T cell-defined tumor antigens
Pierre van der Bruggen, Vincent Stroobant, Nathalie Vigneron, and Benoît Van den Eynde
Human tumor antigens recognized by CD4+ or CD8+ T cells are being defined at a regular pace. We have tried to classify them into four major groups on the basis of their expression pattern. This classification may appear arbitrary and indeed reflects the biases derived from our own studies, which have mostly dealt with melanoma. The interest of such a classification is practical, as the expression pattern of the antigens is the critical factor determining their potential usefulness for cancer immunotherapy. Although we have tried to incorporate most tumor antigens identified so far, we have certainly missed some. We will try to update the data regularly and we encourage investigators to submit additional information to be included in the database.
A first distinction can be made between unique antigens (Table 1) and shared antigens. The shared antigens can be further divided into tumor-specific antigens (Table 2), differentiation antigens (Table 3) and overexpressed antigens (Table 4). The tables provide the following information for each antigen:
(a) a GeneCard link for the encoding gene and/or the parent protein,
(b) the HLA presenting molecule and its frequency in Caucasians,
(c) the peptide sequence and its position in the protein sequence,
(d) the method used to isolate the CTL recognizing the antigen,
(e) a PubMed link to the relevant reference.
Each line corresponds to a peptide, considered to be a tumor antigen based on its recognition by T lymphocytes that also recognize tumor cells expressing the parent protein. As indicated in the penultimate column, such T lymphocytes have been derived in vitro by stimulating lymphocytes either with autologous tumor cells or with antigen-presenting cells pulsed with peptide or engineered to express the relevant gene. The peptide indicated is usually the shortest synthetic peptide recognized by the T cells.
We have included in the database only the antigenic peptides that fulfill the following requirements:
The “validated peptides” we selected needed to meet the following requirements:
(1) Isolation of stable human T lymphocyte clones or lines recognizing the peptide.
(2) Identification of the peptide recognized by the T cells.
(3) Identification of the HLA presenting molecule.
(4) Evidence that the peptide is processed by tumor cells and presented to the specific CTL.
This implies showing recognition of tumor cells expressing the relevant gene and HLA molecule by the T cells. When a polyclonal T cell line is used rather than a clone, it is essential to demonstrate that the CTLs that lyse the tumor cells are the same as those that recognize the peptide. This can be done by “cold target inhibition” experiments using peptide-pulsed cold targets (1). Other means of proof are also possible, such as the testing of stable transfectants of tumor cells with the sequence encoding the parental protein (2) or knocking down the gene encoding the peptide using the si or shRNA technology (3).
In some cases, unusual processing features of the peptide (e.g. by only some proteasome subtypes (4-9)) may explain the lack of recognition of some tumor cells by the CTL, without precluding the “validation” of the peptide, provided it is explained.
When post-translational modifications are involved, characterization of the peptide should include elution of the peptide from the cell surface (10-13). Eluted fractions can then be tested for their ability to activate the CTL. The CTL-sensitizing fraction should correspond to the fraction able to sensitize the CTL when the synthetic peptide of interest is fractionated by HPLC in the exact same conditions. Alternatively, the presence of the peptide of interest in the CTL-sensitizing fraction could also be demonstrated by mass spectrometry.
In the case of CD4 T lymphocytes, which may not recognize tumor cells directly, the fact that the peptide is processed can be shown by testing antigen-presenting cells loaded with the recombinant protein or a control protein produced in the same organism (14, 15), or loaded with lysates of cells transfected or not with the relevant coding sequence.
(5) Performing a peptide sensitization assay.
Characterization of peptides recognized by CD8 T cells should generally include the identification of the shortest peptide recognized and a titration showing a clear recognition of this peptide at doses below 1 µM. Putting together this update, we realized that in recent years, fewer investigators perform this control. In the case of peptides that were identified by the “reverse immunology approach” (an approach that consists in raising T cells against specific peptides corresponding to fragments of conventional proteins), we considered that the peptide titration curves might not be mandatory as the CTL was directly obtained against the peptide of interest. However, we believe that peptide titration curves remain crucial in order to determine the most adequate immunotherapeutic vaccination modalities (cfr. the B*4402-restricted peptide MAGE-A1 KEADPTGHSY example below) and should therefore always be included when describing new potential vaccine candidates.
(6) Describing the pattern of antigen expression.
A certain level of tumor- or tissue-specificity should be documented, as ubiquitous antigens do not qualify as tumor antigens. This can be done with gene expression, protein expression or lymphocyte recognition data, which should ideally be corroborative.
Unique antigens result from point mutations in genes that are expressed ubiquitously (Mutation). The mutation usually affects the coding region of the gene and is unique to the tumor of an individual patient or restricted to very few patients. Some of these mutations may be implicated in tumoral transformation. Such antigens, which are strictly tumor-specific, may play an important role in the natural anti-tumor immune response of individual patients, but most of them cannot be easily used as immunotherapeutic targets because they are not shared by tumors from different patients.
On the other hand, shared antigens are present on many independent tumors. They can be further divided into three groups. One group corresponds to peptides encoded by “cancer-germline” genes, such as MAGE, which are expressed in many tumors but not in normal tissues (Shared Tumor-specific). The only normal cells in which significant expression of such genes has been detected are placental trophoblasts and testicular germ cells. Because these cells do not express MHC class I molecules, gene expression should not result in expression of the antigenic peptides and such antigens can therefore be considered as strictly tumor-specific. The genes encoding such antigens have also been referred to as “Cancer/Testis” (CT) genes.
A second group of shared tumor antigens, named differentiation antigens, are also expressed in the normal tissue of origin of the malignancy (Differentiation). The paradigm is tyrosinase, which is expressed in normal melanocytes and in most melanomas. Antigens of this group are not tumor-specific, and their use as targets for cancer immunotherapy may result in autoimmunity towards the corresponding normal tissue. In the case of melanocytes, the risk of inducing severe side effects appears minimal, and could be limited to the appearance of vitiligo. More serious concerns about autoimmune side effects apply to carcinoembryonic antigen (CEA), an oncofetal protein expressed in normal colon epithelium and in most gut carcinomas. Autoimmune toxicity should not be an issue, however, in situations where the tissue expressing the antigen is dispensable or even resected by the surgeon in the course of cancer therapy, as would be the case for prostate specific antigen (PSA).
It is much more difficult to make predictions regarding the safety of targeting shared antigens of the third group, which are expressed in a wide variety of normal tissues and overexpressed in tumors (Overexpressed). Because a minimal amount of peptide is required for CTL recognition, a low level of expression in normal tissues may mean that autoimmune damage is not incurred. However, this threshold is difficult to define, as is the normal level of expression of those genes for each cell type.
A large series of additional peptides have been described which have not (yet) been included in the tables because formal evidence to fulfill one or several of the aforementioned criteria has not been provided. The relevant references are listed (Potential). A number of viruses, such as the Epstein-Barr virus (EBV) and human papilloma virus (HPV), are associated with human malignancies. The antigenic peptides encoded by viral genes have not been included in the database, despite their high potential as targets for immunotherapy.
The text is divided into the following sections:
- Table 1. Tumor antigens resulting from mutation
- Table 2. Shared tumor-specific antigens
- Table 3. Differentiation antigens
- Table 4. Antigens overexpressed in tumors
- Potential antigens
- Schultz ES, Lethé B, Cambiaso CL, Van Snick J, Chaux P, Corthals J, Heirman C, Thielemans K, Boon T, van der Bruggen P. A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes. Cancer Res 2000; 60: 6272-6275. (PMID: 11103782)
- Vigneron N, Ooms A, Morel S, Ma W, Degiovanni G, Van den Eynde B. A peptide derived from melanocytic protein gp100 and presented by HLA-B35 is recognized by autologous cytolytic T lymphocytes on melanoma cells. Tissue Antigens 2005; 65: 156-162. (PMID: 15713214)
- Tomita Y, Imai K, Senju S, Irie A, Inoue M, Hayashida Y, Shiraishi K, Mori T, Daigo Y, Tsunoda T, Ito T, Nomori H, Nakamura Y, Kohrogi H, Nishimura Y. A novel tumor-associated antigen, cell division cycle 45-like can induce cytotoxic T-lymphocytes reactive to tumor cells. Cancer Sci 2011; 102: 697-705. (PMID: 21231984)
- Vigneron N, Van den Eynde BJ. Proteasome subtypes and the processing of tumor antigens: increasing antigenic diversity. Curr. Opin Immunol 2012; 24: 84-91. (PMID: 22206698)
- Ma W, Vigneron N, Chapiro J, Stroobant V, Germeau C, Boon T, Coulie PG, Van den Eynde BJ. A MAGE-C2 antigenic peptide processed by the immunoproteasome is recognized by cytolytic T cells isolated from a melanoma patient after successful immunotherapy. Int J Cancer 2011; 129: 2427-2434. (PMID: 21207413)
- Corbière V, Chapiro J, Stroobant V, Ma W, Lurquin C, Lethé B, van Baren N, Van den Eynde BJ, Boon T, Coulie PG. Antigen spreading contributes to MAGE vaccination-induced regression of melanoma metastases. Cancer Res 2011; 71: 1253-1262. (PMID: 21216894)
- Dalet A, Stroobant V, Vigneron N, Van den Eynde BJ. Differences in the production of spliced antigenic peptides by the standard proteasome and the immunoproteasome. Eur J Immunol. 2011; 41: 39-46. (PMID: 21182075)
- Chapiro J, Claverol S, Piette F, Ma W, Stroobant V, Guillaume B, Gairin J-E, Morel S, Burlet-Schiltz O, Monsarrat B, Boon T, Van den Eynde B. Destructive cleavage of antigenic peptides either by the immunoproteasome or by the standard proteasome results in differential antigen presentation. J Immunol 2006; 176: 1053-1061. (PMID: 16393993)
- Guillaume B, Chapiro J, Stroobant V, Colau D, Van Holle B, Parvizi G, Bousquet-Dubouch MP, Theate I, Parmentier N, Van den Eynde BJ. Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules. Proc Natl Acad Sci U S A 2010; 107: 18599-18604. (PMID: 20937868)
- Dalet A, Robbins PF, Stroobant V, Vigneron N, Li YF, El-Gamil M, Hanada K, Yang JC, Rosenberg SA, Van den Eynde BJ. An antigenic peptide produced by reverse splicing and double asparagine deamidation. Proc Natl Acad Sci USA 2011; 108: E323-331. (PMID: 21670269)
- Vigneron N, Stroobant V, Chapiro J, Ooms A, Degiovanni G, Morel S, van der Bruggen P, Boon T, Van den Eynde B. An antigenic peptide produced by peptide splicing in the proteasome. Science 2004; 304: 587-590. (PMID: 15001714)
- Skipper JCA, Hendrickson RC, Gulden PH, Brichard V, Van Pel A, Chen Y, Shabanowitz J, Wölfel T, Slingluff CL, Jr, Boon T, Hunt DF, Engelhard VH. 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 1996; 183: 527-534. (PMID: 8627164)
- Hanada K, Yewdell JW, Yang JC. Immune recognition of a human renal cancer antigen through post-translational protein splicing. Nature 2004; 427: 252-256. (PMID: 14724640)
- Chaux P, Vantomme V, Stroobant V, Thielemans K, Corthals J, Luiten R, Eggermont AM, Boon T, van der Bruggen P. Identification of MAGE-3 epitopes presented by HLA-DR molecules to CD4+ T lymphocytes. J Exp Med 1999; 189: 767-777. (PMID: 10049940)
- Zarour HM, Storkus WJ, Brusic V, Williams E, Kirkwood JM. NY-ESO-1 encodes DRB1*0401-restricted epitopes recognized by melanoma-reactive CD4+ T cells. Cancer Res 2000; 60: 4946-4952. (PMID: 10987311)
This resource should be cited as follows:
van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptide database: T cell-defined tumor antigens. Cancer Immun 2013. URL: http://www.cancerimmunity.org/peptide/
Address correspondence to:
Ludwig Institute for Cancer Research
74 avenue Hippocrate, UCL 74.59
The authors wish to acknowledge the contribution of Aline Van Pel, who retired early in 2010. She is replaced by Nathalie Vigneron.
Copyright © 2001 – 2014 by Pierre van der Bruggen et al.
Last updated: May 14, 2014