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Nov 1, 1995 - Lethe, B., Coulie,. P. G., and Boon, T. The tyrosinase gene codes for an antigen recognized by autologous cytotoxic T lymphocytes on HLA-A2 ...
Recognition of Multiple Epitopes in the Human Melanoma Antigen gp100 by Peripheral Blood Lymphocytes Stimulated in Vitro with Synthetic Peptides Michael L. Salgaller, Alireza Afshar, Francesco M. Marincola, et al. Cancer Res 1995;55:4972-4979. Published online November 1, 1995.

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Downloaded from cancerres.aacrjournals.org on July 15, 2011 Copyright © 1995 American Association for Cancer Research

(CANCER RESEARCH 55, 4972-4979,

November 1, 1995)

Recognition of Multiple Epitopes in the Human Melanoma Antigen gplOO by Peripheral Blood Lymphocytes Stimulated in Vitro with Synthetic Peptides Michael L. Salgaller,1 Alireza Afshar, Francesco M. Marincola, Licia Rivoltini,2 Yutaka Kawakami, and Steven A. Rosenberg Surgery Branch. National Cancer Institute. National Institutes of Health, Bethesda. Maryland 20892

ABSTRACT gplOO is a melanocyte lineage-specific antigen recognized by tumorinfiltrating lymphocytes whose adoptive transfer has been associated with tumor regression in patients with metastatic melanoma. The peripheral blood mononuclear cells of five melanoma patients were sensitized in vitro with synthetic peptides to elicit antigen-specific cytotoxic T lymphocyte (CTL) lines against four gplOO epitopes. These epitope-specific CTL lines were generated following weekly in vitro stimulation with the synthetic decamer GI0476 (V-L-Y-R-Y-G-S-F-S-V) or the nonamers 09^0 90% purity) using a solid-phase tech nique. MART-127_15 peptide (A-A-G-I-G-I-L-T-V) was synthesized by

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Fig 1. In vitro expansion of CTL lines generated from patients 1 to 6 stimulated with gplOO-, MART-1-, and Ml influenza-derived peptides. Fold expansion of CTLs produced from PBMCs stimulated weekly with 1 ¿LM Gl()47(, (•),G921I(,(•),and MART-127_15 (A) synthetic peptides (patients 1. 2, 3, and 6), as well as G9,54 (D), G9,,„(O), and M15(1_6Õ> (A) synthetic peptides (patients 4 and 5) pulsed onto autologous PBMCs. Cells were supplemented with 30 ID/ml IL-2 on the third day following establishment of cultures, then subsequently 1 day after each restimulation.

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RECOGNITION OF MELANOMA ANTIGEN BY LYMPHOCYTES

Table 1 Flow emometrie characterization

of gplOO-specific bulk CTLs

RespondersmAbs 1vs. 1vs. 2vs. 2VS. 3vs. 4vi. 4vs. 5vs. G9I549490564283Patient forCD3TCR-a/0CDSCD4HLA-DRPatient G1047(,99°99474887Patient G92809989414688Patient G104769495445091Patient G92809495444888PatientG921io9596454888Patient G92m9695544387Patient G9,,49493584(189

' Percentage of positive cells.

different supply of autologous PBMCs for weekly restimulations. Maximum expansion for patient CTLs varied widely from less than lO'-fold (patient 4) to over 104-fold (patient 1) during 7-10 weeks, and also exhibited differential expansion of the same patient CTLs stimulated with different synthetic epitopes. Expansion of PBMCs stimulated with the M15X_66influenza peptide was in the intermediate range compared to those stimulated with the other synthetic tumor epitopes (data not shown). After 6-10 weeks some bulk CTLs became quiescent, doubling less than once per week, and were cryopreserved at that time. After 10 weeks, those bulk CTLs still expanding were also cryopreserved at that time. At least two separate series of in vitro stimulations of bulk PBMCs were performed for each patient and peptide combination. These demonstrated similar cell growth except in one instance: during a second series of in vitro stimulations, PBMCs from patient 1 grew in culture over 120 days, after which all cells were cryopreserved. For some patients, the availability of autol ogous PMBCs to serve as peptide-pulsed stimulators limited the duration of culture. Since we were unable to elicit gplOO-, MART-1-, or Ml-specific CTLs from the PBMCs of patient 6, this patient is not discussed further. Flow cytometric characterization of bulk CTLs from patients 1, 2, 3, 4, and 5 stimulated by gplOO-, MART-1-derived peptides was performed on day 55 (Table 1). For every patient and condition, CTLs generated were at least 94% CD3+, with 89% or more of that popu lation consisting of TCR-a/ß+T cells. Activation markers were ex pressed by greater than 83% of cells, regardless of the experimental conditions, as evidenced by HLA-DR staining. Interestingly, the per centages of CDS* and CD4+ cells were nearly equivalent. When the peptide used to stimulate bulk cultures was gplOO-derived, there was little variation in the cytometric profile of CTLs. In only one instance, PBMCs of patient 2 stimulated with MART-127_.,5, was there a majority of CD8+ CTLs (66%) in the bulk population. These data suggest that the CTLs generated with such a protocol contain nearly equal populations of CD3+/CD8+ and CD3+/CD4+-activated TCR-a/ ß-expressingT cells, and that such findings are largely independent of the PBMCs or gplOO-derived peptide used. All cytolytic and cytokine data shown in this report used CTLs harvested within 2 weeks of the phenotypic analysis shown above. Generation of CTLs Demonstrating HLA-A2-restricted Lysis of Targets Expressing gplOO-derived Peptides. After about 7 weeks of culture, CTLs from patients 1, 2, 3, 4, and 5 demonstrated strong specific cytolysis against a wide range of gplOO-expressing targets. These data are summarized in Figs. 2 and 3. Cytolytic activity of bulk PBMCs stimulated in vitro with synthetic peptides was first assessed at 4 weeks of culture, but at that time only PBMCs from patients 1, 2, 3, 4, and 5 pulsed only with synthetic peptides MART127_35 or Mlsii_ft6 demonstrated specific cytotoxic activity (greater than 10% lysis of relevant compared to control targets at all effector: responder ratios tested; data not shown). Initially, we tested reactivity to T2 cells pulsed with various antigens and two cultured melanoma clones (624.38mel and 624.28mel, Fig. 2). CTLs from patient 1 displayed lysis of relevant targets at least 3-fold greater than that

observed against nonspecific targets when either G10476, G92SO, or MART-127_35 was used to stimulate the cultures (Fig. 2a). Recogni tion of the cultured melanoma clone 624.38mel (which expresses gplOO, MART-1, and HLA-A2) but not clone 624.28mel (which has lost HLA-A2 expression but expresses gplOO and MART-1; Ref. 16) indicated that patient CTLs recognized gplOO+ targets in a HLA-A2restricted manner. CTLs from patient 1 stimulated with Mljg,^ displayed peptide specificity in a HLA-A2-restricted fashion, yet with higher background that is not uncommonly observed.4 As expected, Ml5H_66-specific cells from patient 1 did not lyse the (HLA-A2+, Ml~) clonal line 624.38mel. Compared to CTLs from patient 1, specific cytolysis was stronger using CTLs from patient 2 stimulated in vitro with any of the four synthetic peptides: percentage lysis of 624.28mel and T2 pulsed with irrelevant peptide was always 3- to 5-fold lower at any E:T ratio (Fig. 2b). Nonspecific lysis against 624.28mel and T2 pulsed with irrelevant peptide was also minimal (never exceeding 19% at the highest E:T ratio) using CTLs from patient 3 (Fig. 2c). Lysis of Ml58_66-pulsed T2 cells by specific CTLs was evident, although the weakest of the three patient CTLs (Fig. 2c). Anti-G9154 CTLs demonstrating similar specificity could be gener ated from both patients 4 and 5, and anti-G92(w CTLs were obtained from patient 4 (Fig. 2, d and e). For patients 4 and 5, we used only M1SK_66as a control for the ability to generate peptide-specific CTLs from PBMCs, and therefore did not attempt to generate anti-MART127_35CTLs. Therefore, for patients 1, 2, and 3, those CTLs stimu lated with MART-127_35 lysed only T2 pulsed with relevant peptide as well as the HLA-A2+, MART-1* melanoma cell line 624.38mel. CTLs from the five patients stimulated with M15K^6so

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Fig. 2. Cytolytic activity against T2 pulsed with synthetic pcptides or melanoma cell lines by CTLs generated by weekly stimulation with gplllO-derived. peptide-pulsed autologous PBMCs. Epitopes given above each graph represent the synthetic peptide used to stimulate each bulk of PBMCs. a. CTLs from patient I stimulated with G1047„, G9,„,„ MART-],,.,,, and Ml.,„_«,; h. CTLs from patient 2 stimulated with Gl()47h. G92KI1.MART-127_,S, and M !;«_„,: c. CTLs from patient 3 stimulated with G9,s„,MART-1,7_,s. and M !.,„_«,; d, CTLs from patient 4 stimulated with G9,54, G9a„,and M15(,^„„; and e, CTLs from patient 5 stimulated with G9154 and Mls„_,,„. For patients 1 to 3, targets included T2-pulscd with G10J7„ (•)and G92(i(, (•).For patients andT25,alone targets(»).Established included T2-pulsed (•)and G9,lw (•).For (O) patients 1 to 4, targets also624.28mel included (O), MART-1 ,-,_„ for patients 1Daudi to 5, targets also included Ml.,«.«, (V),4 or clonal with linesG9154 HLA-A2»gplOO*"624.38mel and HLA-A2" gplOO* as well as (A): the NK-sensitive lymphoma line (A) were assayed with all CTLs as well. Results of a second assay were similar.

952mel. For CTLs from patient 1, 2, and 5, cytolytic activity against the gpKXr cell lines as well as HLA-mismatched targets was always mini mal (less than 13% at any E:T ratio). Even the highest nonspecific lysis observed, that of patient 3 anti-G92x() CTLs versus 397mel and WM115 at 25:1, was still 3-fold less than that of relevant targets. Lack of lysis versus RM, an HLA-A2* breast tumor cell line, suggests that the effect was melanoma specific. Sufficient anti-G920,, CTLs from patient 5, as well as anti-09!^ and anti-09-,,),.,CTLs from patient 4, did not exist to test them against this panel of established cell lines. For CTLs raised

against the MART-127_,5 or Ml^.^, peptide, lysis of these MART-1 and MP cell lines was always minimal (less than 5% or 7%, respec tively; data not shown). For the data shown in Fig. 3, we again included LAK cells as a nonspecific effector in all experiments to demonstrate lysability of nonspecific targets. LAK cell lysis of nonspecific targets (397mel, WM115, RM, and Daudi) ranged from 49 to 65% for CTLs from patients 1 and 2, 21-49% for CTLs from patient 3, and 31-46% for CTLs from patient 5 at an E:T ratio of 25:1 (data not shown).

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RECOGNITION

OF MELANOMA

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Fig. 3. HLA-A2 and gplOO expression are re quired for the cytolylic activity of GI047(1- and G92lul-restricted CTLs. Panel 1, CTLs from patient 1 stimuliiled with G1047i); panel 2. CTLs from patient I stimulated with G9ïS(,;panel 3. CTLs from patient 2 stimulated with Gl()47„;panel 4. CTLs from patient 2 stimulated with G92S„; panel 5, CTLs from patient 3 stimulated with G9-,«,,;and panel 6, CTLs from patient 5 stimulated with G9,54. Established cell line targets included 952mel (•:HLA-A2».gplOO4), 526mel (•;HLAA2*, gplOu*), 3