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T Boon

Publications and source records attributed to T Boon.

At least 55 records · Page 3Linked to original sources
PubMed1999

Identification of a second major tumor-specific antigen recognized by CTLs on mouse mastocytoma P815.

Murine mastocytoma P815 induces CTL responses against at least four distinct Ags (AB, C, D, and E). Recent studies have shown that the main component of the CTL response against the P815 tumor is targeted against Ags P815AB and P815E. The gene P1A has been well characterized. It encodes the P815AB Ag in the form of a nonameric peptide containing two epitopes, P815A and P815B, which are recognized by different CTLs. Here, we report the identification of the P815E Ag. Using a cDNA library derived from tumor P815, we identified the gene coding for P815E. We also characterized the antigenic peptide that anti-P815E CTLs recognize on the MHC class I molecule H-2Kd. The P815E Ag results from a mutation within an ubiquitously expressed gene encoding methionine sulfoxide reductase, an enzyme that is believed to be important in the protection of proteins against the by-products of aerobic metabolism. Surprisingly, immunizing mice i.p. with syngeneic tumor cells (L1210) that were constructed to express B7-1 and P815E did not induce resistance against live P815, even though a strong anti-P815E CTL response was observed with splenocytes from immunized animals.

Amino Acid Sequence↗
PubMed1999

Identification of MAGE-3 epitopes presented by HLA-DR molecules to CD4(+) T lymphocytes.

MAGE-type genes are expressed by many tumors of different histological types and not by normal cells, except for male germline cells, which do not express major histocompatibility complex (MHC) molecules. Therefore, the antigens encoded by MAGE-type genes are strictly tumor specific and common to many tumors. We describe here the identification of the first MAGE-encoded epitopes presented by histocompatibility leukocyte antigen (HLA) class II molecules to CD4(+) T lymphocytes. Monocyte-derived dendritic cells were loaded with a MAGE-3 recombinant protein and used to stimulate autologous CD4(+) T cells. We isolated CD4(+) T cell clones that recognized two different MAGE-3 epitopes, MAGE-3114-127 and MAGE-3121-134, both presented by the HLA-DR13 molecule, which is expressed in 20% of Caucasians. The second epitope is also encoded by MAGE-1, -2, and -6. Our procedure should be applicable to other proteins for the identification of new tumor-specific antigens presented by HLA class II molecules. The knowledge of such antigens will be useful for evaluation of the immune response of cancer patients immunized with proteins or with recombinant viruses carrying entire genes coding for tumor antigens. The use of antigenic peptides presented by class II in addition to peptides presented by class I may also improve the efficacy of therapeutic antitumor vaccination.

Amino Acid Sequence↗
PubMed1999

Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1.

Thirty-nine tumor-bearing patients with metastatic melanoma were treated with 3 subcutaneous injections of the MAGE-3.A1 peptide at monthly intervals. No significant toxicity was observed. Of the 25 patients who received the complete treatment, 7 displayed significant tumor regressions. All but one of these regressions involved cutaneous metastases. Three regressions were complete and 2 of these led to a disease-free state, which persisted for more than 2 years after the beginning of treatment. No evidence for a cytolytic T lymphocyte (CTL) response was found in the blood of the 4 patients who were analyzed, including 2 who displayed complete tumor regression. Our results suggest that injection of the MAGE-3.A1 peptide induced tumor regression in a significant number of the patients, even though no massive CTL response was produced.

Adult↗
PubMed1999

Improved efficacy of dendritic cell vaccines and successful immunization with tumor antigen peptide-pulsed peripheral blood mononuclear cells by coadministration of recombinant murine interleukin-12.

The well-characterized P815 tumor model was used to optimize anti-tumor immunization approaches in mice. Tumor peptides derived from antigens P198 or P1A were targeted to antigen-presenting cells (APC) by ex vivo pulsing. Initial experiments with irradiated pulsed splenic dendritic cells (sDC) injected weekly in the hind footpads for 3 weeks demonstrated cytolytic T lymphocyte (CTL) generation in 10-20% of mice. Because of the importance of interleukin-12 (IL-12) in tumor rejection responses, pulsed sDCs also were given together with recombinant murine IL-12 (rmIL-12). This strategy induced peptide-specific CTL in 100% of the mice. The IL-12 had to be injected in the footpads on days 0, 1 and 2 of each immunization week to achieve an optimal effect. The improvement seen with the addition of IL-12 prompted examination of other sources of APC. Purified resting B cells, lipopolysaccharide (LPS) blasts and nonfractionated splenocytes or peripheral blood mononuclear cells (PBMC) were pulsed with peptide and administered with the same schedule of rmIL-12. Because these cell types appeared to bind peptides less avidly than did DC, increasing peptide doses were used during pulsing. Interestingly, immunization with each of these APC also induced specific CTL in 100% of mice, provided rmIL-12 was coadministered. CTLs were detected both in the spleen and in the peripheral blood. Immunization with irradiated, P1A-pulsed PBMC plus rmIL-12 resulted in protection against challenge with tumors expressing the specific antigen in all mice. The ease by which human patient PBMCs can be prepared provides a straightforward vaccination approach to be used in clinical trials of peptide-based immunization in melanoma.

Animals↗
PubMed1999

A new family of mouse genes homologous to the human MAGE genes.

The human MAGE genes are expressed in a wide variety of tumors but not in normal cells, with the exception of the male germ cells, placenta, and, possibly, cells of the developing embryo. These genes encode tumor-specific antigens recognized by cytolytic T lymphocytes. The MAGE genes are located on the X chromosome, in three clusters denoted MAGE-A, B, and C, mapping at q28, p21.3, and q26, respectively. The function of these genes remains unknown. Because mice offer many advantages for the study of genes that may be involved in embryonic development, we looked for the murine equivalents of the 12 human MAGE-A genes. Using a MAGE-A probe, we isolated 8 new murine genes that are homologous to the MAGE genes. On average, the open reading frames (ORFs) of these 8 closely related genes display a slightly higher degree of nucleotide identity with the MAGE-A ORFs than with the MAGE-B or MAGE-C ORFs. Furthermore, like MAGE-A genes, they encode acidic proteins, whereas the MAGE-B genes encode basic proteins. Accordingly, these 8 murine genes were named Mage-a1 to 8 (approved symbols Magea1 to 8). Mage-a genes were mapped in two different loci on the mouse X chromosome. Mage-a4 and Mage-a7 are located in a region that is syntenic to either Xp21 or Xq28. The 6 other genes are arranged in a cluster located in a region syntenic to Xp22. Like their human counterparts, Mage-a genes were found to be transcribed in adult testis, but not in other tissues. Expression of some Mage-a genes was also detected in tumor cell lines. Two Mage-a genes were found to be expressed in blastocysts.

Animals↗
PubMed1999

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.

Adenoviridae↗
PubMed1999

DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter.

A subset of male germ line-specific genes, the MAGE-type genes, are activated in many human tumors, where they produce tumor-specific antigens recognized by cytolytic T lymphocytes. Previous studies on gene MAGE-A1 indicated that transcription factors regulating its expression are present in all tumor cell lines whether or not they express the gene. The analysis of two CpG sites located in the promoter showed a strong correlation between expression and demethylation. It was also shown that MAGE-A1 transcription was induced in cell cultures treated with demethylating agent 5'-aza-2'-deoxycytidine. We have now analyzed all of the CpG sites within the 5' region of MAGE-A1 and show that for all of them, demethylation correlates with the transcription of the gene. We also show that the induction of MAGE-A1 with 5'-aza-2'-deoxycytidine is stable and that in all the cell clones it correlates with demethylation, indicating that demethylation is necessary and sufficient to produce expression. Conversely, transfection experiments with in vitro-methylated MAGE-A1 sequences indicated that heavy methylation suffices to stably repress the gene in cells containing the transcription factors required for expression. Most MAGE-type genes were found to have promoters with a high CpG content. Remarkably, although CpG-rich promoters are classically unmethylated in all normal tissues, those of MAGE-A1 and LAGE-1 were highly methylated in somatic tissues. In contrast, they were largely unmethylated in male germ cells. We conclude that MAGE-type genes belong to a unique subset of germ line-specific genes that use DNA methylation as a primary silencing mechanism.

Azacitidine↗
PubMed1999

Derivation of tumor-specific cytolytic T-cell clones from two lung cancer patients with long survival.

We derived lung carcinoma cell lines from tumor material resected from a patient with small-cell lung cancer (SCLC) and from a patient with non-small-cell lung cancer (NSCLC). The patient with NSCLC was vaccinated with irradiated autologous tumor cells. The two patients enjoyed an exceptionally favorable clinical evolution and are currently without signs of cancer 10 and 8 yr after their diagnoses, respectively. Autologous mixed lymphocyte-tumor cell cultures (MLTC) were produced with blood lymphocytes stimulated with irradiated autologous tumor cells. The first patient's SCLC cells, which carried a small amount of human leukocyte antigen (HLA) class I molecules, were incubated with interferon-gamma (IFN-gamma) before being used as stimulator cells. A cytolytic T-lymphocyte (CTL) clone was derived that specifically lysed the IFN-gamma-treated SCLC cells but did not lyse untreated tumor cells or autologous lymphoblasts. Clones of autologous tumor-specific CTL, directed against the NSCLC cells of the other patient, were also obtained. These tumor cells carried a higher level of HLA class I molecules and were lysed by the CTL without incubation with IFN-gamma. Altogether, these results indicate that SCLC and NSCLC cancer cells can be recognized by autologous CTL, and might therefore be susceptible to specific immunotherapy.

Carcinoma, Non-Small-Cell Lung↗
PubMed1999

Enhancement by IL-12 of the cytolytic T lymphocyte (CTL) response of mice immunized with tumor-specific peptides in an adjuvant containing QS21 and MPL.

Immunization of cancer patients with tumor-specific antigenic peptides is currently being tested in several clinical studies. We have examined the induction of CTL responses in mice after various modalities of peptide vaccination, to explore protocols that could be applied to humans. Our first model antigen was P198, which results from a point mutation in a normal gene. While two immunizations with peptide P198 in SBAS-1c adjuvant induced measurable CTL responses in less than 10% of DBA/2 mice, the addition of IL-12 to the peptide adjuvant mixture resulted in high CTL responses in nearly all mice. This strong enhancing effect of IL-12 was observed with 1,000 and 300 units and decreased gradually as the doses were reduced to 30 units. When IL-12 was replaced by other cytokines acting on T cells or antigen-presenting cells, such as IFN-gamma, IL-2, IL-6, IL-7, GM-CSF or MCP-3, no significant enhancing effect was observed. The same effect of IL-12 was obtained with peptide P1A, which is a major tumor-specific antigen of mastocytoma P815 and is encoded by a gene that is specifically activated in tumors.

Adjuvants, Immunologic↗
PubMed1998

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 x 10(-7). For the MAGE-3 antigenic peptide presented by HLA-A1, this frequency ranged from 0.4 to 3 x 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.

Antibodies, Neoplasm↗
PubMed1998

An antigen recognized by autologous CTLs on a human bladder carcinoma.

By stimulating blood lymphocytes with autologous bladder carcinoma cells that had been transfected with B7-1, we obtained a panel of CTL clones which lyse specifically the bladder tumor cells in an MHC class I-restricted fashion. Based on inhibition with anti-HLA Abs and the recognition of allogeneic tumor cells, we could distribute our clones in three groups that recognized three distinct Ags. We characterized one of these Ags by screening a cDNA library prepared with the RNA from this bladder tumor line. This new tumor Ag is a peptide presented by HLA-B4403 molecules. It is produced by a point mutation in a gene that is recorded in databases under the name KIAA0205, is ubiquitously expressed, and whose function is unknown. We also found this mutation in the tumor sample that was originally resected from this patient, but the mutation was not found in the 100 or more independent tumors of various histologic types that were tested. This report is the first to describe the isolation of CTL clones directed against human bladder cancer and the molecular characterization of a bladder tumor Ag.

Antigens, Neoplasm↗
PubMed1998

LAGE-1, a new gene with tumor specificity.

Representational difference analysis was used to identify genes that are expressed in a human melanoma cell line and not in normal skin. A cDNA clone that appeared to be specific for tumors was obtained and the corresponding gene was sequenced. This new gene was named LAGE-I. Using a LAGE-I probe to screen a cDNA library from the same melanoma cell line, we identified a closely related gene, which proved to be identical to NY-ESO-I, a gene recently reported to code for an antigen recognized by autologous antibodies in an esophageal squamous cell carcinoma. Gene LAGE-I maps to Xq28. It comprises 3 exons. Alternative splicing produces 2 major transcripts encoding polypeptides of 210 and 180 residues, respectively. Expression of LAGE-I was observed in 25-50% of tumor samples of melanomas, non-small-cell lung carcinomas, bladder, prostate and head and neck cancers. The only normal tissue that expressed the gene was testis. As for MAGE-AI, expression of LAGE-I is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation. The expression of LAGE-I is strongly correlated with that of NY-ESO-I. It is also clearly correlated with the expression of MAGE genes.

Amino Acid Sequence↗
PubMed1998

Identification of a new MAGE gene with tumor-specific expression by representational difference analysis.

Human genes expressed exclusively in tumors and male germ line cells, such as those of the MAGE, BAGE, and GAGE families, encode antigens recognized by T lymphocytes, which are potentially useful for antitumor immunotherapy. To identify new genes of this type, we generated cDNA populations enriched in sequences expressed only in testis and melanoma, using the representational difference analysis approach. A testis cDNA library enriched by subtraction with cDNA from four other normal tissues was hybridized with radiolabeled melanoma cDNA enriched by subtraction with normal skin cDNA. A cDNA fragment sharing significant homology with MAGE genes was identified, and a cosmid containing this new gene, named MAGE-C1, was isolated. MAGE-C1 is composed of four exons and encodes a putative protein of 1142 amino acids. It is about 800 residues longer than the other MAGE proteins due to the insertion of a large number of short repetitive sequences in front of the MAGE-homologous sequence. The MAGE-C1 gene appears to be located on band Xq26, whereas the MAGE-A and MAGE-B genes are located on Xq28 and Xp21, respectively. Like other MAGE genes, MAGE-C1 is expressed in a significant proportion of tumors of various histological types, whereas it is silent in normal tissues except testis. It is probable, therefore, that like other MAGE genes, MAGE-C1 encodes antigens that may constitute useful targets for cancer immunotherapy because of their strict tumoral specificity.

Amino Acid Sequence↗
PubMed1998

Overlapping peptides of melanocyte differentiation antigen Melan-A/MART-1 recognized by autologous cytolytic T lymphocytes in association with HLA-B45.1 and HLA-A2.1.

From the peripheral blood lymphocytes (PBLs) of melanoma patient SK29(AV) we have previously isolated 2 independent cytolytic T lymphocyte (CTL) clones (CTL7/147 and CTL13/211), which lysed autologous tumor cells in association with HLA-B45.1. As demonstrated here, both CTL clones were directed against melanocyte differentiation antigen Melan-A/MART-1, which also was recognized by HLA-A2.1-restricted CTLs from the same patient. By generating and transfecting 3'-deletion mutants of Melan-A/MART-1 cDNA, we localized its peptide-coding regions. The HLA-B45.1-presented peptides were derived from a hydrophobic region of the protein and largely overlapped the peptides recognized by CTLs from the same patient in association with HLA-A2.1. We determined the fine specificity of these CTL clones with synthetic peptides. CTL clone CTL7/147 recognized the 11-mer peptide AEEAAGIGILT (residues 24-34) at the lowest concentrations. The absence of threonine-34 abrogated the recognition by CTL7/147. The truncated peptide AEEAAGIGIL (residues 24-33) proved to be the optimal synthetic peptide for sensitization against lysis by CTL13/211. This indicated that C-terminal threonine-34 was not involved in binding to HLA-B45.1 but, rather, was part of the epitope for CTL7/147. HLA-B45.1-associated peptides of Melan-A/MART-1 were regularly processed and presented by other melanomas and other cell types. Three of 4 independent HLA-A2.1-restricted SK29-CTL clones recognized the 10-mer peptide EAAGIGILTV (residues 26-35) at 10- to 100-fold lower concentrations than the nonamer AAGIGILTV (residues 27-35), previously described as the common immunodominant peptide antigen for all known anti-Melan-A/MART-1 CTLs restricted by HLA-A2.1. Different melanoma peptide antigens currently are applied in therapeutic vaccination studies. Our findings emphasize that restricting to peptides of minimal length might exclude relevant T-cell epitopes.

Amino Acid Sequence↗
PubMed1998

The shared tumor-specific antigen encoded by mouse gene P1A is a target not only for cytolytic T lymphocytes but also for tumor rejection.

A number of human tumor antigens have been characterized recently using cytolytic T lymphocytes (CTL) as screening tools. Some of them are encoded by MAGE-type genes, which are silent in normal tissues except in male germ cells, but are activated in a variety of tumors. These tumor-specific shared antigens appear to be promising targets for cancer immunotherapy. However, the choice of these antigens as targets has been questioned because of the lack of direct evidence that in vivo responses against such antigens can lead to tumor rejection. The antigen encoded by the mouse gene P1A represents the only available animal model system for MAGE-type tumor antigens. We show here that mice immunized by injection of L1210 leukemia cells expressing P1A and B7-1 (L1210.P1A.B7-1) are efficiently protected against a challenge with a lethal dose of mastocytoma P815 tumor cells, which express P1A. Mice immunized with L1210 cells expressing B7-1 but not P1A were not protected. Furthermore, we observed that P1A-transgenic mice, which are tolerant to P1A, were not protected after immunization with L1210.P1A.B7-1. These results demonstrate that the immune response to P1A is the major component of the tumor rejection response observed in normal mice, and support the use of tumor-specific shared antigens as targets for the immunotherapy of human cancer.

Animals↗
PubMed1998

PRAME, a gene encoding an antigen recognized on a human melanoma by cytolytic T cells, is expressed in acute leukaemia cells.

Gene PRAME was found to encode an antigen recognized on a human melanoma cell line by an autologous cytolytic T-lymphocyte clone. This gene is expressed at a high level in a very large fraction of tumours, such as melanomas, non-small-cell lung carcinomas, sarcomas, head and neck tumours and renal carcinomas. It is therefore a candidate for tumour immunotherapy even though some low expression is found in certain normal tissues. We tested by RT-PCR the expression of PRAME on more than 250 bone marrow or blood samples from patients with a haematological malignancy. Approximately 25% of the acute leukaemia samples were positive. Remarkably, all acute myeloblastic leukaemias that carried the chromosomal translocation t(8;21), which fuses the genes AML1 and ETO, expressed PRAME at a high level.

Acute Disease↗
PubMed1998

Mutations of the beta2-microglobulin gene result in a lack of HLA class I molecules on melanoma cells of two patients immunized with MAGE peptides.

Mutations have been identified in the beta2-microglobulin gene of tumor cells of two metastatic melanoma patients who received immunizations with MAGE peptides. One mutation abolishes the start codon whereas the other introduces a premature stop codon. The second beta2-microglobulin allele of both tumors appears to be lost on the basis of sequence data and loss of microsatellite heterozygosity. The lack of beta2-microglobulin gene product results in the absence of HLA class I antigens on the surface of the tumor cells. This may explain why the tumors of both patients progressed despite the immunization treatment and shows the necessity of analyzing in depth the antigen presentation capability of the tumor cells for the interpretation of clinical trials involving anti-tumor vaccination.

Antigens, Neoplasm↗