Michael Arand

GRANULOCYTE-MACROPHAGE-COLONY-STIMULATING FACTOR ENHANCES IMMUNE RESPONSES TO MELANOMA-ASSOCIATED PEPTIDES IN VIVO

Peptide epitopes derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC‐restricted cytotoxic T lymphocytes (CTL). The characterization of multiple CTL‐defined antigenic determinants has opened possibilities of development of antigen‐targeted vaccines. In the present study, we determined CTL reactivity against melanoma‐associated peptides derived from Melan A/MART‐I, tyrosinase, and gp100/Pmel17 in 3 HLA‐A2+ melanoma patients. Then, we assessed the immune responses to synthetic melanoma‐associated peptides injected intradermally. After 3 cycles of immunization with peptide alone, we used systemic GM‐CSF as an adjuvant during the fourth cycle of immunization. Enhanced DTH reactions and CD8+ CTL responses were observed after treatment with systemic GM‐CSF. Immunohistochemical characterization of DTH‐constituting elements revealed infiltrates of CD4+ and CD8+ T lymphocytes and strong expression of IL‐2 and γIFN, suggesting the activation of CD4+ Thl and CD8+ CTL by peptides presented by MHC‐class‐I molecules of dermal APC. Objective tumor regression was documented in all patients. We conclude that systemic GM‐CSF enhances immune responses to melanoma‐associated peptides and supports CTL‐mediated tumor rejection in vivo.

Immunoselection in vivo: independent loss of MHC class I and melanocyte differentiation antigen expression in metastatic melanoma.

Peptides derived from melanocyte differentiation antigens have been identified as targets for MHC class I‐restricted cytolytic T lymphocytes (CTLs) in human melanoma. Regression of antigen‐expressing tumors as well as selection of antigen‐loss variants in the presence of antigen‐specific CTLs have previously been reported. In the present study, we determined the expression of the melanocyte differentiation antigens Melan A/MART‐I and tyrosinase by mRNA analysis and by immunohistochemical staining with the monoclonal antibodies (MAbs) A103 and T311. Co‐expression of Melan A/MART‐I and tyrosinase was detected by both methods in 18/20 melanomas tested. However, immunohistochemistry provided additional information on intensity and microheterogeneity of antigen expression that cannot be detected by mRNA analysis as a molecular basis for the escape from CTL recognition of antigen‐negative tumor cells. Comparative analysis of repeated biopsies of metastatic lesions in 5 HLA‐A2+ patients showed a gradual loss of Melan A/MART‐I expression in 4/5 and of tyrosinase in 2/5 samples in association with tumor progression. However, 3 of these patients had growing antigen‐positive tumors in the presence of antigen‐specific CTLs. This led us to assess the expression of MHC class I, the essential restriction element for CTL recognition, and of HLA‐A2. We found an unexpectedly high frequency of MHC class I‐negative tumors (9/20). Loss of MHC class I expression was detected in 3/5 progressive tumors and isolated loss of HLA‐A2 in 1/5 tumors. Our results suggest that strategies enhancing the expression of MHC class I and tumor‐associated antigens need to be considered in attempts at making vaccination more effective. Int. J. Cancer, 71:142–147, 1997.

Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic‐T‐cell responses: Evidence for immunoselection of antigen‐loss variants in vivo

Antigenic peptides derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC‐restricted cytotoxic T lymphocytes (CTL). CTL directed against peptides derived from the Melan A/MART‐1, tyrosinase and gp100/Pmel17 antigens can be detected in melanoma patients and in healthy controls. The presence of defined antigenic peptides and corresponding precursor CTL in patients with metastatic melanoma opens perspectives for the development of antigen‐specific tumor vaccines. In this study, we examined the expression of Melan A/MART‐1, tyrosinase and gp100/Pmel17 in fresh melanoma tissues of HLA‐A2+ patients and the spontaneous CTL reactivity against antigenic peptides derived from these antigens. Our results demonstrate an inverse correlation of antigen expression and CTL response to Melan A/MART‐1 and tyrosinase were induced by intradermal immunization with synthetic nona‐ or deca‐peptides derived from these antigens. Metastases increasing in size over time showed a loss of Melan A/MART‐1 expression in the presence of CTL in one patient. The regression of a metastasis with persistent tyrosinase expression was observed in the other patient after the induction of CTL, reactive against tyrosinase. We conclude that CTL responses against melanocyte differentiation antigens may mediate regression of antigen‐positive tumors and select for antigen‐loss variants in vivo.

Generation of cytotoxic T‐cell responses with synthetic melanoma‐associated peptides in vivo: Implications for tumor vaccines with melanoma‐associated antigens

Peptide epitopes derived from differentiation antigens of the melanocyte lineage have been identified in human melanomas and normal cultured melanocytes as targets for MHC‐restricted cytotoxic T lymphocytes (CTL). Characterization of multiple CTL‐defined antigenic determinants and the presence of corresponding precursor CTL open perspectives for the development of antigen‐based vaccines. In the present study, we determined the CTL reactivity against melanoma‐associated peptides derived from Melan A/MART‐I, tyrosinase and gp100/Pme117 in 10 HLA‐A2* melanoma patients and 10 healthy individuals. Then, we examined the immunological effects and toxicity of intradermal inoculation of synthetic melanoma‐associated peptides. Six patients with advanced melanoma received weekly intradermal injections of 6 melanoma‐associated peptides and the influenza matrix peptide as a control for 4 consecutive weeks. DTH reactions were observed in 5/6 patients at the injection sites of the tyrosinase signal peptide and of the influenza matrix peptide. No toxic side effects were observed. Changes in CTL reactivity after peptide vaccination were assessed by an MLPC assay for each peptide. Generation of peptide‐specific CTL was documented against Melan A/MART‐I‐derived peptide epitopes, the tyrosinase signal peptide and the influenza matrix peptide after vaccination. A decreasing CTL response against the internal tyrosinase peptide was documented in 1 patient through the course of vaccination and a decrease in DTH reactions. No major tumor regressions were observed. Two patients with rapidly progressive disease before vaccination have shown disease stabilization since vaccinations started. In conclusion, our results demonstrate that peptide alone injected intradermally may generate antigen‐specific DTH reactions and an increase of antigen‐specific CTL reactivity.

Humoral immune responses of cancer patients against "Cancer-Testis" antigen NY-ESO-1: correlation with clinical events.

Humoral immune responses against the “Cancer‐Testis” (CT) antigen NY‐ESO‐1 are frequently observed in patients with NY‐ESO‐1 expressing tumors. This is in contrast to other known tumor antigens (TA) defined by antibody or cytotoxic T cell (CTL) reactivity, i.e., MAGE‐1, MAGE‐3, SSX2, Melan A, and tyrosinase. No NY‐ESO‐1 antibody has been detected in healthy controls and patients with NY‐ESO‐1 negative tumors. In this study, we have assessed the NY‐ESO‐1 serum antibody response in patients with NY‐ESO‐1 positive tumors of different histological types and stages using Western blotting and an ELISA. Of the 12 patients analyzed, 10 had demonstrable NY‐ESO‐1 antibodies at the start of the study. All patients were followed for changes in NY‐ESO‐1 antibody titers during the course of tumor treatment and clinical evolution. In 4 patients, an increase of NY‐ESO‐1 antibody titer was observed with progression of disease or extensive tumor necrosis under treatment. One patient showed a stable NY‐ESO‐1 antibody titer over 3 years along with gradual regression of a large tumor mass. In 5 patients, a decrease of NY‐ESO‐1 antibody was detected: in 1 patient after curative tumor resection, in 3 patients with partial regression of metastatic disease under chemo‐ and immunotherapy, and in another patient with a NY‐ESO‐1 negative tumor relapse. Our results indicate that the induction and maintenance of NY‐ESO‐1 antibody is dependent on the presence of NY‐ESO‐1 expressing tumors. Furthermore, changes in NY‐ESO‐1 antibody titers correlate with the evolution of NY‐ESO‐1 positive disease. Int. J. Cancer (Pred. Oncol.) 84:506–510, 1999.

Structure of Aspergillus niger epoxide hydrolase at 1.8 Å resolution: implications for the structure and function of the mammalian microsomal class of epoxide hydrolases

BACKGROUND Epoxide hydrolases have important roles in the defense of cells against potentially harmful epoxides. Conversion of epoxides into less toxic and more easily excreted diols is a universally successful strategy. A number of microorganisms employ the same chemistry to process epoxides for use as carbon sources. RESULTS The X-ray structure of the epoxide hydrolase from Aspergillus niger was determined at 3.5 A resolution using the multiwavelength anomalous dispersion (MAD) method, and then refined at 1.8 A resolution. There is a dimer consisting of two 44 kDa subunits in the asymmetric unit. Each subunit consists of an alpha/beta hydrolase fold, and a primarily helical lid over the active site. The dimer interface includes lid-lid interactions as well as contributions from an N-terminal meander. The active site contains a classical catalytic triad, and two tyrosines and a glutamic acid residue that are likely to assist in catalysis. CONCLUSIONS The Aspergillus enzyme provides the first structure of an epoxide hydrolase with strong relationships to the most important enzyme of human epoxide metabolism, the microsomal epoxide hydrolase. Differences in active-site residues, especially in components that assist in epoxide ring opening and hydrolysis of the enzyme-substrate intermediate, might explain why the fungal enzyme attains the greater speeds necessary for an effective metabolic enzyme. The N-terminal domain that is characteristic of microsomal epoxide hydrolases corresponds to a meander that is critical for dimer formation in the Aspergillus enzyme.

Sequence similarity of mammalian epoxide hydrolases to the bacterial haloalkane dehalogenase and other related proteins. Implication for the potential catalytic mechanism of enzymatic epoxide hydrolysis

Direct comparison of the amino acid sequences of microsomal and soluble epoxide hydrolase superficially indicates that these enzymes are unrelated. Both proteins, however, share significant sequence similarity to a bacterial haloalkane dehalogenase that has earlier been shown to belong to the α/β hydrolase fold family of enzymes. The catalytic mechanism for the dehalogenase has been elucidated in detail [Verschueren et al. (1993) Nature 363, 693‐698] and proceeds via an ester intermediate where the substrate is covalently bound to the enzyme. From these observations we conclude (i) that microsomal and soluble epoxide hydrolase are distantly related enzymes that have evolved from a common ancestral protein together with the haloalkane dehalogenase and a variety of other proteins specified in the present paper, (ii) that these enzymes most likely belong to the α/β hydrolase fold family of enzymes and (iii) that the enzymatic epoxide hydrolysis proceeds via a hydroxy ester intermediate, in contrast to the presently favoured base‐catalyzed direct attack of the epoxide by an activated water.

Mammalian epoxide hydrolases in xenobiotic metabolism and signalling.

Epoxide hydrolases catalyse the hydrolysis of electrophilic—and therefore potentially genotoxic—epoxides to the corresponding less reactive vicinal diols, which explains the classification of epoxide hydrolases as typical detoxifying enzymes. The best example is mammalian microsomal epoxide hydrolase (mEH)—an enzyme prone to detoxification—due to a high expression level in the liver, a broad substrate selectivity, as well as inducibility by foreign compounds. The mEH is capable of inactivating a large number of structurally different, highly reactive epoxides and hence is an important part of the enzymatic defence of our organism against adverse effects of foreign compounds. Furthermore, evidence is accumulating that mammalian epoxide hydrolases play physiological roles other than detoxification, particularly through involvement in signalling processes. This certainly holds true for soluble epoxide hydrolase (sEH) whose main function seems to be the turnover of lipid derived epoxides, which are signalling lipids with diverse functions in regulatory processes, such as control of blood pressure, inflammatory processes, cell proliferation and nociception. In recent years, the sEH has attracted attention as a promising target for pharmacological inhibition to treat hypertension and possibly other diseases. Recently, new hitherto uncharacterised epoxide hydrolases could be identified in mammals by genome analysis. The expression pattern and substrate selectivity of these new epoxide hydrolases suggests their participation in signalling processes rather than a role in detoxification. Taken together, epoxide hydrolases (1) play a central role in the detoxification of genotoxic epoxides and (2) have an important function in the regulation of physiological processes by the control of signalling molecules with an epoxide structure.

Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site

Epoxide hydrolases are essential for the processing of epoxide‐containing compounds in detoxification or metabolism. The classic epoxide hydrolases have an α/β hydrolase fold and act via a two‐step reaction mechanism including an enzyme–substrate intermediate. We report here the structure of the limonene‐1,2‐epoxide hydrolase from Rhodococcus erythropolis, solved using single‐wavelength anomalous dispersion from a selenomethionine‐substituted protein and refined at 1.2 Å resolution. This enzyme represents a completely different structure and a novel one‐step mechanism. The fold features a highly curved six‐stranded mixed β‐sheet, with four α‐helices packed onto it to create a deep pocket. Although most residues lining this pocket are hydrophobic, a cluster of polar groups, including an Asp–Arg–Asp triad, interact at its deepest point. Site‐directed mutagenesis supports the conclusion that this is the active site. Further, a 1.7 Å resolution structure shows the inhibitor valpromide bound at this position, with its polar atoms interacting directly with the residues of the triad. We suggest that several bacterial proteins of currently unknown function will share this structure and, in some cases, catalytic properties.

Induction of the peroxisome proliferator activated receptor by fenofibrate in rat liver

The process of peroxisome proliferation in rodent liver by hypolipidemic compounds and related substances has recently been shown to be receptor‐madiated. In the present study, we have examined the effect of oral administration of the strong peroxisome proliferator fenofibrate on the hepatic expression level of the peroxisome proliferator activated receptor (PPAR) in rats. Immunoblots of rat liver cytosols and nuclear extracs using antibodies raised against recombinant PPAR/β‐galactosidase fusion proteins revealed a pronounced increase in the amount of PPAR protein in response to fenofibrate treatment. This induction could also be confirmed at the level or RNA by Northern blotting. A time‐course investigation showed a delayed accumulation of mRNA in response to the treatment, starting on day 2 after a latency period of at least one day. Thus, induction of the PPAR as a response to peroxisome proliferators represents one important dimension of the pleiotropic effects of peroxisome proliferators.