Conference on cancer vaccines.
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Animals↗
Biomedical subjects
G Parmiani
Publications and source records attributed to G Parmiani.
Immunology and immunotherapy of human cancer: present concepts and clinical developments.
Immunotherapy of cancer is entering into a new phase of active investigation both at the pre-clinical and clinical level. This is due to the exciting developments in basic immunology and tumour biology that have allowed a tremendous increase in our understanding of mechanisms of interactions between the immune system and tumour cells. This review briefly summarizes the state of the art in basic tumour immunology before discussing the clinical applications of the new concepts in the clinical setting. Clinical approaches are diverse but can now be based on strong scientific rationales. The analysis of the available clinical results suggests that, despite some disappointments, there is room for optimism that both active immunotherapy (vaccination) and adoptive immunotherapy may soon become part of the therapeutic arsenal to combat cancer in a more efficient way.
Antigens, Neoplasm↗
Type I consensus interferon (CIFN) gene transfer into human melanoma cells up-regulates p53 and enhances cisplatin-induced apoptosis: implications for new therapeutic strategies with IFN-alpha.
In this study, we describe the effects produced by the retroviral transduction of human type I consensus IFN (CIFN) coding sequence into the 8863 and 1B6 human melanoma cell lines, derived from a metastatic and a primary human melanoma, respectively. Melanoma cell lines producing approximately 103 IU/ml of IFN were obtained. Interestingly, cisplatin treatment of IFN-producing 8863 and 1B6 melanoma cells resulted in a three- to four-fold increase in the percentage of apoptotic cells with respect to similarly treated parental or control-transduced cell cultures. A similar effect, although less intense, was caused by cultivation of parental melanoma cells in the presence of exogenous CIFN. The increased susceptibility of the IFN-producing melanoma cell lines to cisplatin-induced apoptosis was associated with an IFN-dependent accumulation of p53, which also correlated with a decrease in Bcl-2 expression. Addition of exogenous CIFN to parental melanoma cells resulted in similar although weaker modulations of p53 and Bcl-2 expression. Cisplatin administration to nude mice bearing 3-day-old IFN-producing 8863 tumors resulted in complete tumor regression, while only a partial tumor inhibition was observed upon cisplatin treatment of mice bearing parental or control-transduced 8863 tumors. Starting the cisplatin treatment 7 days after tumor cell injection still resulted in a stronger inhibition of tumor growth in the mice bearing IFN-producing 8863 tumors as compared with parental tumor-bearing mice. A comparable therapeutic effect was obtained after repeated peritumoral administration of 103 IU of exogenous CIFN and cisplatin treatment. Interestingly, a spontaneous tumor regression was observed in nude mice injected with IFN-producing 1B6 cells, in contrast to the progressive tumor growth occurring in mice receiving a similar inoculum of the parental or control-transduced 1B6 melanoma cells. Repeated peritumoral administration of 103 IU of exogenous CIFN to mice bearing parental 1B6 tumors caused only a transient inhibition of tumor growth. These results indicate that type I IFN gene transfer is an effective approach for suppressing the tumorigenic phenotype of human melanoma cells and for increasing the efficacy of anticancer drugs. These observations, together with our previous findings showing the importance of IFN-alpha-T cell interactions in the generation of an antitumor response in mouse models, underline the interest of using type I IFN in gene therapy strategies for the treatment of human melanoma.
Animals↗
Human melanocytes and melanomas express novel mRNA isoforms of the tyrosinase-related protein-2/DOPAchrome tautomerase gene: molecular and functional characterization.
We previously reported that a melanoma antigen, recognized by tumor-specific cytotoxic T lymphocytes, was encoded by intron sequences retained in a partially spliced transcript of the tyrosinase-related protein-2/DOPAchrome tautomerase gene. At difference with the mRNA encoding tyrosinase-related protein-2, this anomalous transcript was not expressed in melanocytes. This study examined whether neoplastic and/or normal cells of the melanocytic lineage could express additional forms of tyrosinase-related protein-2 mRNA. Screening of a melanoma-derived cDNA library with a tyrosinase-related protein-2 probe allowed identification of two novel isoforms. The first, tyrosinase-related protein-2-long tail, corresponds to the dominant transcript detected on melanomas and melanocytes by northern blot analysis. Tyrosinase-related protein-2-long tail is identical to the tyrosinase-related protein-2-encoding published cDNA sequence except for an extended 3'-untranslated region and is originated by alternative polyadenylation. This novel 3'-untranslated region contains an alternatively spliced, tyrosinase-related protein-2 last exon in the second isoform (tyrosinase-related protein-2-8b). The protein encoded by tyrosinase-related protein-2-8b is identical to tyrosinase-related protein-2 in its first 460 amino acids but possesses a different carboxyl-terminus devoid of transmembrane domain. Tyrosinase-related protein-2-long tail exhibited DOPA-chrome tautomerase activity, when transiently transfected into COS-7 cells. On the contrary, no detectable activity was exhibited by tyrosinase-related protein-2-8b. Reverse transcription-polymerase chain reaction analysis indicated that tyrosinase-related protein-2-long tail and tyrosinase-related protein-2-8b are expressed by tyrosinase-related protein-2-positive melanomas and normal melanocytes. Moreover all cell lines positive for tyrosinase-related protein-2 isoforms expressed tyrosinase and, all but one, tyrosinase-related protein-1. These data show that the human tyrosinase-related protein-2/DOPAchrome tautomerase gene can yield different isoforms by alternative poly(A) site usage or by alternative splicing. The pattern of expression of these isoforms suggest that they might play a part in the normal pathway of melanin biosynthesis.
Alternative Splicing↗
Tumour immunotherapy: the adjuvant treatment of the 21st century?
In the course of a century, tumour immunology has revealed a picture of a very complex immune system involving the recognition and eradication of malignancies. Many tumours evade the immune system, and understanding of tumour escape mechanisms is the key to a successful immunotherapy for cancer. A wide array of tumour immunotherapy modalities have been developed, many of which have reached the phase of clinical trials, with some satisfactory results. Based on the available clinical, data and the techniques available for further improvement, we analyse the prospects for the different treatment modalities, and predict an important role for tumour immunotherapy in the near future.
Adjuvants, Immunologic↗
Immunological therapy of human tumors by gene-modified cellular vaccines.
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Animals↗
Differential loss of T cell signaling molecules in metastatic melanoma patients' T lymphocyte subsets expressing distinct TCR variable regions.
In this study we tested the hypothesis that loss of T cell signaling molecules in metastatic melanoma patients' T cells may affect differently T cell subsets characterized by distinct TCR variable regions. By a two-color immunofluorescence technique, expression of zeta-chain, lck, and ZAP-70 was evaluated in CD3+ T cells and in three representative T cell subsets expressing TCRAV2, TCRBV2, or TCRBV18. Partial loss of lck and ZAP-70 was found in CD3+ T cells from PBL of most melanoma patients, but not of healthy donors. The extent of zeta-chain, lck, and ZAP-70 loss depended on the TCRV region expressed by the T cells, and this association was maintained or increased during progression of disease. Coculture of patients' or donors' T cell with melanoma cells, or with their supernatants, but not with normal fibroblasts or their supernatants, down-modulated expression of zeta-chain, lck, and ZAP-70 in a TCRV region-dependent way. Immunodepletion of soluble HLA class I molecules present in tumor supernatants, but not of soluble ICAM-1, blocked the suppressive effect on T cell signaling molecule expression. T cell activation with mAbs to a single TCRV region and to CD28 led to significant and TCRV region-specific re-induction of zeta-chain expression. These findings indicate that extent of TCR signaling molecules loss in T lymphocytes from metastatic melanoma patients depends on the TCRV region and suggest that tumor-derived HLA class I molecules may contribute to induce such alterations.
Adult↗
Vaccination of melanoma patients with interleukin 4 gene-transduced allogeneic melanoma cells.
A human melanoma line genetically modified to release interleukin 4 (IL-4) was utilized to immunize advanced melanoma patients in order to elicit or increase a specific anti-melanoma immune response, which may affect distant lesions. Twelve metastatic melanoma patients were injected subcutaneously at least three times with 5 x 10(7) IL-4 gene-transduced and irradiated allogeneic melanoma cells per dose. Both systemic and local toxicities were mild, consisting of transient fever and erythema, swelling, and induration at the vaccination site. Two mixed but not complete or partial clinical responses were recorded. To assess the immune response of vaccinated patients, both serological and cell-mediated activities were evaluated. Antibodies to alloantigens could be detected in 2 of 11 patients tested. Mixed tumor-lymphocyte cultures were performed, utilizing autologous and allogeneic HLA-A2-matched melanoma lines as simulators and targets. A significant increase in IFN-gamma release was detected in 7 of 11 cases when postvaccination lymphocytes were stimulated by the untransduced allomelanoma cells. However, induction of a specific recognition of autologous melanoma cells by PBLs was obtained after vaccination in only one of six cases studied. This response involved the melanoma peptide Melan-A/MART-1(27-35) that was recognized in an HLA-A2-restricted fashion. These results indicate that vaccination with allogeneic melanoma cells releasing IL-4 locally can expand a T cell response against antigen(s) of autologous, untransduced tumor, although in a minority of patients.
Adult↗
An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions.
It is not known if immune response to T cell-defined human histocompatibility leukocyte antigen (HLA) class I-restricted melanoma antigens leads to an expanded peripheral pool of T cells in all patients, affects cytotoxic T lymphocyte (CTL) generation, and correlates with anti-tumor response in metastatic lesions. To this end, a limiting dilution analysis technique was developed that allowed us to evaluate the same frequency of peptide-specific T cells as by staining T cells with HLA-peptide tetrameric complexes. In four out of nine patients, Melan-A/Mart-1(27-35)-specific CTL precursors (CTLp) were >/=1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO(+) memory T cell subset. In the remaining five patients, a low (<1/40,000) peptide-specific CTLp frequency was measured, and the precursors were only in the CD45RA(+) naive T cell subset. Evaluation of CTL effector frequency after bulk culture indicated that peptide-specific CTLs could be activated in all patients by using professional antigen-presenting cells as dendritic cells, but CTLp frequency determined the kinetics of generation of specificity and the final number of effectors as evaluated by both limiting dilution analysis and staining with HLA-A*0201-Melan-A/Mart-1 tetrameric complexes. Immunohistochemical analysis of 26 neoplastic lesions from the nine patients indicated absence of tumor regression in most instances, even in patients with an expanded peripheral T cell pool to Melan-A/Mart-1 and whose neoplastic lesions contained a high frequency of tetramer-positive Melan-A/Mart-1-specific T cells. Furthermore, frequent lack of a "brisk" or "nonbrisk" CD3(+)CD8(+) T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances. Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell-mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance.
Adult↗
Selective histocompatibility leukocyte antigen (HLA)-A2 loss caused by aberrant pre-mRNA splicing in 624MEL28 melanoma cells.
Histocompatibility leukocyte antigen (HLA)-A2 is used as a restricting element to present several melanoma-associated antigen (MAA)-derived peptides to cytotoxic T lymphocytes (CTLs). HLA-A2 antigen is selectively lost in primary melanoma lesions and more frequently in metastases. Only scanty information is available about the molecular mechanisms underlying this abnormality, in spite of its potentially negative impact on the clinical course of the disease and on the outcome of T cell-based immunotherapy. Therefore, in this study we have shown that the selective HLA-A2 antigen loss in melanoma cells 624MEL28 is caused by a splicing defect of HLA-A2 pre-mRNA because of a base substitution at the 5' splice donor site of intron 2 of the HLA-A2 gene. As a result, HLA-A2 transcripts are spliced to two aberrant forms, one with exon 2 skipping and the other with intron 2 retention. The latter is not translated because of an early premature stop codon in the retained intron. In contrast, the transcript with exon 2 skipping is translated to a truncated HLA-A2 heavy chain without the alpha(1) domain. Such a polypeptide is synthesized in vitro but is not detectable in cells, probably because of the low steady state level of the corresponding mRNA and the low translation efficiency. These results indicate that a single mutational event in an HLA class I gene is sufficient for loss of the corresponding allele. This may account, at least in part, for the high frequency of selective HLA class I allele loss in melanoma cells. Our conclusion emphasizes the need to implement active specific immunotherapy with a combination of peptides presented by various HLA class I alleles. This strategy may counteract the ability of melanoma cells with selective HLA class I allele loss to escape from immune recognition.
Alternative Splicing↗
Immunotherapy for colon cancer.
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Colonic Neoplasms↗
Novel HLA-Cw8-restricted T cell epitopes derived from tyrosinase-related protein-2 and gp100 melanoma antigens.
The identification of T cell epitopes presented by alternative HLA-B and -C alleles may provide a means to counteract the tumor escape mechanism based on the selection of tumor cells no longer susceptible to HLA-A-restricted T cell recognition. Several T cell clones and lines were obtained from T lymphocytes purified from melanoma-infiltrated or noninfiltrated lymph nodes of a patient who remained disease free 8 yr after surgery. Selected T cells recognized the autologous melanoma as evaluated by direct cytolysis and production of cytokines. These effectors were directed against the tyrosinase-related protein-2 (TRP-2) and gp100 melanoma epitopes restricted by HLA-Cw8. The nonamer and decamer peptides containing the sequence ANDPIFVVL (residues 387-395) of TRP-2 and the octamer, nonamer, and decamer peptides containing the sequence SNDGPTLI (residues 71-78) of gp100 reconstituted the epitope for TRP-2- and gp100-specific T cell lines and clones, respectively. However, only the nonameric form of TRP-2 and the nonameric and octameric forms of gp100 were able to induce peptide-specific T cells recognizing the autologous tumor in an HLA-class I-restricted fashion from PBMC of the melanoma patient studied. Together these data indicate that HLA-Cw8 can restrict the recognition of gp100 and TRP-2 epitopes by CTL, and that such peptides could stimulate a patient's PBL, suggesting that these Ags could have contributed to a systemic immunity against melanoma.
Alleles↗
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↗
A superagonist variant of peptide MART1/Melan A27-35 elicits anti-melanoma CD8+ T cells with enhanced functional characteristics: implication for more effective immunotherapy.
In the present study, we show that a singly substituted peptide derived from the epitope MART1(27-35) and containing a Leu in position 1 (LAGIGILTV; 1L) behaves as a superagonist by in vitro inducing specific T cells with enhanced immunological functions. 1L-specific CTLs can be raised from peripheral blood of HLA-A2+ melanoma patients more efficiently than T cells specific for the cognate peptide. These T cells show a greater sensitivity to native MART1(27-35) when compared with CTL variable raised to parental peptide from the same patients. More importantly, anti-1L but not anti-native T cells display high levels of interferon gamma production at early time points, and readily secreted interleukin-2 in response to native epitope endogenously presented by melanoma cells. Additionally, anti-1L T cells are insensitive to the inhibitory effects of MART1(27-35) natural analogues that antagonize the lytic response of CTLs raised to the cognate peptide. Analysis of T-cell receptor variable beta usage suggests that the native and 1L peptides stimulate different components of the MART1(27-35)-reactive T cell population. These data provide rationale to the use of superagonist analogues of tumor antigens for inducing in vivo immunization potentially able to overcome tumor immune escape and mediate a more significant control of tumor growth.
CD8-Positive T-Lymphocytes↗
High frequency of T cell clonal expansions in primary human melanoma. Involvement of a dominant clonotype in autologous tumor recognition.
It was previously found that primary melenomas from HLA-A2-matched patients display an increased expression of a few T cell receptor (TCR) variable-region beta-chain transcripts (BV) compared with autologous peripheral blood lymphocytes (PBL) and uninvolved skin. In order to see whether expansions of clonal/oligoclonal subsets of T cells occurred, complementarity-determining region 3 (CDR3) of BV transcripts overexpressed in the neoplastic infiltrate were cloned and sequenced. Dominant rearrangements were found for BV14, which were commonly increased in the neoplastic lesions of all analysed HLA-A2 melanoma patients, as well as for other overexpressed BV gene families, but none of them could be identified among autologous PBL. No identical CDR3 sequences could be detected in the dominant BV14 rearrangements obtained from the different patients. In one patient a single clonally expanded SLSGTGVNEQF CDR3 clonotype accounted for the entire BV14 relative frequency of expression (24%) in tumor-infiltrating lymphocytes (TIL). Two independent mixed lymphocyte/tumor cultures (MLTC) could be successfully established from TIL of the patient and were found to exert HLA-class-I-restricted cytotoxicity for the autologous melanoma line. BV14 T cells that constituted from 22% to 32% of all T cells present in both MLTC lines, as assessed by flow cytometry, all displayed the same CDR3 clonotype found in vivo and could be shown, by TCR down-modulation experiments, to be involved in autologous tumor recognition. These results support the hypothesis of a tumor-antigen-driven origin of clonally amplified T cells present at high frequency in the in situ neoplastic infiltrate.
Clone Cells↗
Cytotoxic T lymphocyte response against non-immunoselected tumor antigens predicts the outcome of gene therapy with IL-12-transduced tumor cell vaccine.
The colon adenocarcinoma C26, carrying two endogenous tumor-associated antigens (TAA) recognized by CTL, has been transduced with the gene coding for the human folate receptor alpha (FR alpha) as an additional antigen in order to study the efficacy of vaccination against a tumor expressing multiple antigens. A dicistronic vector was used to transduce the IL-12 genes to create C26/IL-12/FR alpha that has been used as a cellular vaccine to treat mice bearing lung metastases of C26/FR alpha. After vaccination mice were partially splenectomized and splenic lymphocytes frozen and used retrospectively to study in vitro CD8 T cell response related to the treatment outcome. Vaccination cured 50% of mice and the effect was CD8 T cell dependent. Mice either cured (responders) or not cured (nonresponders) by vaccination developed tumor-specific CTL. However, analysis of CTL specificity and pCTL frequencies revealed that responders had a predominant CTL activity against endogenous C26-related tumor antigens, whereas nonresponders had CTL that recognized preferentially the FR alpha antigen. CD8 from responder mice were characterized to release high levels of granulocyte-macrophage (GM)-CSF upon antigen stimulation. Tumors obtained from mice that died despite vaccination lost expression of the FR alpha transgene but maintained expression of endogenous C26 antigens. Immunoselection against FR alpha antigen was not observed in tumors from non-vaccinated controls and from CD8-depleted vaccinated mice. Down-regulation of FR alpha antigen expression was due, at least in part, to methylation of retroviral vector long terminal repeat promoter since FR alpha expression was partially restored, ex vivo, by treatment with 5-aza-2'-deoxy-cytidine (aza). These results indicate that CD8 T cell-mediated immunoselection and production of GM-CSF are determining factors for the efficacy of tumor vaccines.
Adenocarcinoma↗
Genes involved in melanoma: an overview of INK4a and other loci.
Melanoma is the most aggressive of the skin cancers and its prognosis is often poor. The only known environmental risk factor for this tumour is ultraviolet light exposure. This fact together with the existence of melanoma-prone families has prompted investigation of genetic risk factors that may be involved in melanoma development. Inactivation of the INK4a/p16 gene is known to play a role in familial cases. Data on genes or loci involved in sporadic melanoma are less definitive and require more detailed research. In addition to the INK4a locus, other genes involved in melanoma development are discussed here, in particular those genes that participate in the same functional pathway, such as CDK4 and Rb, and p53, which is regulated by the alternative product of INK4a. Evidence showing the possible location of melanoma susceptibility genes on chromosomes 1p, 6, 10q and 11q is analysed along with data showing N-ras, betacatenin, c-myc and MC1R involvement. Melanoma is a well-characterized disease in terms of its progression stages; therefore obtaining precise genetic information is crucial in the development of a stepwise model of melanoma pathogenesis.
Animals↗
Translation of a retained intron in tyrosinase-related protein (TRP) 2 mRNA generates a new cytotoxic T lymphocyte (CTL)-defined and shared human melanoma antigen not expressed in normal cells of the melanocytic lineage.
We report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011-restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein (TRP)-2, containing exons 1-4 with retention of intron 2 and part of intron 4 (TRP-2-INT2). The sequence coding for the antigenic epitope is located at the 5' end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3-like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2-INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2-INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011-transduced, TRP-2(+) melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.
Alleles↗