Stability and CTL-activity of P40/ELA Melanoma Vaccine Candidate

The decapeptide ELA (ELAGIGILTV), a Melan-A/MART-1 antigen immunodominant peptide analogue, is an interesting melanoma vaccine candidate alone or in combination with other tumour antigens. P40, the recombinant outer membrane protein A of Klebsiella pneumoniae (kpOmpA), was recently shown to target dendritic cells and to induce peptide-specific CTLs. Here we investigated the adjuvant role of P40 mixed or chemically conjugated to ELA. This compound is an N-terminal glutamic acid-containing peptide. However, it has been reported that the amino group and the gamma-carboxylic group of glutamic acids easily condense to form pyroglutamic derivatives. Usually, to overcome this stability problem, peptides of pharmaceutical interest were developed with a pyroglutamic acid instead of N-terminal glutamic acid, without loss of pharmacological properties. Unfortunately, the pyroglutamic acid derivative (PyrELA) as well as the N-terminal acetyl capped derivative (AcELA) failed to elicit CTL activity when mixed with P40 adjuvant protein. Despite the apparent minor modifications introduced by PyrELA and AcELA, these two derivatives have probably lower affinity than ELA for the class I Major Histocompatibility Complex. Furthermore, this stability problem is worse in the case of clinical grade ELA, produced as an acetate salt, like most of the pharmaceutical grade peptides. We report here that the hydrochloride shows a higher stability than the acetate and may be suitable for use in man.     

A manual subtractive end-group determination of oligopeptides using fluorescamine or o-phthalaldehyde

A new method for the end-group determination of peptides using the fluorogenic reagents fluorescamine or o-phthalaldehyde is described. The method is based on the property that the derivatives of the N-terminal amino group of peptides formed in solution after reaction with either reagent are resistant to acid hydrolysis. The N-terminal amino acid can be determined by simply comparing the amino acid analysis of the original peptide with the fluorescent derivative of the peptide. In general, the decrease of the N-terminal residue in the reacted peptides in 80–90% with fluorescamine and more than 90% with o-phthalaldehyde. Any N-terminal amino acid, with the exception of proline, can thus be determined.     

Primary structure and synthesis of a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea maderae

A peptide which stimulates the contractile activity of the cockroach hindgut was isolated from head extracts of the cockroach, Leucophaea maderae. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N-terminal blocking by pyroglutamic acid. The N-terminal pGlu was removed enzymatically and the unblocked fragment was sequenced with an automated gas-phase peptide sequencer. The structure determined (pGlu-Thr-Ser-Phe-Thr-Pro-Arg-Leu-NH2) was synthesized and shown to be both chemically and biologically identical with the natural product.     

Formation of specific amino acid sequences during carbodiimide-mediated condensation of amino acids in aqueous solution

Carbodiimide-mediated peptide synthesis in aqueous solution at room temperature has been studied with respect to self-ordering of amino acids. Inasmuch as glutamic acid is readily converted into pyroglutamic acid, the peptides formed by copolymerization of glutamic acid with other amino acids are preferentially pyroglutamyl-peptides. Competition experiments were carried out to determine the influence of the amino acid side chains on the reactivity of amino acids and peptides during the dehydration condensation. The results show that the self-ordering process is controlled by both the activated carboxyl component (amino acid or growing peptide) and the incoming amino acid. A condensation of pyroglutamic acid, alanine and another amino acid component Xxx (Xxx = Gly, Val, Leu or Gly-Tyr) preferentially yielded the dipeptide pyroGlu-Ala, but the formation of the tripeptide pyroGlu-Ala-Ala became strongly reduced because of competing reactions. A simple explanation for the observed selectivities is not at hand. Polypeptides were so far only obtained when they were allowed to precipitate in the reaction system. Evidence for the non-random copolymerization of larger peptides is presented as well.     

Identification of melanoma-specific peptide epitopes by HLA-A2.1-restricted cytotoxic T lymphocytes

HLA-A2.1-associated peptides, extracted from human melanoma cells, were used to study epitopes for melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes （CTLs） by epitope reconstitution, active peptide sequence characterization and synthetic peptide verification. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and aUogeneic HLA-A2. 1 positive melanomas, but not HLA-A2.1 negative melanomas or HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by anti-CD3, anti-HLA class I and anti-HLA-A2.1 monoclonal antibodies. HLA-A2.1 molecules were purified from detergent-solubilized human melanoma cells by immunoaffinity column chromatography and further fractionated by reversed phase high performance liquid chromatography. The fractions were assessed for their ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive antigen-processing mutant T2 cells. Three reconstitution peaks were observed in lactate dehydrogenase release assay. Mass spectrometry and ion-exchange high performance liquid chromatography analysis were used to identify peptide epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine amino acid residues. The data from reconstitution experiments confirmed that the synthetic peptides contained epitopes and that the peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL were present in these different melanoma cells. These peptides could be potentially exploited in novel peptide-based antitumor vaccines in immunotherapy for CTL.     

Assessment of immunogenicity of human Melan-A peptide analogues in HLA-A*0201/Kb transgenic mice

Previous studies have shown that substitution of single amino acid residues in human Melan-A immunodominant peptides Melan-A27-35 and Melan-A26-35 greatly improved their binding and the stability of peptide/HLA-A*0201 complexes. In particular, one Melan-A peptide analogue was more efficient in the generation of Melan-A peptide-specific and melanoma-reactive CTL than its parental peptide in vitro from human PBL. In this study, we analyzed the in vivo immunogenicity of Melan-A natural peptides and their analogues in HLA-A*0201/Kb transgenic mice. We found that two human Melan-A natural peptides, Melan-A26-35 and Melan-A27-35, were relatively weak immunogens, whereas several Melan-A peptide analogues were potent immunogens for in vivo CTL priming. In addition, induced Melan-A peptide-specific mouse CTL cross-recognized natural Melan-A peptides and their analogues. More interestingly, these mouse CTL were also able to lyse human melanoma cell lines in vitro in a HLA-A*0201-restricted, Melan-A-specific manner. Our results indicate that the HLA-A*0201/Kb transgenic mouse is a useful animal model to perform preclinical testing of potential cancer vaccines, and that Melan-A peptide analogues are attractive candidates for melanoma immunotherapy.     

Formation of specific amino acid sequences during carbodiimide-mediated condensation of amino acids in aqueous solution,and computer-simulated sequence generation

Carbodiimide-mediated peptide synthesis in aqueous solution has been studied with respect to self-ordering of amino acids. The copolymerisation of amino acids in the presence of glutamic acid or pyroglutamic acid leads to short pyroglutamyl peptides. Without pyroglutamic acid the formation of higher polymers is favoured.The interactions of the amino acids and the peptides, however, are very complex. Therefore, the experimental results are rather difficult to explain. Some of the experimental results, however, can be explained with the aid of computer simulation programs. Regarding only the tripeptide fraction the copolymerisation of pyroGlu, Ala and Leu, as well as the simulated copolymerisation lead to pyroGlu-Ala-Leu as the main reaction product. The amino acid composition of the insoluble peptides formed during the copolymerisation of Ser, Gly, Ala, Val, Phe, Leu and Ile corresponds in part to the computer-simulated copolymerisation data.     

Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes

CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.     

Formation of specific amino acid sequences during carbodiimide-mediated condensation of amino acids in aqueous solution, and computer-simulated sequence generation

Carbodiimide-mediated peptide synthesis in aqueous solution has been studied with respect to self-ordering of amino acids. The copolymerisation of amino acids in the presence of glutamic acid or pyroglutamic acid leads to short pyroglutamyl peptides. Without pyroglutamic acid the formation of higher polymers is favoured. The interactions of the amino acids and the peptides, however, are very complex. Therefore, the experimental results are rather difficult to explain. Some of the experimental results, however, can be explained with the aid of computer simulation programs. Regarding only the tripeptide fraction the copolymerisation of pyroGlu, Ala and Leu, as well as the simulated copolymerisation lead to pyroGlu-Ala-Leu as the main reaction product. The amino acid composition of the insoluble peptides formed during the copolymerisation of Ser, Gly, Ala, Val, Phe, Leu and Ile corresponds in part to the computer-simulated copolymerisation data.     

Isolation and characterization of the glutaminyl cyclases from Solanum tuberosum and Arabidopsis thaliana: implications for physiological functions

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamic acid at the N-terminus of several peptides and proteins. On the basis of the amino acid sequence of Carica papaya QC, we identified cDNAs of the putative counterparts from Solanum tuberosum and Arabidopsis thaliana. Upon expression of the corresponding cDNAs from both plants via the secretory pathway of Pichia pastoris, two active QC proteins were isolated. The specificity of the purified proteins was assessed using various substrates with different amino acid composition and length. Highest specificities were observed with substrates possessing large hydrophobic residues adjacent to the N-terminal glutamine and for fluorogenic dipeptide surrogates. However, compared to Carica papaya QC, the specificity constants were approximately one order of magnitude lower for most of the QC substrates analyzed. The QCs also catalyzed the conversion of N-terminal glutamic acid to pyroglutamic acid, but with approximately 10(5)- to 10(6)-fold lower specificity. The ubiquitous distribution of plant QCs prompted a search for potential substrates in plants. Based on database entries, numerous proteins, e.g., pathogenesis-related proteins, were found that carry a pyroglutamate residue at the N-terminus, suggesting QC involvement. The putative relevance of QCs and pyroglutamic acid for plant defense reactions is discussed.     

An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles

We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8⁺ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.     

pS10147-2, a3.7 kb multicopy plasmid isolated from Streptomyces coelicolor

The following putative precursors of the pseudomurein were isolated from trichloroacetic acid extracts of Methanobacterium thermoautotrophicum: a uridine diphosphate activated derivative of glutamic acid and the uridine diphosphate activated peptides (see text). The activated glutamic acid residue and the three activated pepetides lack the glycan components N-acetylglucosamine and N-acetyltalosaminuronic acid present in the intact pseudomurein. In this case uridine diphosphate should be directly linked to the amino group of a glutamic acid residue, which represents a new mode of amino acid and peptide activation.     

Isolation of N-acetylglutamine, pyroglutamic acid and the butyl ester of pyroglutamic acid from human brain

N-acetyl-l -glutamine, pyroglutamic acid, and the butyl ester of pyroglutamic acid were isolated in pure form from an aqueous extract of human brain. These compounds were isolated by combination of paper and ion exchange chromatography. The isolated substance identified as N-acetyl-l -glutamine did not react with the ninhydrin reagent but yielded glutamic acid and ammonia upon acid hydrolysis. An acetyl hydrazide was identified by paper chromatography from hydrazinolysates of the isolated substance. The glutamic acid liberated by hydrolysis had the l -configuration. The results of elementary analysis of the isolated compound were in full accord with the analysis calculated for synthetic N-acetyl-l -glutamine. A large amount of pyroglutamic acid and a substance identical with the butyl ester of pyroglutamic acid were isolated in pure form. The results of our studies suggest that pyroglutamic and the butyl ester derivative were artifacts formed during the isolation and purification procedures.     

Conformational dimorphism of self-peptides and molecular mimicry in a disease-associated HLA-B27 subtype

An interesting property of certain peptides presented by major histocompatibility complex (MHC) molecules is their acquisition of a dual binding mode within the peptide binding groove. Using x-ray crystallography at 1.4 A resolution, we show here that the glucagon receptor-derived self-peptide pGR ((412)RRRWHRWRL(420)) is presented by the disease-associated human MHC class I subtype HLA-B*2705 in a dual conformation as well, with the middle of the peptide bent toward the floor of the peptide binding groove of the molecule in both binding modes. The conformations of pGR are compared here with those of another self-peptide (pVIPR, RRKWRRWHL) that is also displayed in two binding modes by HLA-B*2705 antigens and with that of the viral peptide pLMP2 (RRRWRRLTV). Conserved structural features suggest that the N-terminal halves of the peptides are crucial in allowing cytotoxic T lymphocyte (CTL) cross-reactivity. In addition, an analysis of T cell receptors (TCRs) from pGR- or pVIPR-directed, HLA-B27-restricted CTL clones demonstrates that TCR from distinct clones but with comparable reactivity may share CDR3alpha but not CDR3beta regions. Therefore, the cross-reactivity of these CTLs depends on TCR-CDR3alpha, is modulated by TCR-CDR3beta sequences, and is ultimately a consequence of the conformational dimorphism that characterizes binding of the self-peptides to HLA-B*2705. These results lend support to the concept that conformational dimorphisms of MHC class I-bound peptides might be connected with the occurrence of self-reactive CTL.     

Thermal condensation of a mixture of six amino acids

Amino acids can be converted to peptides by the process of heating if the mixture is dry and if the appropriate proportion of glutamic acid as the dominant monomer is present. Three thermal peptide fractions of the diffusible fraction were isolated in this study. Trifluoracetic acid (TFA) hydrolysis was utilized to open the blocked N-terminal group before performing the dansylation reaction. When the molecular weight of the thermal peptide increased, the proportion of neutral amino acids increased slightly, with a decrease in the concentration of glutamic acid. Quantitative analysis of amino acid composition is also reported.     

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens

Melanoma-reactive HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pmel17/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A*0201⁺ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pmel17/gp100, gp75/trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLA-A*0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A*0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A*0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP. Received: 31 October 1997 / Accepted: 4 August 1999     

Post-translational modifications of chloroperoxidase from Caldariomyces fumago

The secreted form of the halogenating glycoenzyme, chloroperoxidase, is processed from a precursor containing a 21-residue-long, moderately hydrophobic signal sequence, at an atypical Gln-Glu peptide bond. Following cleavage, the N-terminal glutamic acid readily cyclizes into pyroglutamic acid. Chloroperoxidase contains two high-mannose N-glycosylation sites, identified as Asn12 and Asn213. Other modifications include deamidation of residues Asn13, Asn198, and Gln183 into the corresponding acids. Finally, structural arguments suggest that Cys87 may be the axial heme ligand in the active site of chloroperoxidase.     

Use of selected reaction monitoring mass spectrometry for the detection of specific MHC class I peptide antigens on A3 supertype family members

The development of peptide-based vaccines that are useful in the therapeutic treatment of melanoma and other cancers ultimately requires the identification of a sufficient number of antigenic peptides so that most individuals, regardless of their major histocompatibility complex (MHC)–encoded class I molecule phenotype, can develop a cytotoxic T lymphocyte (CTL) response against one or more peptide components of the vaccine. While it is relatively easy to identify antigenic peptides that are presented by the most prevalent MHC class I molecules in the population, it is problematic to identify antigenic peptides that are presented by MHC class I molecules that have less frequent expression in the population. One manner in which this problem can be overcome is by taking advantage of known MHC class I supertypes, which are groupings of MHC class I molecules that bind peptides sharing a common motif. We have developed a mass spectrometric approach which can be used to determine if an antigenic peptide is naturally processed and presented by any given MHC class I molecule. This approach has been applied to the A3 supertype, and the results demonstrate that some, but not all, A3 supertype family–associated peptides can associate with all A3 supertype family members. The approach also demonstrates the shared nature of several newly identified peptide antigens. The use of this technology negates the need to test peptides for their ability to stimulate CTL responses in those cases where the peptide is not naturally processed and bound to the target MHC class I molecule of interest, thus allowing resources to be focused on the most promising vaccine candidates.     

Identification of a naturally processed cytotoxic CD8 T-cell epitope of coxsackievirus B4, presented by HLA-A2.1 and located in the PEVKEK region of the P2C nonstructural protein

The adaptive immune system generates CD8 cytotoxic T lymphocytes (CTLs) as a major component of the protective response against viruses. Knowledge regarding the nature of the peptide sequences presented by HLA class I molecules and recognized by CTLs is thus important for understanding host-pathogen interactions. In this study, we focused on identification of a CTL epitope generated from coxsackievirus B4 (CVB4), a member of the enterovirus group responsible for several inflammatory diseases in humans and often implicated in the triggering and/or acceleration of the autoimmune disease type 1 diabetes. We identified a 9-mer peptide epitope that can be generated from the P2C nonstructural protein of CVB4 (P2C(1137-1145)) and from whole virus by antigen-presenting cells and presented by HLA-A2.1. This epitope is recognized by effector memory (gamma interferon [IFN-gamma]-producing) CD8 T cells in the peripheral blood at a frequency of responders that suggests that it is a major focus of the anti-CVB4 response. Short-term CD8 T-cell lines generated against P2C(1137-1145) are cytotoxic against peptide-loaded target cells. Of particular interest, the epitope lies within a region of viral homology with the diabetes-related autoantigen, glutamic acid decarboxylase-65 (GAD(65)). However, P2C(1137-1145)-specific cytotoxic T lymphocyte (CTL) lines were not activated to produce IFN-gamma by the GAD(65) peptide homologue and did not show cytotoxic activity in the presence of appropriately labeled targets. These results describe the first CD8 T-cell epitope of CVB4 that will prove useful in the study of CVB4-associated disease.     

Development of a novel T cell-oriented vaccine using CTL/Th-hybrid epitope long peptide and biodegradable microparticles,against an intracellular bacterium

Antigen-specific CD8+ T-lymphocytes (cytotoxic T-lymphocytes: CTL), as well as CD4+ T-lymphocytes (helper T-lymphocytes: Th), simultaneously play an important role in the elimination of intracellular bacteria such as Mycobacterium tuberculosis and Listeria monocytogenes. Administration of T-cell epitope short peptide needs large numbers of peptides for effective vaccination due to its easily degradable nature in vivo. In this respect, biocompatible and biodegradable microparticles combined with CTL/Th-hybrid epitope long peptide (long peptide) have been used to diminish the degradation of loaded peptide. The aim of this study is to develop a novel T cell-oriented vaccine against intracellular bacteria that is composed of long peptide and poly (lactic-co-glycolic acid) (PLGA) microparticles. Mouse bone marrow-derived dendritic cells (BMDCs) were loaded with L. monocytogenes listeriolysin O (LLO)-derived or ovalbumin (OVA)-derived long peptide/PLGA or other comparative antigens. The antigen-loaded BMDCs were injected subcutaneously into the flank of mice twice, and then, the spleens were collected and lymphocyte proliferation and interferon-γ production were evaluated. The median diameter of the PLGA spheres was 1.38 μm. Both LLO- and OVA-long peptide/PLGA showed significantly more robust CTL and Th proliferations with higher interferon-γ production than the long peptide alone or CTL and Th short peptides/PLGA vaccination. Furthermore, the LLO-long peptide/PLGA vaccination showed a significantly lower bacterial burden in spleens compared with the long peptide alone or the CTL and Th short peptides/PLGA vaccination after the challenge of lethal amounts of L. monocytogenes. These results suggest that the novel vaccine taking advantages of CTL/Th-hybrid epitope long peptide and PLGA microparticle is effective for protection against intracellular bacteria.