Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3807
formed as specified above. After an overnight incubation at 4
Southwood, S.; Sette, A.; Robbins, P. F.; Marincola, F. M.;
Salgaller, M. L.; Yannelli, J . R.; Appella, E.; Rosenberg, S. A.
Induction of tumor-reactive CTL from peripheral blood and
tumor-infiltrating lymphocytes of melanoma patients by in vitro
stimulation with an immunodominant peptide of the human
melanoma antigen MART-1. J . Immunol. 1995, 154, 2257-2265.
°
C, unbound peptides were eliminated by centrifugation on
NANOSEP 10K (Pall Filtron, Northborough, MA). Samples
were diluted in PBS-Tw, and aliquoted in Eppendorf micro-
tubes for further incubations (1, 3, 5, 24 and 48 h) at 37 °C.
An aliquot (time 0) was immediately tested for having the
maximal number of formed complexes. The two incubations
with anti-HLA mAb were performed in microplates at 37 °C
for 1 h. Final detection was performed as described above.
T2 Cell Lin e. T2 is a variant of the cell line T1 produced
by fusion of the lymphoma cell CEM and the B lymphoblastoid
cell line 721.174. It expresses low amounts of HLA-A2 and no
HLA-B or -C molecules at the cell surface because a lack of
peptide transporters results in an accumulation of HLA heavy
(
5) J aeger, E.; Bernhard, H.; Romero, P.; Ringhoffer, M.; Arand, M.;
Karbach, J . Ilsemann C, Hagedorn M, Knuth A. Generation of
cytotoxic T-cell responses with synthetic melanoma-associated
peptides in vivo: implications for tumor vaccines with melanoma-
associated antigens. Int. J . Cancer 1996, 66, 162-169.
6) (a) Sette, A.; Vitiello, A.; Reherman, B.; Fowler, P.; Nayersina,
R.; Kast, W. M.; Melief C. J .; Oseroff, C.; Yuan, L.; Ruppert, J .;
sidney, J . del Guercio, M. F.; Southwood, S.; Kubo, R. T.;
Chesnut, R. W.; Grey, H. M.; Chisari, F. V. The relationship
between class I binding affinity and immunogenicity of potential
cytotoxic T cell epitopes. J . Immunol. 1994 153, 5586-5592. (b)
van der Burg, S. H.; Visseren, M. J .; Brandt, R. M.; Kast, W.
M.; Melief, C. J . Immunogenicity of peptides bound to MHC class
I molecules depends on the MHC-peptide complex stability. J .
Immunol. 1996 156, 3308-3314.
7) (a) Parkhurst, M. R.; Salgaller, M. L.; Southwood, S.; Robbins,
P. F.; Sette, A.; Rosenberg, S. A.; Kawakami, Y. Improved
induction of melanoma-reactive CTL with peptides from the
melanoma antigen gp100 modified at HLA-A*0201-binding
residues. J . Immunol. 1996 157, 2539-2548. (b) Rosenberg, S.
A.; Yang, J . C.; Schwartzentruber, D. J .; Hwu, P.; Marincola, F.
M.; Topalian, S. L.; Restifo, N. P.; Dudley, M. E.; Schwarz, S.
L.; Spiess, P. J .; Wunderlich, J . R.; Parkhurst, M. R.; Kawakami,
Y.; Seipp, C. A.; Einhorn, J . H.; White, D. E. Immunologic and
therapeutic evaluation of a synthetic peptide vaccine for the
treatment of patients with metastatic melanoma. Nat. Med.
1998, 4, 321-327.
(
2
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chain in the endoplasmic reticulum. HLA-A2 molecules can
be stabilized at the cell surface by the addition of exogenous
peptides associating with them.
TILs. Human T cell clone-infiltrating melanomas, restricted
by the HLA A2.1 molecule and specific to the MART-1(27-
(
3
5) peptide, namely LT12 (Vâ2), LT11 (Vâ9), LT8 (Vâ5), were
kindly provided by F. Farace (Institut Gustave Roussy,
Villejuif, France). Cells were grown in complete medium. RPMI
1
0% human AB serum containing 50 IU/mL penicillin, 50 µg/
mL streptomycin, nonessential amino acids, 10 mM hepes, 1
mM pyruvate, glutamine, 100 U/ml IL-2 (Boehringer) and
TCGF 3%. The mutant T2 cell line was used as target cell in
cytolytic assays. Cells were grown in complete medium: RPMI
1
0% FCS containing 50 IU/mL penicillin, 50 µg/mL strepto-
mycin, nonessential amino acids, 10 mM hepes, 1 mM pyru-
vate, glutamine.
(8) Valmori, D.; Fonteneau, J . F.; Lizana, C. M.; Gervois, N.;
Li e´ nard, D.; Rimoldi, D.; J ongeneel, V.; J otereau, F.; Cerottini,
J . C.; Romero, P. Enhanced Generation of Specific Tumor-
Reactive CTL in vitro selected Melan-A/MART-1 immunodomi-
nant peptide analogues. J . Immunol. 1998, 160, 1750-1758.
Cytolytic Activity Assa y. Cytolytic activity was detected
by a standard 4-h 51Cr assay. The T2 target cells labeled with
5
1
1
00 µCi of sodium chromate ( Cr, 100 mCi/mL, DuPont-NEN
Research Products, Boston, MA) were incubated for 1 h with
µg/mL peptide, washed twice with 0.9% NaCl medium
(
9) (a) Podlech, J .; Seebach, D. On the preparation of â-amino acids
from R-amino acids using the Arndt-Eistert reaction: scope,
limitations and stereoselectivity. Application to carbohydrate
peptidation. Stereoselective a-alkylations of some â-amino acids.
Liebigs Ann. 1995, 1217-1228. (b) Guichard, G.; Abele, S.;
1
containing 5% FCS and dispatched at 3000 cells/well. Spon-
taneous release never exceeded 25% of the maximum 51Cr
uptake. The percent specific lysis was determined as: 100 ×
2
3
Seebach, D. Preparation of N-Fmoc-protected â - and â -amino
acids and their use as building blocks for the solid-phase
synthesis of â-peptides. Helv. Chim. Acta 1998, 52, 187-206.
(
experimental release - spontaneous release)/(maximum re-
lease - spontaneous release).
(
10) Barlos, K.; Gatos, D.; Kallitsis, J .; Papaphotiu, G.; Sotiriu, P.;
Wenging, Y.; Sch a¨ fer, W. Darstellung gesch u¨ tzter peptid-frag-
mente unter einsatz substituierter triphenylmethyl-harze. Tet-
rahedron Lett. 1989, 30, 394-396.
11) Connan, F.; Hlavac, F.; Hoebeke, J .; Guillet, J .-G.; Choppin, J .
A simple assay for detection of peptides promoting the assembly
of HLA class I molecules. Eur. J . Immunol. 1994, 24, 777-780.
12) Guo, H. C.; J ardetzky, T. S.; Garrett, T. P.; Lane, W. S.;
Strominger, J . L.; Wiley, D. C. Different length peptides bind to
HLA-Aw68 similarly at their ends but bulge out in the middle.
Nature 1992, 360, 364-366.
13) Madden, D. R.; Garboczi, D. N.; Wiley: D. C. The antigenic
identity of peptide-MHC complexes: A comparison of the
conformations of five viral peptides presented by HLA-A2. Cell
1993, 75, 693-708.
14) Weiss, G. A.; Collins, E. J .; Garboczi, D. N.; Wiley: D. C.;
Schreiber, S. L. A tricyclic ring system replaces the variable
regions of peptides presented by three alleles of human MHC
class I molecules. Chem. Biol. 1995, 2, 401-407.
(15) Poenaru, S.; Lamas, J . R.; Folkers, G.; Lopez de Castro, J . A.;
Seebach, D.; Rognan, D. Nonapeptide analogues containing (R)-
Ack n ow led gm en t. Martine Schneider is gratefully
aknowledged for CZE analyses. A.Z. was financially
supported by a postdoctoral grant from Association pour
la Recherche sur le Cancer (ARC). This work was
supported in part by the ARC.
(
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Su p p or tin g In for m a tion Ava ila ble: 1H NMR data of
analogue 4, recognition of MART-1(27-35) and analogue 4 by
clone LT12 at different concentrations, and molecular models
(
2
8
of MART-1(27-35), [Leu ]MART-1(27-35), and analogue 4
complexed toHLA/A2. This material is available free of charge
via the Internet at http://pubs.acs.org.
(
Refer en ces
(
1) Boon, T.; Coulie, P. G.; Van den Eynde, B. Tumor antigens
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mer, R. Melan A/MART-1 immunoreactivity in formalin-fixed
paraffin-embedded primary and metastatic melanoma: fre-
quency and distribution. Melanoma Res. 1998, 8, 337-343.
3) (a) Kawakami, Y.; Eliyahu, S.; Delgado, C. H.; Robbins, P. F.;
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J . C.; Boon, T. A new gene coding for a differentiation antigen
recognized by autologous cytolytic T lymphocytes on HLA-A2
melanomas. J . Exp. Med. 1994, 180, 35-42.
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-hydroxybutanoate and beta-homoalanine oligomers: synthesis
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(
(
16) Bianco, A.; Brock, C.; Zabel, C.; Walk, T.; Walden, P.; J ung, G.
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17) Loftus D. J .; Castelli, C.; Clay, T. M.; Squarcina, P.; Marincola,
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18) Chen, W.; McCluskey, J .; Rodda, S.; Carbone, F. R. Changes at
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4) (a) Kawakami, Y.; Eliyahu, S.; Sakaguchi, K.; Robbins, P. F.;
Rivoltini, L.; Yannelli, J . R.; Appella, E.; Rosenberg, S. A.
Identification of the immunodominant peptides of the MART-1
human melanoma antigen recognized by the majority of HLA-
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peptide residues buried in the major histocompatibility complex
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a
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80, 347-352. (b) Rivoltini, L.; Kawakami, Y.; Sakaguchi, K.;