 |
INTRODUCTION |
CD8+ autologous cytotoxic T lymphocytes
(CTLs)1 specifically
recognize and lyse tumor cells bearing tumor antigens in human and
animal models (1). These antigens are small peptides (8-11 amino acids
(aa) in length) presented at the cell surface in the context of the
major histocompatibility complex (MHC) class I molecules. In humans,
several tumor antigens have been identified on the basis of gene
cloning and/or sequencing of the naturally presented peptide eluted
from MHC class I molecules (2-4). These tumor antigens are potential
therapeutical agents for cancer immunotherapy, and some of them, under
different forms, are currently assayed in vivo (5). MHC
class I-restricted antigenic peptides have been tested for in
vivo vaccination trials in animal models and in humans (6). The
use of peptides offers a number of advantages, especially those of a
highly specific response and a very low toxicity (7). However, the
results obtained with such peptide vaccines have been variable,
dependent on a number of factors (6). Successful immunization was
obtained after vaccination with MUT1 and MUT2 antigens expressed on
murine carcinoma with regression of established metastases (8). Tumor
regression was also observed after vaccination with a synthetic peptide
with enhanced MHC binding derived from human melanoma antigen gp100 (9). Protective immune responses were obtained against viruses after
peptide vaccination (10), but in other models peptide vaccines gave
limited or no results (7, 11, 12).
Indeed, the major factor limiting the efficiency of peptide vaccines
in vivo is, in most cases, their rapid degradation in serum
and other biological fluids (13, 14). Peptides presented naturally by
MHC class I molecules after intracellular processing of endogenous
proteins were shown to be protected, by the MHC, against further
protease degradation (15, 16). When exogenous peptides are used for
vaccination, they may be degraded by extracellular proteases before
reaching, and binding to, MHC molecules. Presentation of exogenous
peptides by MHC molecules might be altered by the presence of serum
proteases (14, 17, 18). A correlation was found between peptide
stability in vitro in serum and peptide persistence in
vivo, in ascitic fluid as well as on the surface of cells
presenting the peptide (18). The stability of peptides in biological
media must therefore be taken into account in the design of synthetic
peptide vaccines, and the development of non-natural peptide antigens
may be of great interest. However, the major difficulty in the design
of such modified peptides lies in the fact that the structural
modifications introduced must not induce a loss in the antigenicity and
immunogenicity of the peptide. Indeed, the activity of an antigenic
peptide depends upon two critical steps: (i) its presentation by the
MHC, determined by the anchor positions (19-21) and (ii) the
recognition of the peptide-MHC complex by a specific T cell receptor
(TcR) (22, 23). These two mechanisms depend very closely on the
structure of the antigen (24). Examples of non-natural (25-29) or
altered peptides (30) that specifically bind MHC molecules have been
described. Many of these peptides were MHC blockers and did not
therefore activate CTLs (25, 28). In the case of cancer vaccines,
peptide analogues should not only bind to MHC but must also activate
specific antitumor CTLs.
In this report, we present the rational design of a non-natural,
peptidase-resistant, and antigenic analogue of human tumor antigen
MAGE-1.A1. Encoded by the MAGE-1 gene expressed in 40% of melanomas
but not in normal tissues (testis excepted) (2), MAGE-1.A1 (EADPTGHSY)
is presented by the MHC class I molecule HLA-A1 (3). Its anchor
positions are Asp (D)3 and Tyr (Y)9 (31). The
residues of MAGE-1.A1 recognized by the different anti-MAGE-1.A1 CTL
clones are located at P1 and in the P4-P8 domain (32). Analysis of the
structural parameters involved in the degradation mechanism in serum of
MAGE-1.A1 and in its interactions with the MHC on one side and the TcR
on the other allowed us to design stable and biologically active non
natural MAGE-1.A1 analogues.
 |
EXPERIMENTAL PROCEDURES |
Peptide Synthesis--
Peptides were synthesized by the
solid-phase method using FMOC chemistry. FMOC aa derivatives were
purchased from Bachem, France. The non natural N- or
-methylated aa derivatives were coupled using
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate instead of
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
used for natural aa derivatives. Peptides were purified by high
pressure liquid chromatography (HPLC) on reversed-phase columns
(RP300-C8 Brownlee Lab and an Applied Biosystems 130 apparatus). The
purity of all peptides was >98%. The identity of the purified
peptides was confirmed by electrospray ionization-mass spectrometry
(ESI-MS) analysis. Stock solutions (2 × 10
3
M in H2O) were prepared and stored at
20 °C.
Human Cell Lines and CTL Clones--
The TAP
BM36.1 (45) and the TAP+ BM21 EBV-transformed lymphomas
(HLA-A1, B35, Cw4), were used in MHC stabilization experiments and CTL
assays, respectively. They were grown in RPMI medium supplemented with
8% heat-inactivated fetal calf serum and antibiotics. Melanoma cell
line MZ2-MEL (derived from patient MZ2), LG2-EBV, and CTL clones 82/30,
253/47, and 258/8 were cultured in Iscove's modified Dulbecco's
medium containing 10% human serum. CTL clones were stimulated weekly
with MZ2-MEL cell line in the presence of LG2-EBV as feeder cells and
human interleukin-2 as described previously (46).
Peptide Degradation Assay--
Peptides were added to preheated
(15 min at 37 °C before the assay) human serum to a final
concentration of 10
3 M and incubated at
37 °C. Human sera were obtained by centrifugation at 2000 × g of blood samples of five healthy donors. MAGE-1.A1 degradation in individual sera was qualitatively and quantitatively identical. All sera were pooled, and subsequent degradation experiments were realized in the pool. At different incubation times, an aliquot (150 µl) was removed and the protease and peptidase activities were
stopped by addition of 15 µl of trifluoroacetic acid (TFA). Precipitated serum proteins were pelleted by centrifugation at 15000 rpm for 10 min at 4 °C. The supernatants were frozen and preserved
at
20 °C until analysis by off-line HPLC and/or on-line HPLC/ESI-MS. In control experiments, MAGE-1.A1 and its substituted analogues did not precipitate in presence of 10% TFA and remained stable under these acidic conditions. Serum alone was precipitated by
10% TFA, and its HPLC profile was recorded to detect peaks corresponding to non-precipitated peptides present in the serum.
HPLC and HPLC/MS Analysis--
Peptides and their degradation
products were separated by reversed-phase HPLC and peaks corresponding
to the different products were quantified. Samples analysis at
different incubation times allowed the study of the degradation
kinetics of the peptides, determination of their half-life as well as
the formation of the different degradation products. The following HPLC
conditions were used: (i) ultrasphere ODS column: 5-mm, 4.6 mm × 25 cm, C18 (Beckman); (ii) gradient: 4-20%
CH3CN (0.08% TFA in H2O, 0.08% TFA in
CH3CN), 0-30 min; (iii) UV detection: 200-300 nm using a
Waters 996 photodiode array detector; (iv) chromatograms at 225 and 280 nm were derived. The sequences of degradation fragments were determined
by on-line HPLC/ESI-MS. Chromatographic conditions were the same as
above except for the gradient (4-15% instead of 4-20%). MS analysis
was performed with a TSQ-700 Finnigan Mat mass spectrometer (San Jose,
CA). The entire HPLC flow was introduced via an atmospheric pressure
ionization interface operating in ESI. The interface conditions were
such that the needle voltage was maintained at 5 kV, producing a
current of 70 µA. The temperature of the heated capillary was
250 °C. Nitrogen was used as both nebulizing gas (80 p.s.i.) and
auxiliary gas (20 p.s.i.). MS data (positive ions) were collected in
the full scale mode (m/z 150-1200) at 2 ms per step.
MHC Class I (HLA-A1) Binding Assay--
Peptide binding to
HLA-A1 was assayed using the peptide-induced MHC stabilization
procedure (47) using the TAP-deficient BM36.1 cell line. Briefly, cells
(2 × 105/sample) were incubated with or without
increasing amounts of peptide (10
9 M to
10
4 M for 4 h in the presence of 20%
inactivated fetal calf serum as a
2-microglobulin source
and peptidase inhibitors (bestatin and captopril, 10
4
M). HLA-A1 molecules were detected at the cell surface by
using the monoclonal antibody W6.32, which reacts with all human MHC class I alleles. After one wash with ice-cold culture medium, cells
were incubated on ice for 1 h with 100 µl of W6.32 culture supernatant or with medium alone for negative control. After one wash
with ice-cold 1% bovine serum albumin-phosphate-buffered saline, cells
were incubated for 1 h with fluorescein isothiocyanate-conjugated goat anti-mouse IgG secondary antibody (Sigma). Cells were then washed
twice, fixed with 1% formaldehyde in bovine serum
albumin-phosphate-buffered saline, and analyzed by flow cytometry
(Becton Dickinson FACScan). For each peptide, a SC50 value
(stabilizing concentration 50: concentration of peptide giving half of
the maximal stabilization effect) was defined, which reflects its
affinity for the studied MHC allele.
CTL Assays--
Lysis of target cells by CTL clones was measured
by chromium release assay. Target cells were 51Cr-labeled
for 1 h at 37 °C in the presence of monoclonal anti-human MHC
class I antibody W6.32 and washed three times. Target cells were then
incubated in V-bottom microwells (103 cells in 50 µl) in
the presence of different concentrations of peptide (50 µl) for 30 min at 37 °C. CTLs were then added at an effector:target ratio of
10:1 (104 cells in 100 µl), and 51Cr release
was measured after 4 h at 37 °C.
Molecular Modeling of HLA-A1-Peptide Interactions--
HLA-A1
(
1 and
2 domains) structure was obtained by modeling the
crystallographic data of the HLA-A2 3D structure (48) (Brookhaven
Protein Data Bank, Brookhaven National Laboratory, Upton, NY), both
molecules sharing more than 85% sequence homology in their peptide
binding grooves. The HLA-A2/Tax nonamer to HLA-A1/MAGE-1.A1 mutations
were introduced manually using the program o. Side chain conformation was based on the most probable rotamer and the probable hydrogen bonds. The structure obtained was minimized by X-PLOR. Additional minimization, 1000 iterations without constraints using Steepest Descent minimization, was performed after transfer of HLA-A1/MAGE-1.A1 data to insightII (Biosym Technologies), and further
studies were performed using insightII. MAGE-1.A1 residues at P2 and P8
were then substituted by their non-natural analogues using HOMOLOGY
(for D-isomers and Aib) and BIOPOLYMER (for NMe-aa) modules
of insightII (Biosym Technologies) followed by minimization using VA09A
(500 iterations without constraints) and STEEPEST DESCENT (1000 iterations without constraints) algorithms.
 |
RESULTS |
MAGE-1.A1 Is Rapidly Degraded in Human Serum in Vitro by Amino- and
Dipeptidylcarboxypeptidases--
MAGE-1.A1 degradation fragments
generated at 37 °C were analyzed by on-line HPLC/MS. UV analysis
(Fig. 1, panel a)
facilitated the quantification and determination of the kinetics for
the appearance and disappearance of each peak (peaks A-F). In the
experimental conditions used, the MAGE-1.A1 half-life (incubation time
necessary for the degradation to half of the initial quantity of
peptide) was less than 30 min (Fig. 2,
panel a). MS analysis (Fig. 1, panel b) of each peak facilitated the determination of the
corresponding MAGE-1.A1 degradation fragment. The mass spectrum of peak
D (m/z = 269) is given as an example in Fig.
1b (inset). In the MAGE-1.A1 sequence, the only
fragment corresponding to a m/z value of 269 is the
dipeptide SY. Similar analysis of peaks A-F led to the identification of all MAGE-1.A1 degradation fragments (Table
I). The kinetics studies (Fig. 2,
panels a, e, and i) allowed
us to establish the degradation pathway of MAGE-1.A1 (Fig.
1c); MAGE-1.A1 was sensitive to two exopeptidase
activities, aminopeptidase and dipeptidyl carboxypeptidase (or
angiotensin-converting enzyme, ACE). The major pathway was initiated by
two successive aminopeptidase cleavages, leading to the heptapeptide
DPTGHSY (peak E), then cleaved by ACE to give the pentapeptide DPTGH
(peak A) and the dipeptide SY (peak D) (degraded itself to amino
acids). Direct action of ACE on MAGE-1.A1 represented a minor
degradation pathway.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1.
Analysis of MAGE-1.A1 degradation by
HPLC/MS. a, HPLC chromatograms of MAGE-1.A1 after 0 or
60 min incubation in serum. After 60 min, six degradation fragments
(peaks A-F) were detected. Peaks
S1 and S2 corresponded to serum components.
b, total ion current chromatogram (TIC) from MS
analysis, showing peaks A-F and MAGE-1.A1. The inset
represents the mass spectrum of peak D; m/z values of 269.1 and 310.1 correspond to the dipeptide SY (269.1) and its acetonitrile
adduct (269.1 + 41.0) classically obtained in MS. c,
MAGE-1.A1 degradation model in human serum showing aminopeptidase
(bold arrows) and ACE
(normal arrows) cleavages.
|
|

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 2.
Degradation kinetics of MAGE-1.A1, analogues
5 ([Aib2]), 8 ([NMeSer8]), and 10 ([Aib2, NMeSer8]). Amount of the
parent peptide or its degradation fragments was determined at different
incubation times in serum by measuring the surface of the corresponding
HPLC peak. Peptide degradation kinetics are shown in upper
panels (a-d). Fragments resulting from
aminopeptidase and ACE degradation are presented in middle
(e-h) and lower (i-l) panels,
respectively.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
MAGE-1.A1 degradation product retention times, characteristic ions, and
deduced sequences
Retention times were deduced from the UV chromatogram (Fig.
1a) corresponding to a sample of MAGE-1.A1 incubated for 30 min in human serum. Masses of MAGE-1.A1 and its degradation fragments
(simply [MH]+ or doubly charged [M + 2H]2+)
were measured by on-line HPLC-MS analysis of the same sample (Fig.
1b). Sequences of degradation fragments were deduced from
masses measured by MS.
|
|
Point Amino Acid Modifications Can Confer Peptidase Resistance to
MAGE-1.A1--
A series of MAGE-1.A1 analogues modified at positions
engaged in one or both of the peptidase-sensitive bonds
Glu1-Ala2 and His7-Ser8
was synthesized and then studied for their degradation properties (Table II). Peptides substituted at
either position 1 or 2 (analogues 1 to 5) showed
increased half-lives but this effect remained poorly effective since it
varied from very weak (analogue 5) to moderate (analogue
3) in a range of 5-25 min increase as compared with
MAGE-1.A1 half-life. However, all these substitutions were efficient
against aminopeptidases, since none of the fragments 2-9 and 3-9
expected from aminopeptidase cleavage of the nonamer were detectable by
UV (see for example analogue 5 in Fig. 2, panel
f) or on-line HPLC/MS (data not shown) analysis. In
contrast, in all cases, the fragment 1-7, generated from ACE cleavage,
was more intense than for unmodified MAGE-1.A1 degradation, showing
that NH2-terminal protection of the peptide rendered its
COOH terminus more sensitive (see analogue 5 in Fig. 2,
panel j). This explains the poor overall gain in
half-life for these analogues. At the COOH terminus, substitutions at
positions 7 or 8 (analogues 6-8) did not
significantly improve MAGE-1.A1 half-life, as expected, since ACE
cleavage represents the minor degradation pathway. However, they
efficiently protected MAGE-1.A1 against ACE, since fragments 1-7 and
8-9 were totally undetectable among the degradation products of the
COOH terminus-protected analogues by UV (see analogue 8, Fig. 2, panel k) or by on-line HPLC/MS (data not
shown) analysis. However, analogues were degraded by aminopeptidases
giving fragments 2-9, 3-9 and 5-9 (see for example analogue
8 in Fig. 2, panel g). As protection of one
extremity sensitized the opposite one to peptidase activity, we
synthesized four analogues (9-12) in which
substitutions at NH2 and COOH terminus were combined. All four disubstituted peptides showed no detectable degradation even after
a 24-h incubation in serum (Table II) (analogue 10 is shown
in Fig. 2, panels d, h, and
l). This shows that disubstitution conferred long term
resistance to both aminopeptidase and ACE and, importantly, that
MAGE-1.A1 was not sensitive to other peptidase activities in serum.
View this table:
[in this window]
[in a new window]
|
Table II
Biochemical properties of MAGE-1.A1 and its substituted analogues
Part a, half-lives were determined as follows: after different
incubation times in human serum, the parent peptide was separated from
its degradation fragments by HPLC, and the corresponding peak was
quantified (Fig. 2, upper panels). Part b, binding
affinities (MHC stabilization assay) are the mean of at least three
independent experiments.
|
|
The HLA-A1 Binding Groove Tolerates Structural Modifications in the
MAGE-1.A1 Sequence--
Binding of the structurally modified analogues
to HLA-A1 was then tested and compared with that of the parent peptide
MAGE-1.A1 (examples are shown in Fig. 3,
panels a, d, g, and j;
results are presented in Table II, right column). The effect of a
substitution on MHC binding depended on both the nature of the chemical
modification and its position in the peptide sequence. Introduction of
Aib at position 2 (analogue 5), a D-isomer at
position 7 (analogue 6), or a NMe group at position 8 (analogue 8) did not alter dramatically HLA-A1 binding of
MAGE-1.A1 (Table II). When two of these substitutions were introduced
in the same sequence, their effects accumulated; the disubstituted
analogues 9 and 10 bound to HLA-A1 with lowered
but still good affinities (Fig. 3, panels a and
d; Table II). Unlike the previous substitutions, D
isomerization at position 2 (analogue 3) or 8 (analogue 7) dramatically decreased HLA-A1 binding. Consequently, the
disubstituted
[D-Ala2,D-Ser8]
peptide (analogue 12) did not bind to HLA-A1. Finally, the
monosubstituted analogues 1 and 4 and
disubstituted analogue 11 exhibited intermediate to weak
(i.e. 30-100 times lower than that of MAGE-1.A1) binding to
HLA-A1 (Fig. 3, panel g; Table II). Thus, out of
the 12 non-natural MAGE-1.A1 analogues, 2 (analogues 9 and
10) were peptidase-resistant with preserved HLA-A1 binding
properties.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 3.
HLA-A1 binding and CTL activation properties
of MAGE-1.A1 and its structurally modified analogues. HLA-A1
binding (left panels) was determined using the
peptide-induced MHC stabilization procedure. Activation of CTL clones
82/30 (middle panels) and 258/8 (right
panels) was measured by 51Cr assay. The
disubstituted analogues 9 [Aib2,
D-His7], 10 [Aib2,
NMe-Ser8], 11 [NMe-Ala2,
NMe-Ser8], and 12 [D-Ala2, D-Ser8]
(closed circles) and their respective
amino-terminal (open squares) and
carboxyl-terminal (open triangles)
monosubstituted analogues are shown in comparison to MAGE-1.A1
(open circles).
|
|
Stable MAGE-1.A1 Analogues Can Activate MAGE-1.A1-specific
CTL--
MAGE-1.A1 mono- and disubstituted analogues were tested for
their capacity to activate anti-melanoma MAGE-1.A1-specific CTL clones
82/30 and 258/8. Residues Gly5, Thr6, and
His7 are recognized by both clones while residues
Pro4 and Ser8 are selectively recognized by
clones 82/30 and 258/8, respectively (32). Results are summarized in
Table III (CTL assay experiments for four
series of mono- and corresponding disubstituted analogues are shown in
Fig. 3, middle and right panels).
View this table:
[in this window]
[in a new window]
|
Table III
Biological activities of MAGE-1.A1 and its stable substituted analogues
The recognition of MAGE-1.A1 or its substituted analogues by CTL clones
82/30 and 258/8 was determined by 51Cr release assay after
incubation of 51Cr-labeled BM21 target cells with the studied
peptides. EC50 are the results of at least two independent
experiments.
|
|
Activation of CTL clone 82/30 tolerated some (panels
b or h) but not all (panel
k) structural modifications at the carboxyl-terminal side of
MAGE-1.A1. Analogues 6 and 8 activated CTLs within a concentration range close (1 order of magnitude or less) to
that of the parent peptide (panels b and
e and Table III). Analogue 7 had a low biological activity
(panel k and Table III), which could be explained
by its low HLA-A1 binding affinity (see Table II). For NH2
terminus-modified MAGE-1.A1 analogues, out of four peptides with a good
HLA-A1 binding affinity, analogue 2 retained a good
biological activity (Table III), analogues 1 and
5 had intermediate activities (panel e
and Table III), and analogue 4 had a weak activity
(panel h and Table III). As expected, analogue
3, a weak HLA-A1 binder (Table II), activated weakly CTL
(Table III). Disubstituted analogues 10 and, to a lesser
extend, 9 showed good capacities to activate clone 82/30
(panels b and e and Table III),
whereas analogues 11 and 12 exhibited only weak
or no biological activity (panels h and
k and Table III).
Activation of CTL clone 258/8 was expectedly affected by structural
modifications at Ser8 since this residue represents a major
and specific TcR-peptide contact. Monosubstituted (analogues
7 and 8) or disubstituted (analogues
10-12) peptides were unable to activate clone
258/8 (see panels f, i, and
l and Table III). Modification at position 7 led to
monosubstituted (analogue 6) or disubstituted (analogue
9) peptides still recognized by clone 258/8
(panel c and Table III). Of the five
NH2 terminus-modified peptides (analogues
1-5), only one (analogue 5) efficiently activated clone 258/8, the others showing intermediate (analogue 1) or very weak (analogues
2-4) activity (Table III).
Since the disubstituted analogue 10 was highly stable in
human serum (half-life > 24 h), had a preserved good HLA-A1 binding affinity (see Table II), and activated efficiently one of two
tested anti-MAGE-1.A1 CTL clones (Table III), we then tested its
capacity to activate a third anti-melanoma MAGE-1.A1-specific CTL
clone, 253/47, that recognizes Glu1 in addition to
Gly5, Thr6, and His7 (32). Analogue
10 activated efficiently anti-MAGE-1.A1 CTL clone 253/47
(Table IV).
View this table:
[in this window]
[in a new window]
|
Table IV
Biological activities of MAGE-1.A1 and
[Aib2,NMe-Ser8]MAGE-1.A1
Recognition of MAGE-1.A1 and [Aib2,NMe-Ser8]MAGE-1.A1
by anti-MAGE-1.A1 CTL clones 253/47, 82/30 and 258/8 was determined by
51Cr release assay. [Aib2,NMe-Ser8]MAGE-1.A1
activated two out of the three anti-MAGE1-A1 CTL clones.
|
|
 |
DISCUSSION |
In a perspective of design of synthetic peptide vaccines, we
showed in this study that non-natural, peptidase-resistant tumor antigen analogues might specifically activate antitumor CTLs. The
rational design of these analogues was based on: (i) the understanding of the interactions of the antigenic peptide with the MHC and the TcR
and (ii) the precise knowledge of the degradation mechanism of the peptide.
On-line HPLC/MS analysis led to the identification and quantification
of degradation fragments and to the determination of the
peptidase-sensitive bonds. Despite the diversity of peptidases present
in serum, we found that MAGE-1.A1 was degraded by exopeptidases (mainly
aminopeptidases, then by ACE, which are both exopeptidases). Indeed,
short peptides are often degraded by exopeptidases rather than
endopeptidases (13, 33). This structure-based approach combined with
the knowledge of the interactions of MAGE-1.A1 with HLA-A1 and TcR
obtained in the present work and from previous structure-activity
relationship studies (32, 34) allowed us to introduce precise and local
substitutions at the sensitive site as opposed to systematic
modifications of all of the peptide positions. Indeed, this point is of
crucial importance for the use of biologically active peptides in the
MHC-peptide-TcR system (Ref. 24 and see below). The non-natural
residues we used (D-amino acids, N-methyl-amino
acids, and Aib) are efficient inhibitors of exopeptidase
(aminopeptidase and ACE) activities (35). We may reasonably postulate
that the disubstituted analogues should also be peptidase-resistant
in vivo (18).
The main successful examples of antigenic mimicry and cross-reactivity
between natural peptide antigens and their non-natural analogues
(retro-, retro-inverso-, or D-peptides) have been described for antibody recognition (36, 37). A major difficulty encountered in
the design of biologically active analogues of MHC-restricted epitopes
is that their activity depends upon the accomplishment of two critical
steps: MHC binding on the one hand and TcR signal triggering on the
other. Several examples of MHC-restricted epitope analogues have been
described. All-retro-inverso analogues (peptides in which all bonds
have been retro-inverted) of MHC class II-restricted epitopes lost
their MHC binding affinity (38). In other studies, binding to MHC class
I was altered for some peptides and not for others following partial
modifications, retro-inverso or reduced bonds, depending on the
position of the modification (26, 27). However, in two studies on MHC
class II-restricted peptides, even when only partial structural
modifications that did not alter MHC binding were introduced,
recognition of non-natural analogues by CTL was dramatically affected
(28, 29). Despite the strong structural analogies between the Fab
antibody fragment and the V
-V
TcR domain (39), the rather common
cross-reactivity found between parent peptides recognized by antibodies
and their non-natural analogues (36) appears to be much less applicable
to MHC-restricted TcR ligands. In fact, this observation is likely to
be explained by the higher stringency of the TcR-epitope interaction in
comparison with the antibody-epitope, since the TcR has dual functions
that must both be fulfilled: (i) the specific recognition of the
antigen and (ii) the activation of the lymphocyte functions. Indeed,
structurally altered ligands, partial agonists or antagonists, that can
only fulfill the first TcR function, but not the second one, may then lead to partial activation or anergy of the lymphocyte (24). Thus,
great care must be taken in the development of antitumor vaccine
strategies to avoid specific inactivation or anergy of antitumor
CTL.
Of the three stabilized analogues (9, 10, and
11) with good HLA-A1 binding, only analogue 10, [Aib2, NMe-Ser8]MAGE-1.A1, activated CTL in
the same range of concentrations as the parent MAGE-1.A1 peptide. In
this analogue, the side chain orientations of the anchoring residues
Asp3 and Tyr9 and, more importantly, that of
the residues pointing out toward the TcR (mainly Glu1,
Pro4, Thr5, His7, Ser8)
were left unmodified by the structural modifications at P2 and P8 (see
Fig. 4). This observation has a dual
consequence in terms of MHC binding and CTL activation. Considering MHC
binding, the drop in affinity confirms that non-anchor residues,
particularly P2 for HLA-A1-restricted peptides (40), may influence
antigen presentation (21). Considering CTL activation, the remarkable correlation observed between the stable analogue 10 and the
natural antigen in their ability to sensitize target cells to lysis by
CTL clone 82/30 (and to lesser extend CTL clone 253/47) and their MHC
affinity indicates unambiguously that the lower CTL activation
properties of analogue 10 are a strict and direct
consequence of its lower MHC presentation and not at all of alteration
in TcR recognition. This point, in perfect accord with the proposed
structural model (see Fig. 4) is critical since it indicates that, once
bound to the MHC, analogue 10 is able to engage the
MAGE-1.A1-specific TcR as the natural ligand does. We can thus
postulate that this synthetic peptide can fully activate the
anti-melanoma CTL functions and therefore acts as a full agonist, an
important parameter in terms of successful vaccine strategies (see
above). Analogue 10, stable after 24 h in serum and
able to activate two of three known MAGE-1.A1-specific CTL clones, may
therefore be considered as a potential activator of anti-melanoma CTL
response in vivo. To overcome the ethical problems
encountered when testing non-natural molecules such as ours in human
cancer vaccine trials, an alternative and attractive approach to follow
would be to use these molecules in ex vivo or in
vitro procedures to generate potent MAGE-1.A1-specific CTL (41).

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 4.
Superimposed structures of MAGE-1.A1
and analogue 10 ([Aib2,NMeSer8]MAGE-1.A1) in the
HLA-A1 binding groove. The 1 and 2
domains are shown as dark gray
ribbons. a, complete side view of MAGE-1.A1
(blue stick) and [Aib2,
NMeSer8]MAGE-1.A1 (green stick).
Aib2 and NMe-Ser8 are shown in
yellow in comparison to Ala2 and
Ser8 shown in red. b and
c, zoomed side views of the
Ser8-Tyr9 domain. In b,
Trp147 (yellow) exchanges a hydrogen bond with
the carbonyl of Ser8 of MAGE-1.A1. In c, the
hydrogen bond between Trp147 and Ser8 is
abolished and two new hydrogen bonds between
Tyr84 and Thr143 (in green) and the
carboxyl terminus of Tyr9 of
[NMeSer8]MAGE-1.A1 are created.
|
|