Inhibition of serine-peptidase activity enhances the generation of a survivin-derived HLA-A2-presented CTL epitope in colon-carcinoma cells.

Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

Phosphinic platinum complexes with 8-thiotheophylline derivatives: synthesis, characterization, and antiproliferative activity.

The platinum mixed-phosphine complexes (SP-4,2)-[PtCl(8-MTT)(PPh3)(PTA)] (2) and cis-[Pt(8-MTT)2(PPh3)(PTA)] (3) (MTTH2 = 8-(methylthio)theophylline, PTA = 1,3,5-triaza-7-phosphaadamantane) have been prepared from the precursor cis-[PtCl2(PPh3)(PTA)] (1), which has been fully characterized by X-ray diffraction determination. Antiproliferative activity tests indicated that the presence of one lipophilic PPh3 and one hydrophilic PTA makes 1-3 more active than the analogues bearing two PPh3 or two PTA. The reactivity of cis-[PtCl2(PPh3)2], cis-[PtCl2(PTA)2], and cis-[PtCl2(PPh3)(PTA)] with the bis(thiopurines) bis(S-8-thiotheophylline)methane (MBTTH2), 1,2-bis(S-8-thiotheophylline)ethane (EBTTH2), and 1,3-bis(S-8-thiotheophylline)propane (PBTTH2) has also been investigated. New binuclear complexes have been prepared and identified by spectroscopic techniques and their antiproliferative activities on T2 and SKOV3 cell lines evaluated.

Synthesis and biological evaluation of new vinyl ester pseudotripeptide proteasome inhibitors.

Here we report the synthesis and biological activities of new tripeptidic-based vinyl ester derivative proteasome inhibitors. Starting from Hmb-Val-Ser-Leu-VE prototype, we investigated P2 position and N-terminal substitution. The more effective inhibitors of the series showed remarkable inhibition and selectivity for the trypsin-like (beta2) subunit and were revealed to be specific for the proteasome. In vitro metabolic stability studies of the new vinyl ester analogues are also reported here.

T-cell engineering by a chimeric T-cell receptor with antibody-type specificity for the HIV-1 gp120.

Immune-based approaches of cell therapy against viral pathogens such as the human immunodeficiency virus type 1 (HIV-1) could be of primary importance for the control of this viral infection. Here, we designed a chimeric cell surface receptor (105TCR) to provide primary human T-lymphocytes with antibody-type specificity for the HIV-1 envelope glycoprotein. This receptor includes the single chain Fv domain of the neutralizing anti-gp120 human monoclonal antibody F105, CD8alpha hinge and the transmembrane and the cytoplasmic domains of TCRzeta. Our results show that 105TCR is expressed at the cellular surface and is capable of recognizing the HIV-1 envelope glycoprotein inducing highly efficient effector T-cell responses, including extracellular signal-regulated kinase phosphorylation and cytokine secretion. Moreover, human primary CD8+ T-lymphocytes transduced by oncoretroviral and lentiviral vectors containing the 105TCR gene are able to mediate in vitro-specific cytolysis of envelope-expressing cells and HIV-1-infected CD4+ T-lymphocytes. These findings suggest that 105TCR is particularly suited for in vivo efficacy studies.

Biologically active platinum complexes containing 8-thiotheophylline and 8-(methylthio)theophylline.

Complexes [Pt(mu-N,S-8-TT)(PPh(3))(2)](2) (1), [Pt(mu-S,N-8-TT)(PTA)(2)](2) (2), [Pt(8-TTH)(terpy)]BF(4) (3), cis-[PtCl(8-MTT)(PPh(3))(2)] (4), cis-[Pt(8-MTT)(2)(PPh(3))(2)] (5), cis-[Pt(8-MTT)(8-TTH)(PPh(3))(2)] (6), cis-[PtCl(8-MTT)(PTA)(2)] (7), cis-[Pt(8-MTT)(2)(PTA)(2)] (8), and trans-[Pt(8-MTT)(2)(py)(2)] (9) (8-TTH(2) = 8-thiotheophylline; 8-MTTH = 8-(methylthio)theophylline; PTA = 1,3,5-triaza-7-phosphaadamantane) are presented and studied by IR and multinuclear ((1)H, (31)P[(1)H]) NMR spectroscopy. The solid-state structure of 4 and 9 has been authenticated by X-ray crystallography. Growth inhibition of the cancer cells T2 and SKOV3 induced by the above new thiopurine platinum complexes has been investigated. The activity shown by complexes 4 and 9 was comparable with cisplatin on T2. Remarkably, 4 and 9 displayed also a valuable activity on cisplatin-resistant SKOV3 cancer cells.

Red blood cell-mediated delivery of recombinant HIV-1 Tat protein in mice induces anti-Tat neutralizing antibodies and CTL.

The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model. HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs). The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin-avidin-biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat.Balb/c mice were then immunized with 10 microg of soluble Tat in complete Freund's adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5 microg soluble Tat in incomplete Freund's adjuvant or with 20 ng of RBC-coupled Tat, respectively. Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies. In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat. These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies.

Peptide analogues of a subdominant epitope expressed in ebv-associated tumors: synthesis and immunological activity.

H-Cys-Leu-Gly-Gly-Leu-Leu-Thr-Met-Val-OH (CLG) peptide is an EBV subdominant epitope that represents the target of HLA-A2 restricted CTL responses. The CLG peptide has low affinity for HLA-A2 and does not produce stable complexes, both factors that determine weak CTL responses. In contrast, the [Tyr(1), Ala(3)]CLG (YLA) analogue showed high affinity for HLA-A2 molecules and efficiently stimulated CLG-specific CTL precursors. Nevertheless, this modified epitope showed low enzymatic stability. To further improve the immunotherapeutical potential of this "improved epitope", we have synthesized and tested YLA analogues containing different modifications next to the scissile peptide bond. Among the analogues we found three peptides, with higher enzymatic resistance, that efficiently stimulate CTL responses. These peptides may be used for EBV-specific immunotherapies.

Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogues: chemical behaviour and biological activity.

Copper complex formation equilibria of glycyl-L-histidyl-L-lysine (Gly-His-Lys, GHK) and of two synthetic analogues, where the histidine residue was replaced with a synthetic amino acid (L-spinacine or L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid), have been carefully investigated using different experimental techniques: potentiometry, solution calorimetry, UV-VIS spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopies. All the ligands formed complexes having different stoichiometries and stabilities; evidence for the formation of binuclear species is also shown. The structures of the main complexes are discussed. It is suggested that the lateral lysine amino group participates in complex formation, but only at alkaline pH values: at physiological pH this group is protonated and available for possible interactions with cellular receptors. The above tripeptides have been tested for their enzymatic stability in human serum: the synthetic compounds showed no significant degradation for at least 3 h. Finally, their activity as growth factor has been studied in vitro. The two synthetic analogues showed an activity comparable to or even higher than that of GHK, thus suggesting their possible use as additives in cell culture media, even in the presence of serum. Relevant information on the GHK action mechanism as cell growth factor has been obtained: the formation of copper complexes, driven by the first (Gly) residue, appears necessary while the second residue (His) does not appear to play a specific role; the presence of the free side chain of the third residue (Lys) appears to be of fundamental importance.

c-myc overexpression activates alternative pathways for intracellular proteolysis in lymphoma cells.

Burkitt's lymphoma (BL) is a highly malignant B-cell tumour characterized by chromosomal translocations that constitutively activate the c-myc oncogene. Here we show that BL cells are resistant to apoptosis and do not accumulate ubiquitin conjugates in response to otherwise toxic doses of inhibitors of the proteasome. Deubiquitinating enzymes and the cytosolic subtilisin-like protease tripeptidylpeptidase II are upregulated in BLs, and could be rapidly induced by the overexpression of c-myc in normal B cells carrying oestrogen-driven recombinant Epstein-Barr virus. Apoptosis was induced by inhibiting tripeptidylpeptidase II, suggesting that the activity of this protease may be required for the survival of BL cells. We thus show that there is a regulatory link between c-myc activation and changes in proteolysis that may affect malignant transformation.

Supra-agonist peptides enhance the reactivation of memory CTL responses.

Single amino acid substitutions at TCR contacts may transform a natural peptide Ag in CTL ligands with partial agonist, antagonist, or null activity. We obtained peptide variants by changing nonanchor amino acid residues involved in MHC class I binding. These peptides were derived from a subdominant HLA-A2-presented, latent membrane protein 2-derived epitope expressed in EBV-infected cells and in EBV-associated tumors. We found that small structural changes produced ligands with vastly different activities. In particular, the variants that associated more stably to HLA-A2/molecules did not activate any CTL function, behaving as null ligands. Interestingly, T cell stimulations performed with the combination of null ligands and the natural epitope produced significantly higher specific CTL reactivation than reactivation of CTLs induced by the wild-type epitope alone. In addition, these particular variants activated memory CTL responses in the presence of concentrations of natural epitope that per se did not induce T cell responses. We show here that null ligands increased ZAP-70 tyrosine kinase activation induced by the natural epitope. Our results demonstrate for the first time that particular peptide variants, apparently behaving as null ligands, interact with the TCR, showing a supra-agonist activity. These variant peptides did not affect the effector T cell functions activated by the natural epitope. Supra-agonist peptides represent the counterpart of antagonists and may have important applications in the development of therapeutic peptides.

Design of dimeric peptides obtained from a subdominant Epstein-Barr virus LMP2-derived epitope.

The latent membrane protein 2 (LMP2) is expressed in EBV-associated tumours. LMP2 is a target of HLA-A2 restricted EBV-specific CTL responses and consequently it may represent a good target for specific CTL-based immunotherapies. However, the efficacy of such therapy is limited by the poor immunogenicity of the protein that induces weak cytotoxic T lymphocyte (CTL) responses directed against the CLGGLLTMV (CLG) epitope. Indeed, the CLG peptide presents low affinity for HLA-A2 and does not produce stable complexes. Therefore we synthesized and tested CLG-dimeric analogues with the purpose of characterizing new compounds with the capacity to bind HLA-A2 molecules. By these studies we have identified a few peptides which, compared to the natural epitope, showed higher affinity for HLA-A2 molecules and superior capacity to form a complex. These dimeric peptides may have the potential to induce efficient CTL responses directed to the natural epitope.

Selective amino acid substitutions of a subdominant Epstein-Barr virus LMP2-derived epitope increase HLA/peptide complex stability and immunogenicity: implications for immunotherapy of Epstein-Barr virus-associated malignancies.

The latent membrane protein 2 is an immunogenic antigen expressed in Epstein-Barr virus (EBV)-associated tumors and consequently it may represent a target for specific cytotoxic T lymphocyte (CTL)-based immunotherapies. However, the efficacy of such a therapy is limited by the poor immunogenicity of the protein that induces weak CTL responses directed to the CLGGLLTMV (CLG) epitope only in the minority of EBV-seropositive donors. We have now demonstrated that selective peptide stimulation of peripheral blood lymphocytes induced CLG-specific CTL in all donors, suggesting that this epitope can be a suitable target for specific immunotherapies. We found that the CLG peptide has a low affinity for HLA-A*0201 and does not produce stable complexes, both factors that are likely to determine the strength of CTL responses to this epitope. Therefore, we synthesized and tested CLG analogues carrying single or combined amino acid substitutions to increase HLA/peptide stability. Among the analogues tested we identified two peptides which, compared to the natural epitope, showed higher affinity for HLA-A*0201 molecules, and produced stable complexes. These peptides demonstrated a potent, specific stimulatory capacity and could be used for selective CTL-based therapies.

The lifespan of major histocompatibility complex class I/peptide complexes determines the efficiency of cytotoxic T-lymphocyte responses.

Major histocompatibility complex (MHC)/peptide association and stability are determined by specific amino acid interactions between peptide antigens and the MHC groove, and are regarded as a critical feature in ensuring efficient monitoring by T cells. In this investigation we examined the relationship between MHC/peptide stability and the immunostimulatory capacity of MHC/peptide complexes. For this purpose we compared synthetic peptide analogues derived from the immunodominant HLA-A11-presented IVTDFSVIK (IVT) epitope, for their capacity to reactivate IVT-specific memory cytotoxic T-lymphocyte (CTL) responses. The analogues differentiated from the wild-type epitope by single amino acid substitution at position 2. All peptides showed similar affinity for HLA-A11 molecules and were recognized by IVT-specific CTL clones, but induced HLA-A11 complexes at the cell surface with different lifespan. This model offered the possibility of comparing the capacity of an immunogenic epitope to stimulate a unique population of T-cell precursors depending on the lifespan of its presentation at the cell surface. We demonstrated that stable HLA-A11/peptide complexes efficiently stimulate IVT-specific CTL responses, while HLA-A11/peptide complexes with short lifespan do not. The precise identification of the role of amino acid residues in the formation of stable MHC/peptide complexes may be relevant for the design of wild-type-derived epitopes with high immunogenicity. These analogues may have important applications in the immunotherapy of infectious diseases and immunogenic tumours.

A single specific amino acid residue in peptide antigens is sufficient to activate memory CTL: potential role of cross-reactive peptides in memory T cell maintenance.

In the present study, we examined the structural requirements of peptide Ags for productive interactions with the TCR of CTL. For this purpose, we used as a model a previously identified immunodominant epitope that represents the target of EBV-specific HLA-A11-restricted CTL responses. By the use of peptides having minimal sequence homology with the wild-type epitope, we demonstrated that it is possible to selectively expand and reactivate memory CTL precursors without triggering the lytic mechanisms of wild-type specific effectors. In fact, stimulation of PBL from EBV-seropositive donors by polyalanine analogues, sharing only the putative TCR contact residue with the natural epitope, exclusively induced clonal expansion and reactivation of EBV-specific memory CTL precursors. Interestingly, these polyalanine peptides failed to trigger the cytotoxic function of CTLs specific for the wild-type viral epitope. This clearly indicates that reactivation of memory CTL precursors and triggering of the cytotoxic function have different requirements. The same phenomenon was observed using as stimulators naturally occurring peptides carrying the appropriate TCR contact residue. These data strongly suggest that cross-reactive peptides may play an important role in the expansion and reactivation of CTL clones from the memory T cell pool, and may be involved in long-term maintenance of T cell memory.

High structural side chain specificity required at the second position of immunogenic peptides to obtain stable MHC/peptide complexes.

Peptides binding to HLA-A11 contain a hydrophobic or a small polar amino acid at position 2 and a lysine at the carboxy terminus. Synthetic peptides carrying natural and unnatural amino acids in position 2 were used to determine the requirements for formation of stable HLA-A11/peptide complexes. By kinetic analysis we demonstrate that a stereospecific interaction between the side chain residue in position 2 and a subsite of pocket B is required to obtain stable HLA/peptide complexes. This specific interaction is mediated by a methyl group or by an ethyl group bound to the asymmetric Cbeta atom with the correct configuration. Experiments performed with different peptide sequences suggest that the presence of adequate anchor residues may be sufficient to produce stable HLA/peptide complexes.

Epitope-dependent selection of highly restricted or diverse T cell receptor repertoires in response to persistent infection by Epstein-Barr virus.

The T cell receptor (TCR) repertoires of cytotoxic responses to the immunodominant and subdominant HLA A11-restricted epitopes in the Epstein-Barr virus (EBV) nuclear antigen-4 were investigated in four healthy virus carriers. The response to the subdominant epitope (EBNA4 399-408, designated AVF) was highly restricted with conserved Vbeta usage and identical length and amino acid motifs in the third complementarity-determining regions (CDR3), while a broad repertoire using different combinations of TCR-alpha/beta V and J segments and CDR3 regions was selected by the immunodominant epitope (EBNA4 416-424, designated IVT). Distinct patterns of interaction with the A11-peptide complex were revealed for each AVF- or IVT-specific TCR clonotype by alanine scanning mutagenesis analysis. Blocking of cytotoxic function by antibodies specific for the CD8 coreceptor indicated that, while AVF-specific TCRs are of high affinity, the oligoclonal response to the IVT epitope includes both low- and high-affinity TCRs. Thus, comparison of the memory response to two epitopes derived from the same viral antigen and presented through the same MHC class I allele suggests that immunodominance may correlate with the capacity to maintain a broad TCR repertoire.

HLA-A11-mediated protection from NK cell-mediated lysis: role of HLA-A11-presented peptides.

The capacity of MHC class I to protect target cells from NK is well established, but the mechanism by which these molecules influence NK recognition and the physical properties associated with this function remain poorly defined. We have examined this issue using as a model the HLA-A11 allele. HLA-A11 expression correlated with reduced susceptibility to NK and interferon-activated cytotoxicity in transfected sublines of the A11-defective Burkitt's lymphoma WW2-BL and the HLA class I A,B-null C1R cell line. Protection was also achieved by transfection of HLA-A11 in the peptide processing mutant T2 cells line (T2/A11), despite a very low expression of the transfected product at the cell surface. Induction of surface HLA-A11 by culture of T2/A11 cells at 26 degrees C or in the presence of beta 2m did not affect lysis, whereas NK sensitivity was restored by culture in the presence of HLA-All-binding synthetic peptides derived from viral or cellular proteins. Acid treatment rendered T2/A11 and C1R/A11 cells sensitive to lysis, but protection was restored after preincubation with peptide preparations derived from surface stripping of T2/A11 cells. Similar peptide preparations from T2 cells had no effect. The results suggest that NK protection is mediated by HLA-A11 molecules carrying a particular set of peptides that are translocated to the site of MHC class I assembly in the ER in a TAP-independent fashion.

Polymorphonuclear neutrophils pulsed with synthetic peptides efficiently activate memory cytotoxic T lymphocytes.

Polymorphonuclear neutrophils (PMNs), traditionally considered effector cells in the inflammatory response, have recently been regarded as potential regulators of the immune response. In the present study we investigate whether PMNs are efficient antigen-presenting cells for reactivation of memory cytotoxic T lymphocytes (CTLs). PMNs were pulsed with synthetic peptides derived from Epstein-Barr virus (EBV) antigens. We have used the IVTDFSVIK (IVT) peptide derived from the Epstein-Barr virus-encoded nuclear antigen 4 protein, corresponding to the immunodominant epitope of HLA-AII-restricted CTL responses, and the CLGGLLTMV (CLG) peptide derived from the latent membrane protein 2 antigen, representing a subdominant epitope of HLA-A2-restricted CTL responses. The data indicate that peptide-pulsed PMNs selectively activate specific CTL responses to both immunodominant and subdominant epitopes. The efficiency of CTL induction by PMNs was comparable to that observed with the conventional method of EBV-specific CTL reactivation with the autologous lymphoblastoid cell line, as well as with peptide-pulsed monocyte-enriched adherent cells. On the contrary, unactivated peptide-pulsed lymphocytes failed to induce an epitope-specific CTL response. These results demonstrate that PMNs efficiently present antigens to memory virus-specific CTLs and suggest that they may have a role as antigen-presenting cells.

Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides.

Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein-Barr virus (EBV)-specific memory CTL responses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation with the autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivites directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416-424 of EBV nuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC: peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific response upon stimulation with the autologous B95.8 virus-transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.