Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis - PubMed (original) (raw)

. 2001 Jan 1;193(1):73-88.

doi: 10.1084/jem.193.1.73.

N J Beekman, S A Bres-Vloemans, P Verdijk, P A van Veelen, A M Kloosterman-Joosten, D C Vissers, G J ten Bosch, M G Kester, A Sijts, J Wouter Drijfhout, F Ossendorp, R Offringa, C J Melief

Affiliations

Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis

J H Kessler et al. J Exp Med. 2001.

Abstract

We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A(*)0201-presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved "reverse immunology" strategy. Next to motif-based HLA-A(*)0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A(*)0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA(100-108); SLYSFPEPEA, PRA(142-151); ALYVDSLFFL, PRA(300-309); and SLLQHLIGL, PRA(425-433)) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A(*)0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.

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Figures

Figure 1

Figure 1

In vitro proteasome-mediated digestions of four 27-mer PRAME polypeptides containing potential HLA-A*0201–restricted epitopes. 20S proteasomes isolated from an EBV-transformed B cell line were coincubated with 27-mer PRAME peptides at 37°C for the indicated time points. Digestion mixtures were analyzed by mass spectrometry as described in Materials and Methods. Generated digestions fragments are depicted as shaded sequences. The digestion of 27-mer PRAME 90–116 is represented in A, digestion of PRAME 133–159 is depicted in B, in C the digestion of PRAME 290–316 is showed, and D represents the digestion of PRAME 415–441. Notes: 1 IC50 as determined in competition binding assay (see Table ); 2 peptides binding with high or intermediate affinity to HLA-A*0201 are shown. Predicted epitopes used for CTL induction are printed in bold; 3 digestion fragments are shaded and ordered according to their COOH terminus; 4 digestion fragments generated for <1% at 1 h digestion or <3% at 4 and 24 h incubation and not relevant for epitope prediction are not shown; 5 intensity is expressed as percentage of total summed mass-peak intensities of digested 27-mer at the indicated incubation time.

Figure 2

Figure 2

Proteasome-mediated cleavage patterns of 27-mer peptides encompassing 19 high affinity HLA-A*0201 binding PRAME peptides. 20S proteasomes isolated from an EBV-transformed B cell line were coincubated with 27-mer PRAME peptides at 37°C for the indicated time points. Digestion mixtures were analyzed by mass spectrometry as described in Materials and Methods. Major and minor cleavages sites at 1 h digestion are depicted. Notes: 1 all 19 high affinity binding peptides (IC50 < 6 μM) are listed and ranked according to their binding affinity for HLA-A*0201; 2 start aa position in PRAME of the HLA-A*0201 binding peptide. 3 IC50 (in μM) as determined in competition binding assay (see Table ); 4 start and end aa position in PRAME of 27-mer polypeptide encompassing the high affinity binding peptide; 5 aa sequence of 27-mer polypeptide (aa sequence of HLA-A*0201 binding peptide is printed in bold and shaded); 6 major (bold arrows) and minor (thin arrows) cleavage sites at 1 h incubation are depicted, classified according to the following definitions. Major site: fragments containing as COOH terminus the residue NH2-terminal from the cleavage are present for ≥5% at 1 h incubation. Minor site: fragments containing as COOH terminus the residue NH2-terminal from the cleavage are present for <5% at 1 h incubation. 7 Generation by digestion of fragments containing the correct COOH terminus of the HLA-A*0201 binding peptide. Generation at 1 h digestion or after a longer incubation period is separately indicated. Classification: (++) present for ≥5%, (+) present for <5%, (−) no fragments containing the correct COOH terminus were found. 8 Generation by digestion of fragments containing the intact HLA-A*0201 binding peptide and/or NH2-terminal elongated precursors of the peptide. Classification: (++) present for ≥5% at 1 h incubation, (+) present for <5% at 1 h or only detectable after 4 or 24 h, (−) no fragments containing the HLA-A*0201 binding peptide were found. 9 Epitope prediction based on digestion results. Classification: (+) most likely an epitope, (+/−) doubtful epitope, (−) not an epitope.

Figure 4

Figure 4

Recognition by CTL clones of four endogenously processed PRAME epitopes presented on melanoma cell lines in an HLA class I–restricted and proteasome-dependent fashion. (Top) Lysis of 51Cr-labeled melanoma cell line Mel603, expressing PRAME but lacking HLA-A*0201 expression (•), was tested vs. lysis of M453 (▪) and FM3 (▴), both expressing PRAME and HLA-A*0201 together. CTL clones no. 551 anti-PRA100–108, no. 314 anti-PRA142–151, no. 460 anti-PRA300–309, and no. 1257 anti-PRA425–433 were used at E/T ratios ranging from 25 to 0.75. (Middle) Lysis of 51Cr-labeled M453 was tested after 1 h preincubation with HLA class I blocking mAb W6.32 (black bars) or an IgG2a control Ab (gray bars). (Bottom) Lysis of 51Cr-labeled FM3 was tested after 17 h treatment with 10 μM of the proteasome inhibitor lactacystin (black bars) or without treatment (gray bars). As control, the lactacystin-treated cells were loaded with the relevant peptide (white bars). Results of one representative experiment of at least three performed are shown.

Figure 3

Figure 3

HLA-A*0201–restricted peptide specificity and sensitivity of CTL clones raised against four PRAME peptides. (A) Lysis by CTL clones no. 551 anti-PRA100–108, no. 314 anti-PRA142–151, no. 460 anti-PRA300–309, and no. 1257 anti-PRA425–433 of 51Cr-labeled T2 cells loaded with 5 μM of the relevant peptide (▴) vs. an irrelevant HLA-A*0201 binding peptide (•) at different E/T ratios ranging from 50 to 0.75. (B) Lysis by the same set of CTL clones of 51Cr-labeled T2 cells pulsed for 1 h with titrated concentrations of relevant peptide (▴). The CTL clones were used at an E/T ratio of 10. Results of one representative experiment out of three performed are shown.

Figure 5

Figure 5

Lysis of PRAME transfected renal cell carcinoma cell line MZ1851 by CTL anti-PRA300–309. CTL no. 460 directed against PRA300–309 was tested on 51Cr-labeled MZ1851 (HLA-A*0201+ but lacking PRAME expression) transfected with PRAME cDNA (▴) vs. MZ1851 transfected with the empty vector (•). CTL no. 460 was used at E/T ratios ranging from 50 to 0.75. Results of one representative experiment of three performed are shown.

Figure 6

Figure 6

Lysis of tumor cell lines from multiple histologic origins by CTL anti-PRAME in a PRAME-specific and HLA-A*0201–restricted fashion. (Top) Lysis of 51Cr-labeled renal cell carcinoma cell lines MZ1851, expressing HLA-A*0201 but PRAME negative (•), MZ1257 (PRAME+ and HLA-A*0201+) (▪), and MZ1774 (PRAME+ and HLA-A*0201+) (▴) was compared. (Middle) Lysis of 51Cr-labeled lung carcinoma cell lines. GLC02, expressing PRAME but HLA-A*0201 negative (•) and GLC36 (▴) expressing both PRAME and HLA-A*0201 was compared. (Bottom) 51Cr-labeled mammary carcinoma cell lines MCF7 (HLA-*0201+ and PRAME+) (•) and ZR-75-1, expressing PRAME but lacking HLA-A*0201 (▴), were tested. The CTL clones no. 551 anti-PRA100–108, no. 314 anti-PRA142–151, no. 460 anti-PRA300–309, and no. 1257 anti-PRA425–433 were used at E/T ratios ranging from 25 to 0.75. Results of one representative experiment of at least three performed are shown.

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