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© 2002 American Society for Clinical Oncology
Phase I Trial of Adoptive Immunotherapy With Cytolytic T Lymphocytes Immunized Against a Tyrosinase EpitopeMemorial-Sloan Kettering Cancer Center, New York, NY
To the Editor:In the February 15, 2002, issue of the Journal of Clinical Oncology, Mitchell et al1 reported the results of a phase I trial in which melanoma patients were infused with autologous T cells that had been stimulated in vitro against peptide YMNGTMSQVN. Although this is the amino acid sequence predicted from the tyrosinase DNA sequence for codons 369 to 377, it is well established that the peptide actually expressed by melanoma cells is YMDGTMSQVN, having undergone posttranslational deamination at the third amino acid (asparagine Work by Skipper et al2 showed that T cells against the YMN peptide do not cross-react with melanoma expressing the YMD peptide (although both bind to HLA-A2.1), suggesting that the third amino acid of this peptide is critical for interaction with the T-cell receptor. This may explain why the T cells expanded by the YMN peptide showed limited cytotoxicity against melanoma cells (Mitchell et al,1 Fig 1). This is also sufficient to explain why the investigators were unable to detect circulating cytotoxic T lymphocytes against melanoma or demonstrable targeting of indium-111–labeled T cells in patients after reinfusion of the expanded T cells. We find the clinical responses described in Fig 3 difficult to discern, which may possibly be explained by the inability of the infused cells to bind the native peptide. A typographical error appears to occur in the Introduction that could cause confusion and errors in clinical or laboratory experiments using this article as a reference. The first amino acid of the posttranslationally modified peptide is incorrectly identified as a threonine (T), rather than tyrosine (Y). REFERENCES
1. Mitchell MS, Darrah D, Yeung D, et al: Phase I trial of adoptive immunotherapy with cytolytic T lymphocytes immunized against a tyrosinase epitope. J Clin Oncol 20: 1075-1086, 2002
2. Skipper JC, Hendrickson RC, Gulden PH, et al: An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J Exp Med 183: 527-534, 1996
ResponseKarmanos Cancer Institute, Detroit, MI In Reply:We are grateful for the opportunity to reply to the comments of Drs Wolchok and Chapman about the relevance and importance of the encoded epitope sequence and their interpretation of our published data.1 Also, although it was not called into question, we would like to clarify our numbering of the tyrosinase epitope as 369 to 377 rather than as 368 to 376, as it is often presented. The first important point is that we conducted preliminary experiments to determine whether immunization with the unmodified epitope led to cross-reactive cytotoxic T lymphocytes (CTLs) capable of lysing melanoma cells. As shown in Fig 1, which was typical of several different experiments, immunization in vitro with YMNGTMSQV led to CTLs cross-reactive against the other epitope but not vice versa. This is what prompted us to test YMNGTMSQV in the study, with full knowledge of the article by Skipper et al,2 especially because in their report only a single CTL clone was evaluated, whereas our cell product was a polyclonal "bulk" culture.
Lysis of melanoma cells in our hands, and probably in most others’, is always less than that of heavily peptide-loaded T2 cells. However, that lysis is specific, as shown by far lower lysis of control HLA-matched target cells lacking the epitope (MALME 3) (Figs 2 and 3).
Perhaps most relevant to this discussion, Visseren et al3 obtained tyrosinase-specific CTL bulk cultures from an HLA-A2 donor against YMNGTMSQV-sensitized target cells. The bulk cultures had strong CTL activity against JY cells loaded with YMNGTMSQV peptide and melanoma cells SK-23 and 453 at low effector-to-target ratios without the addition of exogenous tyrosinase peptide. Ten CTL clones were isolated from the bulk culture that recognized YMNGTMSQV peptide, and eight of the 10 clones cross-reacted with melanoma cell lines SK-23, 453, and Mi 3046/2, in a tumor necrosis factor release assay. Lytic activity of three clones was tested further on the melanoma tumor panel and shown to be identical with that of the bulk culture. The melanoma cell panel was also lysed efficiently by CTL from five further bulk cultures generated from normal donors. The authors concluded that the YMNGTMSQV peptide was naturally processed and presented on melanoma cells, but the reactivity could equally well have been explained by cross-reactivity to YMDGTMSQV on the melanomas. Reduction of the size of the computed tomography scans in the final publication, which was a decision made by the Journal, undoubtedly made it difficult for Wolchok and Chapman to fully appreciate the change in the lesions, which was also observed on physical examination. The mediastinal nodes disappeared after treatment as we noted in the text, and the inguinal and iliac nodes dramatically shrank and became more discrete masses. The scans were read officially by disinterested university radiologists, not by the investigators. We would be pleased to provide either the full-size photographs or a copy of the original computed tomography scans to be certain Wolchok and Chapman see the changes we described. With regard to the numbering of the peptide, the original article by Brichard et al4 describing the cloning of the tyrosinase cDNA (clone 123.B2) presents a cDNA whose amino acid residues 369 to 377 are given as YMNGTMSQV in the direct submission sequence provided to Genbank. The authors note that in the sequence reported by Kwon et al5 in 1987, there is a three-base pair deletion beginning at base 124, which accounts for the numbering of the peptide beginning at 368 rather than 369 in some publications. Finally, we thank Wolchok and Chapman for correcting the typographical error substituting T for Y in the sequence of the YMDGTMSQV peptide in the Introduction. We believe that further refinement of our approach and the addition of other elements such as cytokines to the regimen may well yield more convincing results than our phase I study. Nevertheless, if the article stimulated in other readers the same degree of enthusiasm as we generated in Wolchok and Chapman, we may have achieved one of our aims. REFERENCES
1. Mitchell MS, Darrah D, Yeung D, et al: Phase I trial of adoptive immunotherapy with cytolytic T lymphocytes immunized against a tyrosinase epitope. J Clin Oncol 20: 1075-1086, 2002
2. Skipper JC, Hendrickson RC, Gulden PH, et al: An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J Exp Med 183: 527-534, 1996 3. Visseren MJ, van Elsas A, van der Voort EI, et al: CTL specific for the tyrosinase autoantigen can be induced from healthy donor blood to lyse melanoma cells. J Immunol 154: 3991-3998, 1995[Abstract]
4. Brichard V, Van Pel A, Wolfel T, et al: The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 178: 489-495, 1993
5. Kwon BS, Haq AK, Pomerantz SH, et al: Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus. Proc Natl Acad Sci U S A 84: 7473-7477, 1987 (erratum published in Proc Natl Acad Sci U S A 85:6352, 1988)
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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aspartic acid).
