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

BACKGROUND: Genomic 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 METHODS: An open-label prospective phase II trial ('PREDICT') in patients with MAGE-A3-positive unresectable stage IIIB-C/IV-M1a melanoma. RESULTS: Of 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. CONCLUSION: Treatment 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.

Recurrent T cell receptor rearrangements in the cytotoxic T lymphocyte response in vivo against the p815 murine tumor.

P815 is a murine mastocytoma of DBA/2 origin which, although immunogenic, rapidly develops as a tumor in immunocompetent syngeneic hosts. In this report, we have studied, by a molecular approach, the in vivo alpha/beta T cell response to P815. Both situations of tumor growth after engraftment of naive animals or tumor rejection by preimmunized animals have been analyzed. The spectrum of T cell receptor beta chain rearrangements in the tumor-infiltrating lymphocytes was found to be highly variable among individual tumor-bearing mice. However, two rearrangements, one using V(beta)1 and J(beta)1.2 segments and one using the V(beta)1 and J(beta)2.5 segments, with conserved junctional regions, reproducibly emerge in most individuals. These two rearrangements thus correspond to "public" (recurrent) T cell clones, as opposed to "private" ones, which emerge in a seemingly stochastic fashion in immunized animals. Importantly, these public cells are observed in situations of either growth or rejection of the tumor. Quantification provides a clear increase in public T cells in secondary responses, but no obvious correlation provides between their level and primary tumor rejection. The V(beta)1- J(beta)1.2 rearrangement is borne by CTL directed against an antigen derived from P1A, a nonmutated mouse self protein which is expressed in P815 but not in normal mouse tissues except testis. A recurrent, public T cell response can thus be observed to an antigen derived from a self protein expressed by a tumor.

The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.

Lymphocytes of melanoma patients can be restimulated in vitro with autologous tumor cells to generate antitumor cytolytic T lymphocytes (CTL). Previous reports have indicated that, when such CTL are obtained from HLA-A2 melanoma patients, they often display broad reactivity on A2 melanoma cell lines. Such antitumor CTL clones, which appeared to recognize the same antigen, were isolated from two patients. We report here the cloning of a cDNA that directs the expression of the antigen recognized by these CTL. This cDNA corresponds to the transcript of the tyrosinase gene. The gene was found to be active in all tested melanoma samples and in most melanoma cell lines. Among normal cells, only melanocytes appear to express the gene. The tyrosinase antigen presented by HLA-A2 may therefore constitute a useful target for specific immunotherapy of melanoma. But possible adverse effects of antityrosinase immunization, such as the destruction of normal melanocytes and its consequences, will have to be examined before clinical pilot studies can be undertaken.

An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins.

T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.

A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas.

It has been reported previously that antitumor cytolytic T lymphocyte (CTL) clones can be isolated from blood lymphocytes of HLA-A2 melanoma patients, after stimulation in vitro with autologous tumor cells, and that some of these CTL clones lyse most HLA-A2 melanomas. A first antigen recognized by such CTL clones was previously shown to be encoded by the tyrosinase gene. We report here the identification of another gene that also directs the expression of an antigen recognized on most melanomas by CTL clones that are restricted by HLA-A2. The gene, designated Melan-A, is unrelated to any known gene. It is 18 kb long and comprises five exons. Like the tyrosinase gene, it is expressed in most melanoma tumor samples and, among normal cells, only in melanocytes.

A peptide recognized by human cytolytic T lymphocytes on HLA-A2 melanomas is encoded by an intron sequence of the N-acetylglucosaminyltransferase V gene.

A cytolytic T lymphocyte (CTL) clone that lyses many HLA-A2 melanomas was derived from a population of tumor-infiltrating lymphocytes of an HLA-A2 melanoma patient. The gene coding for the antigen recognized by this CTL was identified by transfection of a cDNA library. It is the gene which has been reported to code for N-acetylglucosaminyltransferase V (GnT-V). Remarkably, the antigenic peptide recognized by the CTL is encoded by a sequence located in an intron. In contrast to the fully spliced GnT-V mRNA, which was found in a wide range of normal and tumoral tissues, the mRNA containing the intron region coding for the antigen was not found at a significant level in normal tissues. This mRNA was observed to be present in about 50% of melanomas. Our results suggest that a promoter located near the end of the relevant intron is activated in melanoma cells, resulting in the production of an mRNA coding for the antigen.

New tumor antigens recognized by T cells.

A series of tumor cell antigens that are recognized by cytolytic T lymphocytes has been characterized this year. Besides the antigens derived from proteins specifically expressed in tumors, many melanoma antigens derive from melanocytic differentiation proteins. In addition, antigens unique to individual tumors result from mutations in ubiquitously expressed genes.

Safety and immunogenicity of the PRAME cancer immunotherapeutic in metastatic melanoma: results of a phase I dose escalation study.

PURPOSE: The PRAME tumour antigen is expressed in several tumour types but in few normal adult tissues. A dose-escalation phase I/II study (NCT01149343) assessed the safety, immunogenicity and clinical activity of the PRAME immunotherapeutic (recombinant PRAME protein (recPRAME) with the AS15 immunostimulant) in patients with advanced melanoma. Here, we report the phase I dose-escalation study segment. PATIENTS AND METHODS: Patients with stage IV PRAME-positive melanoma were enrolled to 3 consecutive cohorts to receive up to 24 intramuscular injections of the PRAME immunotherapeutic. The RecPRAME dose was 20, 100 or 500 µg in cohorts 1, 2 and 3, respectively, with a fixed dose of AS15. Adverse events (AEs), including predefined dose-limiting toxicity (DLT) and the anti-PRAME humoral response (ELISA), were coprimary end points. Cellular immune responses were evaluated using in vitro assays. RESULTS: 66 patients were treated (20, 24 and 22 in the respective cohorts). AEs considered by the investigator to be causally related were mostly grade 1 or 2 injection site symptoms, fatigue, chills, fever and headache. Two DLTs (grade 3 brain oedema and proteinuria) were recorded in two patients in two cohorts (cohorts 2 and 3). All patients had detectable anti-PRAME antibodies after four immunisations. Percentages of patients with predefined PRAME-specific-CD4+T-cell responses after four immunisations were similar in each cohort. No CD8+ T-cell responses were detected. CONCLUSIONS: The PRAME immunotherapeutic had an acceptable safety profile and induced similar anti-PRAME-specific humoral and cellular immune responses in all cohorts. As per protocol, the phase II study segment was initiated to further evaluate the 500 µg PRAME immunotherapeutic dose. TRIAL REGISTRATION NUMBER: NCT01149343, Results.

Diet- and diabetes-induced changes of ob gene expression in rat adipose tissue.

ob gene regulation is as yet unknown. We first examined whether the ob gene is under physiological control by the nutritional state. Fasting produced a sharp (95%) decrease of ob mRNA in epididymal and inguinal fat pads from 24 h onward. Refeeding rapidly (3-6 h) re-induced ob gene expression and corrected it within 24 h. Similar changes in fatty acid synthase (FAS) and GLUT4 mRNAs were observed, whereas phosphoenolpyruvate carboxykinase (PEPCK) mRNA showed an opposite evolution. We next examined the potential role of insulin. In adipose tissue of streptozotocin-diabetic rats, ob mRNA levels were decreased by 80%. Insulin treatment (4 days) only marginally increased ob mRNA, but restored euglycemia and overcorrected FAS, GLUT4 and PEPCK expression. In conclusion, we provide evidence for a physiological regulation of ob gene by variations in the nutritional state. We also show that ob expression is impaired in streptozotocin-diabetic rats and only slightly restored by insulin treatment, which suggests that ob gene is not or only minimally regulated by the hormone.

Hypoleptinemia in patients with anorexia nervosa: loss of circadian rhythm and unresponsiveness to short-term refeeding.

Leptin is a protein encoded by the ob gene that is expressed in adipocytes and regulates eating behavior via neuroendocrine mechanisms. Plasma leptin levels have been shown to correlate with weight and body fat in normal, obese and anorexic subjects. In the last of these populations, the dynamic profile of plasma leptin levels during short-term refeeding has never been assessed. We thus investigated basal plasma leptin levels in 29 female patients with anorexia nervosa (AN) (age 21.9 +/- 1.4 years, body mass index (BMI) 15.2 +/- 0.3 kg/m2) and in 80 normal female controls (age 21.2 +/- 0.2 years, BMI 20.3 +/- 0.3 kg/m2, mean +/- S.E.M.). Basal plasma leptin levels in AN were decreased by 77% compared with controls (2.5 +/- 0.2 vs 11.1 +/- 0.7 ng/ml, P < 0.0001). In both AN subjects and controls, plasma leptin levels correlated significantly with BMI (r2 = 0.448, P < 0.0001 and r2 = 0.339, P < 0.0001 respectively). Five AN patients (four female, one male, age 22.0 +/- 4.7 years, BMI 14.2 +/- 0.4 kg/m2, body fat 4.3 +/- 0.9 kg or 11.0 +/- 1.9% of body weight, basal metabolic rate (BMR) 958 +/- 122 kcal/day) were studied during a 3-day refeeding period and compared with eight control subjects (two male, six female, age 25.7 +/- 1.2 years, BMI 21.3 +/- 0.8 kg/m2, body fat 15.1 +/- 0.9 kg or 24.6 +/- 1.7%, BMR 1455 +/- 78 kcal/day) submitted to 36-h fasting. The amount of calories administered was based on BMR + 20% (carbohydrate 60%, protein 17%, fat 23%). In contrast to the rise in leptin levels that occurred during refeeding after a prolonged fast period in normal subjects, plasma leptin levels remained low and unchanged throughout the 3 days of renutrition in AN patients. The circadian rhythm of leptin was also completely abolished. This contrasted with the preserved circadian variations of cortisol, whose mean levels were increased. In conclusion, we confirmed that plasma leptin levels are low in AN and correlate with body weight. We further demonstrated that plasma leptin levels do not respond to short-term refeeding in anorexic patients in whom circadian variations are not restored, which suggests that the acute regulation of leptin by positive changes in energy balance is not preserved under a critical threshold of body fat.

Biological and clinical developments in melanoma vaccines.

The identification of antigens recognised on human tumours by autologous T-lymphocytes has opened the way for vaccination strategies involving defined tumour antigens. These vaccinations are therapeutic, i.e. they involve patients with detectable disease. Tumour regressions have been observed in a minority of melanoma patients in Phase I/II trials. Some of these regressions have been complete and long lasting. Improving the efficacy of therapeutic vaccines will critically depend on their capacity to trigger a robust immune response, on the development of appropriate methods to monitor these antitumour immune responses to vaccination and on a better understanding of the mechanisms used by tumours to escape immune attack. Finally, the initiation of large randomised Phase III trials will determine the impact of these vaccines on melanoma treatment.

Breast cancer vaccines: a clinical reality or fairy tale?

The characterization of tumor antigens recognized by immune effector cells has opened the perspective of developing therapeutic vaccines in the field of breast cancer. The potential advantages of the vaccines are: (i) the induction of a robust immune response against tumors that are spontaneously weekly immunogenic; (ii) the tumor specificity for some antigens; (iii) the good tolerance and safety profile and (iv) the long-term immune memory, critical to prevent efficiently tumor recurrence. Most trials evaluating breast cancer vaccines have been carried out in patients with extended metastatic breast cancer, characterized by aggressive tumors, resistant to standard cytotoxic treatments, so that clinical efficacy was difficult to achieve. However, some significant immune responses against tumor antigens induced upon vaccinations were recorded. The aim of this review is to analyze the activity of vaccination strategies in current clinical trials. Data of clinical activity have been observed by using vaccines targeting HER2/neu protein, human telomerase reverse transcriptase, carcinoembryonic antigen and carbohydrate antigen given after stem cell rescue. The review discusses possible future directions for vaccine development and applications in the adjuvant setting.

Individual differences in the orientation of the cytolytic T cell response against mouse tumor P815.

We reported previously that the mouse tumor P815 expresses four distinct antigens (A, B, C, D) recognized by syngeneic cytolytic T lymphocytes (CTL). A fifth P815 antigen (E) was identified by means of a CTL clone derived from tumor-infiltrating lymphocytes. We compared a number of mice for the orientation of their CTL response with respect to the various P815 antigens. Lymphocytes from mice inoculated subcutaneously with living P815 cells were stimulated in vitro with tumor cells and the resulting CTL were tested against targets expressing either antigens A and B or antigens C, D and E. Many mice had an asymmetrical response, some producing CTL directed almost exclusively against antigens A, B and others producing CTL directed almost exclusively against C, D. E. When mice were inoculated into two separate sites, different orientations in the responses of the two local lymph nodes were often observed, suggesting that individual differences in the orientation of the anti-P815 CTL response do not result from preexisting differences between the animals. Asymmetrical CTL responses persisted in mice that were given a second injection of tumor cells. A possible interpretation of our results is that the major component of the CTL response is made of the progeny of a very small number of CTL precursors that happen to be the first to be stimulated by the tumor antigens.

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.

Identification of a MAGE-1 peptide recognized by cytolytic T lymphocytes on HLA-B*5701 tumors.

"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 antigens that have been used in therapeutic vaccination trials of cancer patients. We report the identification of a new MAGE-1-encoded peptide that is recognized by a cytolytic T-lymphocyte (CTL) clone on human leukocyte antigen (HLA)-B*5701. The sequence of the peptide, corresponding to position 102-112 of the MAGE-1 protein sequence, is ITKKVADLVGF. When tumor cells expressing MAGE-1 were transfected with HLA-B*5701, they were lyzed by the CTL clone, indicating that the peptide is processed in tumor cells and can, therefore, be used as a target for anti-tumoral vaccination.

A tyrosinase nonapeptide presented by HLA-B44 is recognized on a human melanoma by autologous cytolytic T lymphocytes.

The human tyrosinase gene has been reported previously to code for two distinct antigens recognized on HLA-A2 melanoma cells by autologous cytolytic T lymphocytes (CTL). By stimulating lymphocytes of melanoma patient MZ2 with a subclone of the tumor cell line of this patient, we obtained a CTL clone that lysed this subclone but did not lyse other subclones of the same melanoma cell line. The sensitive melanoma subclone was found to express a much higher level of tyrosinase than the others, suggesting that the antigen recognized by the CTL might be encoded by tyrosinase. Transfection of a tyrosinase cDNA demonstrated that the CTL clone indeed recognized a tyrosinase product presented by HLA-B*4403. The relevant antigenic peptide corresponds to residues 192-200 of the tyrosinase protein. Lymphoblastoid cells of the B*4402 subtype were not recognized by the CTL following incubation with the peptide. Nevertheless, by stimulating in vitro lymphocytes of a healthy HLA-B*4402 donor with autologous adherent cells pulsed with the same peptide, we obtained a CTL clone which recognized tumor cells expressing tyrosinase and HLA-B*4402. As HLA-B44 is expressed in 24% of Caucasians, the tyrosinase-B44 antigen may constitute a useful target for specific immunotherapy of melanoma.

Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes.

A number of cytolytic T lymphocyte (CTL) clones derived from several melanoma patients have been found to recognize a majority of melanomas from HLA-A2 patients. We have reported previously that two such CTL clones recognize a product of the tyrosinase gene that is presented by HLA-A2. Here we show that one of these CTL clones recognizes a peptide encoded by the first nine amino acids of the putative signal sequence of tyrosinase. The other CTL clone recognizes a different tyrosinase peptide corresponding to amino acids 368-376. Both peptides contain consensus motifs of HLA-A2 binding peptides.

Genes coding for tumor antigens recognized by human cytolytic T lymphocytes.

In order to define the antigens recognized by cytolytic T lymphocytes (CTLs) on autologous tumors, we derived tumor-specific CTL clones from autologous mixed lymphocyte tumor cell cultures. The gene coding for a tumor rejection antigen expressed on a melanoma was isolated by transfecting genomic DNA of the tumor into an antigen-loss variant of the melanoma. Transfectants were identified on the basis of their ability to stimulate tumor necrosis factor release by the CTL clone. The gene that transferred the expression of the antigen was named MAGE-1. It is a new gene, silent in normal tissues with the exception of testis, but expressed in several types of tumors. The antigen recognized by the CTL clone is a nonapeptide derived from the protein encoded by gene MAGE-1, and presented by the HLA class I molecule HLA-A1. Using two other antimelanoma CTL clones, we identified the tyrosinase gene as coding for an antigen presented by HLA-A2 on this type of tumors. The identification of these tumor rejection antigens open new possibilities for the specific immunotherapy of cancer.

Analysis of antigens recognized on human melanoma cells by A2-restricted cytolytic T lymphocytes (CTL).

We have pursued our analysis of potential tumor-rejection antigens recognized on human melanoma by autologous cytolytic T lymphocytes (CTL). We reported previously that 3 distinct antigens (A,B,C) were recognized on melanoma cell line SK29-MEL in association with HLA-A2. Selection for melanoma-cell variants resistant to anti-A CTL revealed that antigen A consists of at least 2 determinants (Aa, Ab) which can be lost separately. Genetic linkage between Aa and Ab was suggested by concomitant loss of Aa and Ab in an immunoselected tumor-cell variant. This variant was also resistant to an autologous CTL clone restricted by HLA-B45, indicating that this CTL may also recognize a determinant of antigen A. Of 11 allogeneic HLA-A2 melanoma cell lines that were tested, 5 expressed both Aa and Ab, 1 expressed only Aa, and 1 only Ab. None of them was lysed by anti-B or anti-C CTL clones. A CTL clone derived from another HLA-A2-melanoma patient was found to have exactly the same lytic pattern as the anti-Ab CTL of the first patient. This suggested that it may be possible to elicit an anti-Ab response in many HLA-A2 patients. We conclude that there are at least 2 distinct antigens presented in association with HLA-A2 that are common to many melanomas and therefore constitute promising targets for specific immunotherapy.