MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial.

BACKGROUND: Immunotherapy offers a promising novel approach for the treatment of cancer and both adoptive T-cell transfer and immune modulation lead to regression of advanced melanoma. However, the potential synergy between these two strategies remains unclear. METHODS: We investigated in 12 patients with advanced stage IV melanoma the effect of multiple MART-1 analog peptide vaccinations with (n = 6) or without (n = 6) IMP321 (LAG-3Ig fusion protein) as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs at day (D) 0 (Trial registration No: NCT00324623). All patients were selected on the basis of ex vivo detectable MART-1-specific CD8 T-cell responses and immunized at D0, 8, 15, 22, 28, 52, and 74 post-reinfusion. RESULTS: After immunization, a significant expansion of MART-1-specific CD8 T cells was measured in 83% (n = 5/6) and 17% (n = 1/6) of patients from the IMP321 and control groups, respectively (P < 0.02). Compared to the control group, the mean fold increase of MART-1-specific CD8 T cells in the IMP321 group was respectively >2-, >4- and >6-fold higher at D15, D30 and D60 (P < 0.02). Long-lasting MART-1-specific CD8 T-cell responses were significantly associated with IMP321 (P < 0.02). At the peak of the response, MART-1-specific CD8 T cells contained higher proportions of effector (CCR7⁻ CD45RA⁺/⁻) cells in the IMP321 group (P < 0.02) and showed no sign of exhaustion (i.e. were mostly PD1⁻CD160⁻TIM3⁻LAG3⁻2B4⁺/⁻). Moreover, IMP321 was associated with a significantly reduced expansion of regulatory T cells (P < 0.04); consistently, we observed a negative correlation between the relative expansion of MART-1-specific CD8 T cells and of regulatory T cells. Finally, although there were no confirmed responses as per RECIST criteria, a transient, 30-day partial response was observed in a patient from the IMP321 group. CONCLUSIONS: Vaccination with IMP321 as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs induced more robust and durable cellular antitumor immune responses, supporting further development of IMP321 as an adjuvant for future immunotherapeutic strategies.

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania.

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.

Effect of metoclopramide on angiotensins, aldosterone, and atrial peptide during hypoxia.

The coupling of aldosterone with renin is altered during acute hypoxemia. We measured the various components of the renin-angiotensin system and the plasma levels of immunoreactive atrial natriuretic factor (iANF) during room air and hypoxic gas-mixture breathing before and after administration of metoclopramide, a competitive antagonist of dopamine. Seven resting volunteers were studied 1 wk apart under room air and hypoxic conditions (inspired O2 fraction 0.12). During hypoxemia, the release of aldosterone induced by metoclopramide was significantly smaller. This change was associated with a slight increase in iANF and with a decrease in plasma angiotensin II levels, without any change in immunoreactive blood angiotensin I concentrations. Plasma electrolytes and blood acid-base status did not show relevant changes, nor did blood pressure and heart rate. We conclude that the decreased aldosterone concentrations seen under hypoxemia are related to decreased angiotensin II levels. Other influences, such as elevated ANF, may also mediate this effect.

Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation.

In neurons, soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1-24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Here we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a-Syx1a complex.

Studies on bioactive components from Chinese medicinal plants

Several novel bioactive components isolated from Chinese medicinal plants will be presented. These include novel maytansinoid tumor, inhibitors, some new ent-kaurane and rosane diterpenoids from Mallotus anomalus Meer et Chun (Euphorbiaceae), as well asnovel insecticide, stemona alkaloids from Stemona parviflora C. H. Wright (Stemonaceae). Both are native plants of Hainan island, Chine. 2D NMR techniques such as mono and hetero-COSY, NOESY, COLOC as well as H-NMR line broadening effect were utilized for structure elucidation. The separation techniques, struture elucidations and bioassay results will be reported.

Some progress on the chemistry of natural bioactive terpenoids form Chinese medicinal plants

(1) Pseudolaric acids - Novel diterpenes, Pseudolaric acid A, B, C and D were isolated from Pseudolarix kaempferi Gorden (pinaceae). Their structures were assigned by spectroscopic data and chemical correlations. In the contineous studies, the absolute configurations, the conformations in the solutions, the framentation mechanisms of MS and assigments of all NMR spectral signals were also reported. They showed the antifungal and cytotoxic activities. (2) Daphnane diterpenes - In the further studies on the plants of Thymelaeaceae, besides 10 known diterpenes, 16 new daphnane diterpenes were isolated from Daphne genkwa, D. tangutica, D. giraldii, Wikstroemie chamaedaphne. They showed the antifertilities activities. (3) Tripterygium diterpenes 14 new diterpenes were isolated from Triperygium wilfordii, T. regeli and T. hypoglaucum. Some of them showed the antitumor activities. The CD spectra showed that A/B ring of all compoundshave trans configuration as same as tripdiolide and triptolide determined by X-ray diffraction (4) Pregnane glycosides from Marsdenia koi - Two new pregnane glycosides marsdenikoiside A and marsdenikoiside B which can terminate the early pregnancy were isolated from Marsdeia koi. Their structures were elucidated by hydrolysis and spectroscopic methods.

Bioactive glycosides from Chinese medicines

Glycosides are the bioactive components of many famous Chinese medicines. Here reported are some bioactive glycosides we discovered from Chinese medicines in recent years. (1) Pheolic glycosides from Chinese medicines: Gastrodia elata, acontium austroynanense and Helicia erratica, three bioactive phenolic glycosides were discovered and two of them have been developed into new drugs. (2) Terpenoidal glycosides: a) Monoterpenoid: the sweroside from Swertia mollensis has been developed intro an anti-hepatitis drug; b) Diterpenoid: Phlomis betonicoides contains sweet glycoides; c) Triterpenoid: many biologically active triterpenoid glycosides were isolated from Panax plants and Siraitia grosvenorii. (3) Steroidal glycosides: a) C21-steroid: Cynanchum otophyllum and C. atratrum contain anti-epilepsy and-tumor glycosides; b) C27-steroid Hemostatic saponins were found in Paris polyphylla.

Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein.

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.

New inhibitors of Helicobacter pylori urease holoenzyme selected from phage-displayed peptide libraries.

Urease is an important virulence factor for Helicobacter pylori and is critical for bacterial colonization of the human gastric mucosa. Specific inhibition of urease activity has been proposed as a possible strategy to fight this bacteria which infects billions of individual throughout the world and can lead to severe pathological conditions in a limited number of cases. We have selected peptides which specifically bind and inhibit H. pylori urease from libraries of random peptides displayed on filamentous phage in the context of pIII coat protein. Screening of a highly diverse 25-mer combinatorial library and two newly constructed random 6-mer peptide libraries on solid phase H. pylori urease holoenzyme allowed the identification of two peptides, 24-mer TFLPQPRCSALLRYLSEDGVIVPS and 6-mer YDFYWW that can bind and inhibit the activity of urease purified from H. pylori. These two peptides were chemically synthesized and their inhibition constants (Ki) were found to be 47 microM for the 24-mer and 30 microM for the 6-mer peptide. Both peptides specifically inhibited the activity of H. pylori urease but not that of Bacillus pasteurii.

Heterologous desensitization of the glucagon-like peptide-1 receptor by phorbol esters requires phosphorylation of the cytoplasmic tail at four different sites.

Glucagon-like peptide-1 stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor that activates the adenylyl cyclase pathway. We previously demonstrated that heterologous desensitization of the receptor by protein kinase C correlated with phosphorylation in a 33-amino acid-long segment of the receptor carboxyl-terminal cytoplasmic tail. Here, we determined that the in vivo sites of phosphorylation are four serine doublets present at positions 431/432, 441/442, 444/445, and 451/452. In vitro phosphorylation of fusion proteins containing mutant receptor C-tails, however, indicated that whereas serines at position 431/432 were good substrates for protein kinase C (PKC), serines 444/445 and 451/452 were poor substrates, and serines 441/442 were not substrates. In addition, serine 416 was phosphorylated on fusion protein but not in intact cells. This indicated that in vivo a different PKC isoform or a PKC-activated kinase may phosphorylate the receptor. The role of phosphorylation on receptor desensitization was assessed using receptor mutants expressed in COS cells or Chinese hamster lung fibroblasts. Mutation of any single serine doublet to alanines reduced the extent of phorbol 12-myristate 13-acetate-induced desensitization, whereas substitution of any combination of two serine doublets suppressed it. Our data thus show that the glucagon-like peptide-1 receptor can be phosphorylated in response to phorbol 12-myristate 13-acetate on four different sites within the cytoplasmic tail. Furthermore, phosphorylation of at least three sites was required for desensitization, although maximal desensitization was only achieved when all four sites were phosphorylated.

Direct analysis of peptide binding to cell-associated MHC class I molecules.

Exogenously added synthetic peptides can mimic endogenously produced antigenic peptides recognized on target cells by MHC class I-restricted cytolytic T lymphocytes. While it is assumed that exogenous peptides associate with class I molecules on the target cell surface, direct binding of peptides to cell-associated class I molecules has been difficult to demonstrate. Using a newly developed binding assay based on photoaffinity labeling, we have investigated the interaction of two antigenic peptides, known to be recognized in the context of H-2Kd or H-2Db, respectively, with 20 distinct class I alleles on living cells. None of the class I alleles tested, with the exception of H-2Kd or H-2Db, bound either of the peptides, thus demonstrating the exquisite specificity of peptide binding to class I molecules. Moreover, peptide binding to cell-associated H-2Kd was drastically reduced when metabolic energy, de novo protein synthesis or protein egress from the endoplasmic reticulum was inhibited. It is thus likely that exogenously added peptides do not associate with the bulk of class I molecules expressed at the cell surface, but rather bind to short-lived molecules devoid of endogenous peptides.

Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts.

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Expression of a plant-derived peptide harboring water-cleaning and antimicrobial activities.

Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment-friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components of the extract and whether they may be produced in recombinant form are unknown. Here we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Staphylococcus, Streptococcus, and Legionella species. Thus, this polypeptide displays the unprecedented feature of combining water purification and disinfectant properties. Identification of an active principle derived from the seed extracts points to a range of potential for drinking water treatment or skin and mucosal disinfection in clinical settings.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.