Heterogeneous T-cell response to MAGE-A10(254-262): high avidity-specific cytolytic T lymphocytes show superior antitumor activity

MAGE-encoded antigens, which are expressed by tumors of many histological types but not in normal tissues, are suitable candidates for vaccine-based immunotherapy of cancers. Thus far, however, T-cell responses to MAGE antigens have been detected only occasionally in cancer patients. In contrast, by using HLA/peptide fluorescent tetramers, we have observed recently that CD8(+) T cells specific for peptide MAGE-A10(254-262) can be detected frequently in peptide-stimulated peripheral blood mononuclear cells from HLA-A2-expressing melanoma patients and healthy donors. On the basis of these results, antitumoral vaccination trials using peptide MAGE-A10(254-262) have been implemented recently. In the present study, we have characterized MAGE-A10(254-262)-specific CD8(+) T cells in polyclonal cultures and at the clonal level. The results indicate that the repertoire of MAGE-A10(254-262)-specific CD8(+) T cells is diverse both in terms of clonal composition, efficiency of peptide recognition, and tumor-specific lytic activity. Importantly, only CD8(+) T cells able to recognize the antigenic peptide with high efficiency are able to lyse MAGE-A10-expressing tumor cells. Under defined experimental conditions, the tetramer staining intensity exhibited by MAGE-A10(254-262)-specific CD8(+) T cells correlates with efficiency of peptide recognition so that "high" and "low" avidity cells can be separated by FACS. Altogether, the data reported here provide evidence for functional diversity of MAGE-A10(254-262)-specific T cells and will be instrumental for the monitoring of peptide MAGE-A10(254-262)-based clinical trials.     

Specific cytolytic T-cell responses to human CEA from patients immunized with recombinant avipox-CEA vaccine.

Avipox viruses are replication-defective members of the poxvirus family. Avipox-derived vectors such as ALVAC (canarypox) and fowlpox have the ability to infect mammalian cells, including human cells, but do not replicate. The first clinical trial of an avipox recombinant vaccine for patients with advanced carcinomas has recently been conducted using the ALVAC vector and the human carcinoembryonic antigen (CEA) transgene (designated ALVAC-CEA; J. L. Marshall et al, J. Clin. Oncol., 17: 332-337, 1999). The T-cell responses elicited by patients before and after vaccination with the ALVAC-CEA recombinants are characterized in this report. Pre- and postvaccination peripheral blood mononuclear cells (PMBCs) of the eight patients positive for HLA-class I A2 allele, were incubated with the HLA-A2-CEA peptide CAP-1 and interleukin 2. In no cases using prevaccination PMBCs could cultures be established that had the ability to lyse C1R-A2 target cells pulsed with the CAP-1 peptide. However, T-cell cultures from seven of eight of these same patients, obtained from PBMCs after ALVAC-CEA vaccination, were shown to lyse C1R-A2 cells only when pulsed with CAP-1. Moreover, all seven of these T-cell cultures were shown to lyse allogeneic human carcinoma cell lines (SW1463 and SW480) that were both A2+ and expressed CEA; an allogeneic tumor cell line (LS174T) expressing CEA that was negative for A2 expression was not lysed. HLA-A2+ and CEA+ autologous tumor cells were also capable of being lysed by CEA-specific T cells from this patient. Analysis of this CTL line also revealed the expression of several homing and adhesion-associated molecules. Fluorescence-activated cell sorter analysis of the T-cell lines established from patients after ALVAC-CEA vaccination revealed that most were CD8+/CD4-, but many also had a CD8+/CD4+ component. Analyses of T-cell receptor Vbeta usage of several of the CEA-specific CTL lines showed a relatively diverse Vbeta pattern. These studies demonstrate for the first time the ability to vaccinate cancer patients with an avipox recombinant and derive T cells that are capable of lysing allogeneic and autologous tumor cells in a MHC-restricted manner. These studies thus form the rationale to use such replication-deficient recombinant vaccines in future cancer vaccine trials.     

Frequent downregulation of Fas (CD95) expression and function in melanoma

Membrane-bound Fas ligand (FasL, Apo-1L, CD95L) induces rapid apoptosis of Fas (CD95)-sensitive cells on interaction with Fas, and is an important effector molecule of cytolytic T lymphocytes (CTLs). Melanomas are immunogenic and induce the production of specific CTLs, but are usually able to escape immune destruction. We investigated Fas expression and function in 53 cutaneous melanocytic lesions and 13 melanoma cell lines grown in vitro. Immunohistochemical analysis of Fas expression in cutaneous melanocytic lesions showed moderate to high levels of Fas in common benign melanocytic naevi, but low to undetectable levels in atypical naevi, primary (superficial spreading melanoma, nodular melanoma) and cutaneous melanoma metastases. Fluorescence-activated cell sorting (FACS) analysis of Fas expression in melanoma cell lines revealed undetectable or low levels of cell surface Fas expression in five of the 13 melanoma cell lines. Analysis of Fas signalling by quantification of cell death following exposure to recombinant FasL showed that a reduction in Fas expression results in resistance to FasL-mediated cell death. Furthermore, two of the 13 melanoma cell lines were found to be resistant to FasL-mediated cell death despite conserved Fas expression. Thus seven of the 13 melanoma cell lines were found to have impaired Fas signalling. Taken together, our results indicate that downregulation of Fas expression and resistance to Fas-mediated apoptosis are frequent in melanoma.     

Analysis of the cytolytic T lymphocyte response of melanoma patients to the naturally HLA-A*0201-associated tyrosinase peptide 368-376.

The human tyrosinase gene codes for two distinct antigens that are recognized by HLA-A*0201-restricted CTLs. For one of them, tyrosinase peptide 368-376, the sequence identified by mass spectrometry in melanoma cell eluates differs from the gene-encoded sequence as a result of posttranslational modification of amino acid residue 370 (asparagine to aspartic acid). Here, we used fluorescent tetrameric complexes ("tetramers") of HLA-A*0201 and tyrosinase peptide 368-376 (YMDGTMSQV) to characterize the CD8+ T-cell response to this antigen in lymphoid cell populations from HLA-A2 melanoma patients. Taking advantage of the presence of significant numbers of tetramer-positive CD8+ T cells in tumor-infiltrated lymph node cells from a melanoma patient, we derived polyclonal and monoclonal tyrosinase peptide 368-376-specific CTLs by tetramer-guided flow cytometric sorting. These CTLs efficiently and specifically lysed HLA-A*0201- and tyrosinase-positive melanoma cells. As assessed with tyrosinase peptide variants, the fine antigen specificity of the CTLs was quite diverse at the clonal level. Flow cytometric analysis of PBMCs stained with tetramers showed that tyrosinase peptide 368-376-specific CD8+ T cells were hardly detectable in peripheral blood of melanoma patients. However, significant numbers of such cells were detected after short-term stimulation of CD8+ lymphocytes with tyrosinase peptide 368-376 in 6 of 10 HLA-A2 melanoma patients. Taken together, these findings emphasize the significant contribution of the natural tyrosinase peptide 368-376 to the antigenic specificities recognized by the tumor-reactive CTLs that may develop in HLA-A2 melanoma patients.     

Liver metastasis formation by laminin-1 peptide (LQVQLSIR)-adhesion selected B16-F10 melanoma cells

Laminin-1, a major basement membrane glycoprotein, promotes tumor cell malignancy. Incubation of B16-F10 melanoma cells with a peptide containing an active sequence in laminin-1, designated AG-73 (leu-glu-val-glu-leu-ser-ile-arg; LQVQLSIR), enhances in vitro adhesion, migration, invasion and gelatinase production and in vivo lung colonization and metastases to the liver. In the current study, we have tried to define the mechanism of enhancement of liver metastases induced by AG-73 using B16-F10 murine melanoma cells selected for adhesion on AG-73-coated dishes. Cells were sequentially selected for adhesion more than 30 times and then characterized. AG-73 selected cells had much longer cytoplasmic processes and occasionally formed nodular aggregates. AG-73 selected cells attached 1.2- to 1.5-fold better to both AG-73 and laminin-1, were able to invade through the Matrigel-coated filter up to 6-fold more, grew s.c. 1.5-2 times faster, produced twice the number of lung colonies, and showed more liver nodules (12 of 28 vs. 1 of 27) than parental cells. Our data demonstrate that the enhanced malignant phenotype of B16-F10 cells can be observed in the absence of added peptide with the adhesion-selected cells. Int. J. Cancer 71:436-441, 1997. © 1997 Wiley-Liss Inc.     

Adjuvant immunization of HLA-A2-positive melanoma patients with a modified gp100 peptide induces peptide-specific CD8+ T-cell responses.

PURPOSE: To measure the CD8+ T-cell response to a melanoma peptide vaccine and to compare an every-2-weeks with an every-3-weeks vaccination schedule. PATIENTS AND METHODS: Thirty HLA-A2-positive patients with resected stage I to III melanoma were randomly assigned to receive vaccinations every 2 weeks (13 vaccines) or every 3 weeks (nine vaccines) for 6 months. The synthetic, modified gp100 peptide, g209-2M, and a control peptide, HPV16 E7, were mixed in incomplete Freund's adjuvant and injected subcutaneously. Peripheral blood mononuclear cells obtained before and after vaccination by leukapheresis were analyzed using a fluorescence-based HLA/peptide-tetramer binding assay and cytokine flow cytometry. RESULTS: Vaccination induced an increase in peptide-specific T cells in 28 of 29 patients. The median frequency of CD8+ T cells specific for the g209-2M peptide increased markedly from 0.02% before to 0.34% after vaccination (P <.0001). Eight patients (28%) exhibited peptide-specific CD8+ T-cell frequencies greater than 1%, including two patients with frequencies of 4.96% and 8.86%, respectively. Interferon alfa-2b-treated patients also had significant increases in tetramer-binding cells (P <.0001). No difference was observed between the every-2-weeks and the every-3-weeks vaccination schedules (P =.59). CONCLUSION: Flow cytometric analysis of HLA/peptide-tetramer binding cells was a reliable means of quantifying the CD8+ T-cell response to peptide immunization. This assay may be suitable for use in future trials to optimize different vaccination strategies. Concurrent interferon treatment did not inhibit the development of a peptide-specific immune response and vaccination every 2 weeks, and every 3 weeks produced similar results.     

Contribution of Abcc10 (Mrp7) to in vivo paclitaxel resistance as assessed in Abcc10(-/-) mice

Recently, we reported that the ATP-binding cassette transporter 10 (ABCC10), also known as multidrug resistance protein 7 (MRP7), is able to confer resistance to a variety of anticancer agents, including taxanes. However, the in vivo functions of the pump have not been determined to any extent. In this study, we generated and analyzed Abcc10(-/-) mice to investigate the ability of Abcc10 to function as an endogenous resistance factor. Mouse embryo fibroblasts derived from Abcc10(-/-) mice were hypersensitive to docetaxel, paclitaxel, vincristine, and cytarabine (Ara-C) and exhibited increased cellular drug accumulation, relative to wild-type controls. Abcc10(-/-) null mice treated with paclitaxel exhibited increased lethality associated with neutropenia and marked bone marrow toxicity. In addition, toxicity in spleen and thymus was evident. These findings indicate that Abcc10 is dispensable for health and viability and that it is an endogenous resistance factor for taxanes, other natural product agents, and nucleoside analogues. This is the first demonstration that an ATP-binding cassette transporter other than P-glycoprotein can affect in vivo tissue sensitivity toward taxanes.     

Vaccine-induced CD8+ T-cell responses to MAGE-3 correlate with clinical outcome in patients with melanoma.

PURPOSE: Vaccine-induced antitumor CD8+ T-cell responses are believed to play an important role in increasing resistance to melanoma. The following study was conducted to examine whether these responses are associated with improved clinical outcome in melanoma vaccine-treated patients. EXPERIMENTAL DESIGN: We measured CD8+ T-cell responses to gp100, MART-1, MAGE-3, and tyrosinase by enzyme-linked immunospot assay in peripheral blood of 131 HLA-A*01- or HLA-A*02-positive melanoma patients before and after immunization to a polyvalent, shed antigen, melanoma vaccine, and correlated the results with clinical outcome. RESULTS: Fifty-six percent of patients had a vaccine-induced CD8+ T-cell response to at least one of the four antigens. Recurrences were significantly reduced in patients with vaccine-induced responses to MAGE-3 (hazard ratio, 0.42; 95% confidence interval, 0.18-0.99; P = 0.03) by the Cox proportional hazard model but were unrelated to responses to the other three antigens. Patients with a preexisting response to any of the four antigens were significantly more likely to have a further vaccine-boosted response to that same antigen (P < 0.0001-0.036). CONCLUSIONS: There was a correlation between vaccine-induced CD8+ T-cell responses to melanoma-associated antigens and improved clinical outcome, but the correlation depended on the antigen against which the response is directed. The only significant correlation was with responses to MAGE-3.     

T-cell responses against tyrosinase 368-376(370D) peptide in HLA*A0201+ melanoma patients: randomized trial comparing incomplete Freund's adjuvant, granulocyte macrophage colony-stimulating factor, and QS-21 as immunological adjuvants.

We conducted a randomized trial in HLA*A0201+ patientswith American Joint Committee on Cancer stage III or IV melanoma immunized with tyrosinase 368-376(370D) peptide and gp100 209-217(210M) peptide to compare the potency of three different adjuvants. Patients received 3 monthly immunizations with 500 microg of each peptide either with incomplete Freund's adjuvant (IFA), QS-21, or granulocyte macrophage colony-stimulating factor (GM-CSF). The primary end point was induction of IFN-gamma release by CD8+ T cells against tyrosinase and gp100 peptides measured by enzyme-linked immunospot assays without in vitro prestimulation measured pretreatment, 2 and 8 weeks after the third vaccination. Four of 9 and 4 of 8 patients immunized using QS-21 and GM-CSF, respectively, developed increased frequencies of CD8+ T cells against tyrosinase 370D peptide compared with 0 of 9 patients immunized using IFA (P = 0.045). T-cell responses against a gp100-related peptide showed similar results, but their relevance to T-cell reactivity against native gp100 209-217 is uncertain. These results show that: (a) QS-21 and GM-CSF are superior to IFA as immunological adjuvants for vaccination against tyrosinase 370D peptide; and (b) with appropriate adjuvants, increased frequencies of peptide-specific T cells after vaccination can be detected by enzyme-linked immunospot without prolonged prestimulation in vitro.     

Generation of human T-cell responses to an HLA-A2.1-restricted peptide epitope derived from alpha-fetoprotein.

Alpha-fetoprotein (AFP) is often derepressed in human hepatocellular carcinoma. Peptide fragments of AFP presented in the context of major histocompatibility molecules could serve as potential recognition targets by CD8 T cells, provided these lymphocytes were not clonally deleted in ontogeny. We therefore wished to determine whether the human T-cell repertoire could recognize AFP-derived peptide epitopes in the context of a common class I allele, HLA-A2.1. Dendritic cells genetically engineered to express AFP were capable of generating AFP-specific T-cell responses in autologous human lymphocyte cultures and in HLA-A2.1/Kb transgenic mice. These T cells recognize a 9-mer peptide derived from the AFP protein hAFP(542-550) (GVALQTMKQ). Identified as a potential A2.1-restricted peptide epitope from a computer analysis of the AFP sequence, hAFP(542-550) proved to have low binding affinity to A2.1, but slow off-kinetics. AFP-specific CTL- and IFN-gamma-producing cells recognize hAFP(542-550)-pulsed targets. Conversely, hAFP(542-550) peptide-generated T cells from both human lymphocyte cultures and A2.1/Kb transgenic mice recognized AFP-transfected targets in both cytotoxicity assays and cytokine release assays. These lines of evidence clearly demonstrate that AFP-reactive clones have not been deleted from the human T-cell repertoire and identify one immunodominant A2.1-restricted epitope. These findings also clearly establish AFP as a potential target for T-cell-based immunotherapy.     

Prospective assessment of a gene signature potentially predictive of clinical benefit in metastatic melanoma patients following MAGE-A3 immunotherapeutic (PREDICT)

Genomic profiling of tumor tissue may identify gene signatures (GS) predictive of tumor response to treatment. We prospectively evaluated a GS linked to clinical benefit following MAGE-A3 immunotherapeutic in patients with advanced melanoma. One-year overall survival was similar in patients with and without the GS: the GS was not predictive of outcome after MAGE-A3 immunotherapeutic treatment.BackgroundGenomic profiling of tumor tissue may aid in identifying predictive or prognostic gene signatures (GS) in some cancers. Retrospective gene expression profiling of melanoma and non-small-cell lung cancer led to the characterization of a GS associated with clinical benefit, including improved overall survival (OS), following immunization with the MAGE-A3 immunotherapeutic. The goal of the present study was to prospectively evaluate the predictive value of the previously characterized GS.Patients and methodsAn open-label prospective phase II trial (‘PREDICT’) in patients with MAGE-A3-positive unresectable stage IIIB-C/IV-M1a melanoma.ResultsOf 123 subjects who received the MAGE-A3 immunotherapeutic, 71 (58.7%) displayed the predictive GS (GS+). The 1-year OS rate was 83.1%/83.3% in the GS+/GS- populations. The rate of progression-free survival at 12 months was 5.8%/4.1% in GS+/GS- patients. The median time-to-treatment failure was 2.7/2.4 months (GS+/GS-). There was one complete response (GS-) and two partial responses (GS+). The MAGE-A3 immunotherapeutic was similarly immunogenic in both populations and had a clinically acceptable safety profile.ConclusionTreatment of patients with MAGE-A3-positive unresectable stage IIIB-C/IV-M1a melanoma with the MAGE-A3 immunotherapeutic demonstrated an overall 1-year OS rate of 83.5%. GS- and GS+ patients had similar 1-year OS rates, indicating that in this study, GS was not predictive of outcome. Unexpectedly, the objective response rate was lower in this study than in other studies carried out in the same setting with the MAGE-A3 immunotherapeutic. Investigation of a GS to predict clinical benefit to adjuvant MAGE-A3 immunotherapeutic treatment is ongoing in another melanoma study.This study is registered at www.clinicatrials.gov NCT00942162.     

Human dendritic cells genetically engineered to express a melanoma polyepitope DNA vaccine induce multiple cytotoxic T-cell responses.

PURPOSE: To assess the therapeutic potential of a melanoma polyepitope vaccine in human cells. Polyepitope DNA vaccines encoding T-cell epitopes have been demonstrated in murine systems to generate multiple cytotoxic T-cell responses to different antigens. Here, for the first time we demonstrate the ability of a melanoma polyepitope to stimulate lymphocytes from normal human donors to simultaneously generate multiple antigen-specific responses. EXPERIMENTAL DESIGN: Human dendritic cells (DC), transduced with a melanoma-polyepitope cDNA, were used to activate autologous lymphocytes from na?ve donors as an in vitro model of DNA vaccination. Lymphocytes were primed with polyepitope or mock-transduced DC, boosted with peptide, then measured for antigen-specific cytotoxicity. RESULTS: Lymphocytes primed with polyepitope-transduced DC and boosted with peptide generated multiple cytotoxic responses. By contrast lymphocytes primed with mock-transfected DCs and boosted with peptide gave no specific cytotoxicity. However, when lymphocytes were repeatedly stimulated with polyepitope-transduced DCs immunodominance was seen with CTLs being generated to only one epitope, MART(27-35). CONCLUSIONS: We show in a human system that a melanoma polyepitope primes CTL to multiple epitopes. However, repeated stimulation by the polyepitope restricts the response to only the MART1 epitope. Thus, although polyepitope vaccines are an effective way of priming multiple na?ve T-cell responses, continual boosting with polyepitope vaccines may, as a result of immunodominance, restrict the CTL. These findings have important implications for the use of DNA polyepitope vaccines in cancer immunotherapy.     

Paucity of functional T-cell memory to melanoma antigens in healthy donors and melanoma patients.

The functional characteristics of CD8+ T cells specific for melanoma antigens (MAs) have often been defined after in vitro culture using nonprofessional antigen-presenting cells. We have examined CD8+ T-cell immunity to MAs and a viral antigen (influenza) in uncultured T cells of healthy donors and melanoma patients using autologous, mature, monocyte-derived dendritic cells (DCs) pulsed with peptide antigens and viral vectors. Antigen-specific IFN-gamma-producing T cells reactive with HLA-A*0201-restricted peptides from four melanoma antigens (MelanA/MART-1, MAGE-3, tyrosinase, and gp100) were detected only at low frequencies (<30 per 2 x 10(5) peripheral blood mononuclear cells for each of the MAs) from HLA-A2.1-positive healthy donors (n = 12) and patients with stages III/IV melanoma (n = 8). Detection of MA-specific, but not influenza matrix peptide (Flu-MP)-specific, T cells required a high concentration (10 microg/ml) of the peptide in this assay. Furthermore, these T cells did not recognize endogenously processed antigen on tumor cell lines or cells infected with viral vectors capable of expressing MAs. The use of autologous, mature DCs led to a significant increase in the number of Flu-MP, but not MA-specific, T cells in 16-h ELISPOT assays for both melanoma patients and healthy donors. In 1-week cocultures with DCs pulsed with 10 microg/ml peptide, MelanA/MART-1-specific T cells did not readily proliferate or differentiate into lytic effectors, in contrast to strong influenza-specific lytic responses. Therefore, despite distinct memory responses to influenza antigens, melanoma patients and healthy controls have a paucity of MA-reactive memory T cells, failing to rapidly generate IFN-gamma-secreting lytic effectors in short-term assays, even when stimulated by DCs.     

Effect of bis(bilato)-1,2-cyclohexanediammineplatinum(II) complexes on lung metastasis of B16-F10 melanoma cells in mice

New platinum(II) complexes, bis(bilato)-1,2-cyclohexanediammineplatinum(II) which were lipophilic and water-miscible, were tested for antitumor activity against lung nodules from intravenously injected B16-F10 melanoma cells in C57BL/6 mice by intravenous administration of the complexes in water suspension form. Among them, DACHP(litho)2 and DACHP(urso)2 had high antitumor activity but others had no activity. The antitumor activity of DACHP(urso)2 was increased significantly by injecting it three times; T/C was over 280% with 100-day survivors of 3 of 6 mice tested. Large amounts of total platinum were found in lung and liver tissues by atomic absorption spectroscopy after single intravenous injection of DACHP(urso)2 suspension in ICR mice.     

Transient resistance to B16F10 melanoma growth and metastasis in CD43-/- mice

CD43, the major transmembrane sialoglycoprotein of neutrophils, monocytes, T lymphocytes and platelets, is highly glycosylated and its high sialic acid content contributes to the strongly negative charge of cells. In this study the role of CD43 in melanoma development was addressed using CD43 -/- mice (null mutated for the corresponding gene or knockout [KO]). Growth of B16F10 melanoma was retarded in the KO mice compared with the wild-type CD43+/+ control (WT). A marked difference in lung colonization and other metastatic foci was observed in the KO and WT mice up to 15 days after intravenous injection of tumour cells. The initial resistance of KO mice was reversed with time, and in the long term there was no difference in the survival rate of the two animal groups. Transient resistance was attributed to increased adhesion of thrombin-activated platelets and leukocytes to melanoma and endothelial cells in KO mice. In the KO mice tumour emboli were found in the central portion of the lung more than at the lung periphery immediately after intravenous injection, in contrast to the WT mice. Activation of melanoma adhesion receptors by thrombin or TRAP stimulated lung colonization in WT but not KO mice. Therefore, the correlation of tumour embolism and metastasis in short-term experiments depends on the nature and stability of interactions between the tumour and the blood/endothelial cells of the host.     

Cytogenetic studies in T-cell acute lymphoblastic leukemia (1981-2002)

Chromosomal analysis was successfully performed in 34 of the 37 patients with T-cell acute lymphoblastic leukemia (ALL) seen at the University Hospital in Brest (France) between 1981 and 2002. A normal karyotype was observed in 29.4% of the patients. Numerical changes were rare, 79.2% of the abnormal karyotypes being pseudodiploid. All 24 abnormal karyotypes had at least a structural rearrangement. Translocations involving band 14q11, that contains the T-cell receptor (TCR) alpha and delta-genes, were observed in 8 patients; in 3 of them, a new partner chromosomal band was found. The short arms of chromosomes 11 and 12 were involved in 3 and 2 translocations respectively. Three patients had a del(6q). Our results are in agreement with those of the literature. Most of the recurrent abnormalities are different from those of B-lineage ALL. Some are known to involve TCR genes whereas others can lead to the discovery of new genes that are important to T-lineage leukemogenesis.