Synthesis of a trimeric gp120 epitope mimic conjugated to a T-helper peptide to improve antigenicity

Article abstract of DOI:10.1021/ja1083915

A fully synthetic trivalent mimotope of gp120 conjugated to pan allelic HLA DR binding epitope was prepared using solid-phase peptide synthesis and optimized copper-catalyzed azide-alkyne cycloaddition. The methodology efficiently provides chemically uniform heteromultimeric peptide constructs with enhanced binding, avidity, and specificity toward an established HIV-neutralizing human antibody, MAb b12. The versatile synthetic strategy serves as a powerful platform for the development of synthetic peptides as potential HIV-1 vaccine candidates.

Full text of DOI:10.1021/ja1083915

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pubs.acs.org/JACS
Synthesis of a Trimeric gp120 Epitope Mimic Conjugated to a T-Helper
Peptide To Improve Antigenicity
Joan G. Schellinger,† Lieza M. Danan-Leon,‡ Jessica A. Hoch,† Aemro Kassa,§ Indresh Srivastava,§
David Davis,|| and Jacquelyn Gervay-Hague*,†
†Chemistry Department and ‡Department of Molecular and Cellular Biology, Campus Mass Spectrometry Facility, University of
California at Davis, 1 Shields Avenue, Davis, California 95616, United States
§Novartis Vaccines and Diagnostics, Inc., 45 Sidney Street, 3105, Cambridge, Massachusetts 02139, United States
Department of Virology, Biomedical Primate Research Centre, Rijswijk, The Netherlands
S Supporting Information
b
ABSTRACT: A fully synthetic trivalent mimotope of gp120
conjugated to pan allelic HLA DR binding epitope was
prepared using solid-phase peptide synthesis and optimized
copper-catalyzed azide-alkyne cycloaddition. The metho-
dology efficiently provides chemically uniform heteromulti-
meric peptide constructs with enhanced binding, avidity,
and specificity toward an established HIV-neutralizing hu-
man antibody, MAb b12. The versatile synthetic strategy
serves as a powerful platform for the development of syn-
thetic peptides as potential HIV-1 vaccine candidates.
Figure 1. Design of the trimeric mimotope-PADRE immunogen.
water-soluble on the basis of the hydrophobicity score.20 The
espite the tremendous success brought about by combined
15-mer peptide with the sequence NWPRWWEEFVDKHSS
D
antiretroviral therapy in reducing HIV-1-related mortality
was identified using MAb IgG1 b12-selected phage and gp120
competition.21 The potent, broadly neutralizing anti-HIV MAb
b1222-24 is known to bind to a discontinuous epitope overlap-
ping the CD4 binding site of the HIV-1 envelope surface protein
gp120.9,10 Because gp120 is known to be trimeric on the viral
surface, the design features three copies of the mimotope. In
addition to the stimulation of an antigen-specific B-cell response
with the trimeric mimotope,25 an immunogenic T-helper (TH)
epitope is also included. Conjugation to pan allelic HLA DR bin-
ding epitope (PADRE), a known TH epitope with the sequence
aKXVAAWTLKAAa, is intended to stimulate TH cells for a
sustained antibody immune response (Figure 1).26-29
There are myriad synthetic approaches available for achieving
multivalent presentation of biologically active ligands, including
the multiple antigenic peptide (MAP) system.14 While this
methodology allows elaborate synthetic assembly of multiple
peptides on the core, difficulties in achieving quantitative cou-
plings and incomplete amino acid side-chain deprotection arise
in sterically crowded dendrimer networks, resulting in undefined
structures and heterogeneous products.30 Meijer and co-
workers31 have reported an attractive alternative approach that
uses native chemical ligation (NCL) of peptides derived from
phage technology by rebuilding the phage’s multivalent
architecture using well-designed dendritic wedges as synthetic
scaffolds. While NCL is one of the most powerful tools for
worldwide, an effective and safe HIV-1 vaccine is still needed for
the ultimate eradication of the virus and control of the AIDS pan-
demic.1,2 A promising vaccine strategy centers on the design and
synthesis of antigenic peptides that mimic HIV envelope protein
epitopes.3-7 The expectation is that these so called mimotopes8
would be capable of eliciting an immune response leading to the
production of broadly neutralizing antibodies. HIV envelope
protein has several conserved neutralizing epitopes, which are
defined by human monoclonal antibodies (MAbs). We have
focused our attention on MAb b12, which recognizes a discon-
tinuous conformational epitope of HIV envelope.9,10 While
phage-display technology has proven effective for the selection
of mimotopes, soluble peptide constructs are only weakly immu-
nogenic relative to fusion proteins with the same sequence.6 The
attenuated immunogenicity has been attributed to the intrinsic
flexibility, relatively small size, and limited binding interface of
monomeric peptides.11,12 Moreover, phage-encoded peptides
are displayed in multiple copies as part of a filamentous protein
coat, suggesting that multimeric mimotope constructs may be
required for a potent immune response.3,13-15 Our research
efforts have focused on developing a chemical synthesis platform
for the construction of chemically uniform multimeric mimo-
topes with improved antigenicity as part of a larger program
directed toward vaccine development.
New mimotopes are continually emerging,16-19 but many
of these peptides are predicted to be hydrophobic, limiting
the types of formulations in which they can be used. Our initial
studies have focused on a mimotope that is predicted to be
Received: September 16, 2010
Published: February 22, 2011
r
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Scheme 1. General Scheme for the Construction of Trimeric Mimotope-PADRE Using SPPS and the CuAAC Reaction
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peptide and protein chemical synthesis,32 one major limitation of
this approach is the need for an N-terminal cysteine-containing
peptide. Concerns over the presence of cysteine, which may
create unpredictable conformational changes of the peptide and/
or undesirable conjugates to native peptides, have stimulated the
development of alternative synthetic platforms.33
resulting in a homogeneous reaction medium and higher
reaction rates.50
We initiated the synthesis of the azide-functionalized mimo-
tope and the trialkyne PADRE using solid-phase peptide synth-
esis (SPPS) with conventional Fmoc-tBu orthogonal protection
and a versatile tentagel resin with a hydroxymethylbenzoic acid
(HMBA) linker (Scheme 1). 1-(Mesitylene-2-sulfonyl)-3-nitro-
1,2,4-triazole (MSNT) and 1-methylimidazole (MeIm) were
used as coupling agents to incorporate the first amino acid
residue,51 and this was followed by iterative microwave-assisted
Fmoc deprotection and amide coupling using 1-hydroxybenzo-
triazole (HOBT)/N,N0-diisopropylcarbodiimide (DIC) as acti-
vators.52 To functionalize the PADRE and the mimotope, the
free amino N-termini of the protected peptides on bead were
coupled with a trialkyne PEG linker (1) and an azide PEG linker
(2), respectively, using the same microwave irradiation condi-
tions and coupling agents as employed to give the resin-bound
functionalized peptides. Amino acid protecting groups were
removed by treating the resin with 95% trifluoroacetic acid
(TFA), 2.5% triisopropylsilane (TIPS), and 2.5% H2O. Cleavage
from the resin was accomplished with 1:3 1 M NaOH/dioxane
followed by immediate neutralization using 1 M HCl (Scheme 1).
Desalting was achieved using a Sep-Pak C18 cartridge, providing
the crude peptides, which were subjected to HPLC purifica-
tion to give >90% pure trialkyne-functionalized PADRE (3) and
Our approach utilizes a well-known bioorthogonal strategy,
the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition
(CuAAC) reaction, also called click chemistry.34-39 The non-
native, highly selective, and exergonic reaction of azide and
alkyne functionalities40-42 and the peptidomimetic nature of
the triazole cycloaddition product and its proteolytic stability43-45
make CuAAC attractive. However, this methodology is not
without its limitations, as copper catalysis can lead to unwanted
side reactions, particularly in biological settings.46-48 Never-
theless, our previous success with conjugating the recombinant
antibody fragment scFv (∼25 kDa protein) to form divalent scFv
(di-scFv) led us in this direction. Formation of di-scFv involves
the use of an azide and an alkyne poly(ethylene glycol) (PEG)
linker to functionalize the protein in preparation for liga-
tion via the cycloaddition reaction.49 This methodology also
allows for the construction of heteromultimers, wherein one
arm can be conjugated to a different peptide such as PADRE.
Another advantage of this approach is that the azide- and
alkyne-functionalized peptides have similar solubility profiles,
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Figure 2. Direct binding of construct 6, mimotope, and PADRE to (a) MAb b12 and (b) MAb 48d, as shown by SPR. Sensorgram comparisons show
enhanced binding, valency effect, and specificity of the trimeric mimotope-PADRE 6 toward MAb b12.
azide-functionalized mimotope (4), as confirmed by mass spec-
trometry (MS) (see the Supporting Information).
information on the SPR experiments can be found in the
Supporting Information. As shown in Figure 2a, 6 showed the
fastest on rate, featuring enhanced binding toward MAb b12
relative to the monomeric mimotope and PADRE, which served
as a negative control. During dissociation (300-660 s), construct
6 exhibited a significant analyte response with a very low off rate,
which is a clear indication of increased avidity.57
To evaluate the binding specificity for MAb b12, the peptides
were screened against a random anti-HIV antibody (MAb 48d),
which was captured to equivalent levels (10 000 RU) on a CM5
chip. Comparison of the increased response from the sensor-
grams toward MAb b12 binding (Figure 2a) to the lack of any
response from the sensorgrams toward MAb 48d binding
(Figure 2b) clearly indicates that trimeric mimotope-PADRE
6 binds specifically to MAb b12. Taken together, these data
demonstrate that 6 shows increased binding, avidity, and speci-
ficity toward MAb b12, which may be attributed to the multi-
valent presentation of the mimotope.
Presented herein is a versatile synthetic platform for preparing
multivalent peptide constructs having two different peptide
components. The high efficiency of the 1,3-dipolar cyclization
under conditions that alleviate oxidation of susceptible amino
acid side chains was critical to the success of this approach. While
the hetero-multivalent design presented here includes peptides
designed to stimulate both B-cell and TH-cell immune response,
one could conceivably join any combination of peptides using
this same strategy, providing a powerful tool for structure-
activity relationship analyses. We look forward to reporting the
results of such studies in the near future.
The most convenient CuAAC reaction conditions involve the
use of CuSO4 and sodium ascorbate as a reductant along with an
appropriate chelating ligand that maintains the Cu(I) oxidation
state.53-55 Unfortunately, our initial experiments utilizing bath-
ophenanthrolinedisulfonic acid as a ligand were not fruitful, as
only oxidized peptides were retrieved, even under oxygen-free
conditions. However, we were fortunate to learn that Finn and co-
workers56 have recently offered a solution to overoxidation that
employs tris(3-hydroxypropyltriazoylylmethyl)amine (THPTA)
(5), which serves as a scavenger for radical species, along with
aminoguanidine as an additive to prevent cross-linking and aggrega-
tion products. We were hopeful that this approach would be well-
suited for mimotope multimerization in view of the high tryptophan
content of our peptides.
We followed the Finn protocol by reacting 3 and 4 with 5 mM
sodium ascorbate, 5 mM aminoguanidine, 0.1 mM CuSO4, and
0.5 mM ligand 5 in 10 mM phosphate buffer (pH 7.4) containing
5% DMSO for 18 h (Scheme 1). After desalting and buffer-
exchange procedures, the lyophilized material gave 3.2 mg (70%)
of a white fluffy solid that was analyzed by high-resolution MS.
After deconvolution, the spectrum gave an observed monoiso-
topic mass peak at m/z 8386.9900 (Figure S4 in the Supporting
Information), which corresponds to the desired trimeric mimo-
tope-PADRE cycloaddition product 6 (theoretical mass = m/z
8387.0104). The recovered excess starting materials showed
minimal oxidation, even after overnight reaction.
We then initiated surface plasmon resonance (SPR) experi-
ments to compare the binding affinity and specificity of the
trimeric mimotope-PADRE construct 6 to the HIV broadly
neutralizing MAb b12. To validate the effect of valency, molar
concentrations were adjusted to equalize the number of ligands
or the total binding sites in solution. A blank chip was used as a
reference cell for all sensorgrams shown in Figure 2. Detailed
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details on the
b
synthesis of the peptide and linkers and the multimerization,
including SPR experiments. This material is available free of
charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
(25) Purcell, A. W.; McCluskey, J.; Rossjohn, J. Nat. Rev. Drug
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Corresponding Author
gervay@chem.ucdavis.edu
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’ ACKNOWLEDGMENT
This work was supported by the National Institutes of Health
(HIVRAD 1PO1 AI066287). Funding for the NMR spectro-
meters was provided by the NIH (RR11973). We also thank
Jonel P. Saludes for helpful insights and Hayden Peacock for
lending THPTA ligand used for preliminary work.
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