Pascal Chaux

Tumor‐specific shared antigenic peptides recognized by human T cells

Summary: The first tumor‐specific shared antigens and the cancer‐germline genes that code for these antigens were identified with antitumor cytolytic T lymphocytes obtained from cancer patients. A few HLA class I‐restricted antigenic peptides were identified by this ‘direct approach’. A large set of additional cancer‐germline genes have now been identified by purely genetic approaches or by screening tumor cDNA expression libraries with the serum of cancer patients. As a result, a vast number of sequences are known that can code for tumor‐specific shared antigens, but most of the encoded antigenic peptides have not yet been identified. We review here recent ‘reverse immunology’ approaches for the identification of new antigenic peptides. They are based on in vitro stimulation of naive T cells with dendritic cells that have either been loaded with a cancer‐germline protein or that have been transduced with viruses carrying cancer‐germline coding sequences. These approaches have led to the identification of many new antigenic peptides presented by class I or class II molecules. We also describe some aspects of the processing and presentation of these antigenic peptides.

Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats

Tumors are tolerated by the immune system notwithstanding the expression of tumor‐associated antigens. PROb tumor cells, derived from a rat colon carcinoma, are rejected by tumor‐immune hosts but give rise to progressive tumors in naive hosts. Paradoxically, these tumors are heavily infiltrated by dendritic cells that express MHC class II and ICAM‐1. These tumor‐infiltrating dendritic cells (TiDCs) could be expected to process and present to T cells the antigens released by the adjacent tumor cells. Indeed, we report here that TiDCs, compared with splenic dendritic cells, are poor stimulators of primary allogeneic T‐cell proliferation and cytokine [interleukin‐2 (IL‐2) and interferon‐γ] production. Most of them (89–97%) do not express B7, an essential co‐stimulatory signal for T cells, even after a culture period allowing B7 up‐regulation on epidermal Langerhans cells. GM‐CSF in association with tumor necrosis factor‐α or IL‐4, or cell‐associated CD40‐ligand, all known to be potent stimulators of B7 expression on other dendritic cells, did not restore B7 expression by TiDCs. After a first exposure to TiDCs, allogeneic T‐cell response to a second challenge to splenic dendritic cells was decreased. The failure of most dendritic cells infiltrating PROb tumors to express B7, even after stimulation, may contribute to their poor capacity to stimulate T cells and could play a role in the immune tolerance allowing tumor growth. Int. J. Cancer 72:619–624, 1997.

Estimation of the frequencies of anti‐MAGE‐3 cytolytic T‐lymphocyte precursors in blood from individuals without cancer

Attempts to detect a cytolytic T‐lymphocyte (CTL) response in melanoma patients vaccinated with MAGE‐3 peptides have been negative so far, even though some tumor regressions have been observed. The detection of such responses may require very sensitive detection assays for CTL precursors. To this end, we set up a method whereby a large number of CD8+ T‐cell microcultures are stimulated with autologous antigen‐presenting cells incubated with a peptide, in the presence of interleukin (IL)‐6 and IL‐12 during the first week, and IL‐2 and IL‐7 from the second week. We report here that not only monocyte‐derived dendritic cells but also activated T cells incubated with the MAGE‐3 antigenic peptide presented by HLA‐A2 were effective in activating specific CTL precursors present in the blood of individuals without cancer. These precursors were detected in the CD8+CD45RA+ subpopulation of T cells. Among the CD8+ T‐lymphocyte population of blood donors, the frequency of CTL precursors specific for the MAGE‐3.A2 antigen ranged from 4 to 17 × 10−7. For the MAGE‐3 antigenic peptide presented by HLA‐A1, this frequency ranged from 0.4 to 3 × 10−7. Knowing that several parameters of this procedure still have to be optimized, we will begin to use it to evaluate the CTL precursor frequencies of cancer patients before and after injection of MAGE peptides. Int. J. Cancer 77:538–542, 1998.

A MAGE-A4 peptide presented by HLA-A2 is recognized by cytolytic T lymphocytes

The MAGE‐encoded antigens that are recognized by cytolytic T lymphocytes (CTL) are shared by many tumors and are strictly tumor specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes. We have used a method to identify CTL epitopes, which selects naturally processed peptides. CD8+ T cells, obtained from individuals without cancer, were stimulated with autologous dendritic cells infected with a recombinant adenovirus containing the MAGE‐A4 coding sequence. Responder cell microcultures that specifically lysed autologous EBV‐transformed B cells infected with vaccinia‐MAGE‐A4 were cloned using autologous stimulator cells infected with a Yersinia enterocolitica carrying the MAGE‐A4 sequence. An anti‐MAGE‐A4 CTL clone was obtained and the epitope was found to be decapeptide GVYDGREHTV (amino acids 230 – 239) presented by HLA‐A2 molecules. The CTL clone lysed HLA‐A2 tumor cells expressing MAGE‐A4. This is the first reported antigenic peptide encoded by MAGE‐A4. It may be valuable for cancer immunotherapy because MAGE‐A4 is expressed in 51 % of lung carcinomas and 63 % of esophageal carcinomas, whereas about 50 % of Caucasians and Asians express HLA‐A2.

A MAGE-3 peptide presented by HLA-DR1 to CD4(+) T cells that were isolated from a melanoma patient vaccinated with a MAGE-3 protein

“Cancer-germline” genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific Ags, which have been used in therapeutic vaccination trials of cancer patients. MAGE-3 is expressed in 74% of metastatic melanoma and in 50% of carcinomas of esophagus, head and neck, bladder, and lung. We report here the identification of a new MAGE-3 peptide, which is recognized by three different CD4+ T cell clones isolated from a melanoma patient vaccinated with a MAGE-3 protein. These clones, which express different TCRs, recognize on HLA-DR1 peptide ACYEFLWGPRALVETS, which corresponds to the MAGE-3267–282 and the MAGE-12267–282 protein sequences. One of the T cell clones, which expresses LFA-1 at a high level, lysed tumor cells expressing DR1 and MAGE-3. Another of these DR1-restricted CD4+ clones recognized not only the MAGE-3/12 peptide but also homologous peptides encoded by genes MAGE-1, 2, 4, 6, 10, and 11.

A MAGE-1 peptide recognized on HLA-DR15 by CD4(+) T cells.

Antigens encoded by MAGE genes and recognized by T cells are of interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Several MAGE‐1 peptide that are recognized by CD8+ cytolytic T lymphocytes have been used in therapeutic vaccination trials. To obtain anti‐tumor immune response, vaccines combining peptides recognized by CD8+ and peptides recognized by CD4+ T cells might be optimal. We focused therefore on the identification of MAGE peptides recognized by CD4+ T cells. We report here the identification of MAGE‐1 epitope EYVIKVSARVRF, which is presented to CD4+ T lymphocytes by HLA‐DR15. This HLA allele is present in 29 % of Asians and 17 % of Caucasians.

A novel approach to identify antigens recognized by CD4 T cells using complement-opsonized bacteria expressing a cDNA library

In patients with hematological malignancies receiving HLA-matched stem cell transplantation, T cells specific for minor histocompatibility antigens play a major role in graft rejection, induction of graft-versus-host disease and beneficial graft-versus-leukemia reactivity. Several human minor histocompatibility antigens recognized by T cells have been identified, but only two are presented by HLA class II molecules. In search of an efficient approach to identify antigenic peptides processed through the HLA class II pathway, we constructed a cDNA library in bacteria that were induced to express proteins. Bacteria were opsonized with complement to enforce receptor-mediated uptake by Epstein–Barr virus immortalized B cells that were subsequently used as antigen-presenting cells. This approach was validated with an HLA class II-restricted antigen encoded by gene DBY. We were able to identify bacteria expressing DBY diluted into a 300-fold excess of bacteria expressing a nonrelevant gene. Screening of a bacterial library using a DBY-specific CD4 T cell clone resulted in the isolation of several DBY cDNAs. We propose this strategy for a rapid identification of HLA class II-restricted antigenic peptides recognized by CD4 T cells.

Towards vaccination with defined tumor antigens

Following the identification of the first tumor antigens recognized by CTL on melanoma cells, concern was expressed that tumors of other histological types might be less likely to express such antigens. However, the results obtained now with other types of tumors suggest that there is no fundamental difference in this respect between melanoma and other tumors. As new antigens are discovered, an increasing proportion are found to be the result of mutations, several of which may play a role in tumoral transformation or progression. Some of these tumor antigens are currently in the early stages of clinical study. There can be little doubt that the coming years will witness a large number of clinical trials involving peptides, proteins, DNA, and recombinant defective viruses, while increasingly sensitive tools are developed to measure the induction of CTL responses in immunized patients.