click Conjugation. To a suspension of resin 5
in a dry THF-DMF mixture (1:1) was added copper iodide (CuI, 1 mg, 5%
M), a solution of each azides R–N3 (10 equiv., see Scheme 2) in dry
THF-DMF (1:1) and DIPEA (5 equiv.). After stirring overnight, the resins
were washed twice with DMF and CH2Cl2.
Release of ELA-mimetics 7-18 from Resin. Final deprotection and
cleavage from the resins were carried out using
triisopropylsilane (TIS) mixture (95:2.5:2.5, v/v/v) for
a
TFA-H2O-
h. The
4
peptidomimetics were precipitated using cold Et2O and then filtered. The
filtrates were washed with cold Et2O, and the peptidomimetics were
extracted from the residues with H2O-CH3CN mixtures containing 0.1%
TFA. The resulted solutions were frozen and lyophilized to afford the
ELA-mimetics 7-18 as white solids. These crude products were checked
for purity by RP-HPLC and purified by semi-preparative RP-HPLC.
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000038
ChemMedChem
FULL PAPER
Preparation of Resin-Bound ELA-mimetics 20-22 and 24. To the H-TV
dipeptide construct on Wang resin (270 mg, 0.13 mmol) pre-swelled in a
solution of 1% AcOH in DMF was added Fmoc-Leu-H[34] (0.40 mmol) and
NaBH3CN (59 mg, 0.94 mmol). The resin was shaken for 2 h after which
time it was filtered off and successively washed with MeOH, DMF,
CH2Cl2 and again DMF. Coupling with the AMBA residue was then
performed, and the preparation of these resin-bound ELA-mimetics was
continued according to the general procedures described for the
preparation of resins 5 and 6.
fellowship, and E.S.J.E by a studentship from the MRC and
Cardiff University's I3 Interdisciplinary Research Group. Dr
Céline Douat is gratefully acknowledged for her contributions to
the design and synthesis of the peptidomimetics and for her co-
supervisorship on M.T.’s doctoral research studies, Mathew
Clement and Kristin Ladell for their contributions to the
production of T-cell lines and FACS analysis, Prof Linda
Wooldridge (University of Bristol, UK) for her supervision and
advice concerning the T-cell cytokine production experiments,
and Dr Nathalie Marchand-Geneste for her advices in the
realization of the docking modeling procedure.
Incorporation of the Phthalimide Moiety on Resin-Bound ELA-mimetics
19 and 22-24.[35] To the corresponding resin-bound peptidomimetic
intermediates without the terminal -Ala residue and suspended in DMF
was added phthalimide anhydride (5 equiv.) and DIPEA (5 equiv.). After
1 h, each resulting resin was filtered off and washed according to our
standard procedure (vide infra). Each resin was again swollen in DMF,
and HBTU (1.5 equiv.), hydroxybenzotriazole (HOBt, 1.5 equiv.) and
DIPEA (3 equiv.) were successively added. After stirring overnight, the
resins were filtered off and washed according to our standard procedure.
Keywords: immunotherapy • T-cell receptor • antigenic
peptidomimetics • organic haptens • click chemistry
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The authors thank the Servier Laboratories and the Société
Française de Chimie Thérapeutique for financial support and
M.T.’s research assistantship. This work was also supported by
the Biotechnology and Biological Sciences Research Council
(grant BB/H001085/1 to A.K.S.), and by the National Institute of
General Medical Sciences, NIH (grant R35GM118166 to B.M.B).
J.J.M was supported by an NHMRC CDF2 APP1131732
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This article is protected by copyright. All rights reserved.

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10.1002/cmdc.202000038
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Click to fit. A computer-aided construction of a short series of mimetics of the tumor-derived Melan-A/MART-126(27L)-35 antigenic
peptide ELAGIGILTV was achieved by clicking aromatic azido haptens on a propargylated GIGI-mimicking central platform. Out of a
total of only 18 peptidomimetcis thus synthesized, one of the most heavily modified constructs harboring a p-nitrobenzyl moiety
elicited significant Melan-A-specific T-cell responses.
9
This article is protected by copyright. All rights reserved.