Clinical outcome of patients with various advanced cancer types vaccinated with an optimized cryptic human telomerase reverse transcriptase (TERT) peptide: results of an expanded phase II study.

BACKGROUND: TERT (telomerase reverse transcriptase) plays a critical role in tumor cell growth and survival. In an expanded phase II study, we evaluated the immunological and clinical responses to the TERT-targeting Vx-001 vaccine in patients with advanced solid tumors. METHODS: HLA-A*0201-positive patients received two subcutaneous injections of the optimized TERT(572Y) peptide followed by four injections of the native TERT(572) peptide, every 3 weeks. Peptide-specific immune responses were evaluated by enzyme-linked immunosorbent spot at baseline, and after the second and the sixth vaccinations. RESULTS: Fifty-five patients were enrolled and 34 (62%) completed the six vaccinations. A TERT-specific T-cell immune response was observed in 55% and 70% of patients after the second and the sixth vaccinations, respectively. The disease control rate (DCR) was 36% [95% confidence interval (CI) 24% to 49%], including one complete and one partial response. Immunologically responding patients had a better clinical outcome than nonresponders [DCR: 44% versus 14% (P = 0.047); progression-free survival (PFS): 5.2 versus 2.2 months (P = 0.0001) and overall survival: 20 versus 10 months (P = 0.041)]. Multivariate analysis revealed that the immunological response was an independent variable associated with increased PFS (hazard ratio = 3.35; 95% CI 1.7-6.7). CONCLUSION: Vx-001 vaccine was well tolerated and induced a TERT-specific immunological response, which was significantly correlated with improved clinical outcome.

3D solution structure of [Tyr3]octreotate derivatives in DMSO: structure differentiation of peptide core due to chelate group attachment and biologically active conformation.

The solution models of [Tyr3]octreotate (DPhe1-Cys2-Tyr3-DTrp4-Lys5-Thr6-Cys7-Thr8-COOH, disulfide bridged) (I), its analogs functionalized with an open chain tetraamine chelator, N4-[Tyr3]octreotate (II), and the N4-(Asp)2-[Tyr3]octreotate (III) peptide have been determined through 2D 1H NMR spectroscopy in DMSO. Chemical shift analysis has been performed in an attempt to elucidate structural changes occurring during attachment of the tetraamine to the peptide backbone. NMR-derived geometrical constraints have been used in order to calculate high resolution conformers of the above peptides. Conformational analysis of the three synthetic analogues, have shown that these somatostatin analoges adopt a predominant antiparallel beta-sheet conformation characterized by a beta-like turn spanning residues DTrp4 and Lys5 which is supported in the case of N4-(Asp)2-[Tyr3]octreotate and N4-[Tyr3]octreotate by medium range NOEs. These data indicate that the above-mentioned molecules adopt a rather constrained structure in the 4-residue loop Tyr3-Thr6. Additionally, the C-terminal of [Tyr3]octreotate, comprising Cys7 and Thr8, appears to form a turn-like structure manifested by characteristic side-chain NOEs between Lys5 and Thr8, which have not been detected for the other two compounds. These data are discussed in the light of previous structural data of Sandostatin (octreotide) and suggest that attachment of the N4-chelator and two Asp residues at the N-end of [Tyr3]octreotate impose considerable structural changes and affect the binding properties of these peptides. Indeed, the IC50 values determined during competition binding assays against the sst2 (somatostatin subtype 2 receptor) suggest that the presence of the N4 group enhances receptor affinity, while extension of peptide chain by two negatively-charged Asp residues impairs receptor affinity at approximately one order of magnitude.

Synthetic peptides as putative therapeutic agents in transplantation medicine: application of PEPSCAN to the identification of functional sequences in the extracellular domain of the interleukin-2 receptor beta chain (IL-2Rbeta).

A desired treatment strategy in transplantation medicine is the selective targeting of alloreactive T cells without impairing antileukemic and antiviral activities. One approach is the synthesis of peptides that interfere with the binding of interleukin-2 (IL-2) to its high affinity receptor (IL-2R). This blocks the activation and proliferation of the antigen-activated T cells and the secretion of IL-2. The latter binds to its receptor, via the extracellular domain of the IL-2Rbeta chain, while its cytoplasmic domain is required for intracellular signal transduction. In this study, the PEPSCAN method was applied in order to identify antigenic sequences (epitopes) in the extracellular domain of the IL-2Rbeta. Based on the primary amino acid (aa) sequence of the IL-2Rbeta, a total of 239 overlapping dodecapeptides, spanning the entire sequence of IL-2Rbeta, were synthesized by PEPSCAN and their immunoreactivity was tested by ELISA using monoclonal antibodies (mAbs) specific for IL-2Rbeta such as TU11, Mikbeta1, HuMikbeta1 and TU27. TU11 recognized a linear epitope located in the region 85R-Q(96). None of the 239 synthetic peptides was recognized by TU27. Mikbeta1 (and HuMikbeta1) recognized a discontinuous epitope formed by aa located in the IL-2Rbeta domains L(106) to P(148) and E(170) to A(202). Subsequently, synthetic peptides corresponding to the identified putative epitopic sequences were prepared by solid phase synthesis and their immunogenicity in vivo was assessed by raising polyclonal antibodies. Given that Mikbeta1 and HuMikbeta1 inhibit binding of IL-2 on the IL-2Rbeta, we addressed the question of whether the identified antigenic sequences serve as putative IL-2 binding domains. Synthetic peptides corresponding to these sequences were tested for their ability to compete with IL-2 for binding and, thereby, inhibit IL-2-induced proliferation of mitogen-stimulated human peripheral blood T cells. Sequences 107M-E(118) and 178Y-Q(199) probably represent functional IL-2 binding domains on IL-2Rbeta, since these synthetic peptides significantly inhibited the proliferation of activated T cells and secretion of IL-2.

Studies on position 1 of angiotensin II: effects on affinity and duration of action from alkyl amide substitution.

The synthesis and the biological activities of [asparagine]angiotensin II analogues with alkyl-substituted amide groups are reported. This study was performed in order to elucidate further the importance and the influence of the side chain in position 1 of angiotensin II. The two synthesized analogues [1-(N4,N4-dipropyl)asparagine]- and [1-(N4,N4-diisopropyl)asparagine]angiotensin II were compared to [1-asparagine]angiotensin II (hypertensin, Ciba) and to [1-(N4,N4-dimethyl)asparagine]angiotensin II in vitro and in vivo. All compounds had full intrinsic activity, but their potency decreased with increasing alkyl size of the substituted carboxamide group. Despite their reduced potency, the alkylated analogues showed enhanced duration of action on rabbit aorta strips. The relative potencies of the series hypertensin, dimethyl, dipropyl, and diisopropyl analogues on rabbit aorta strips were 100, 46, 16, and 9%, respectively. In the rat blood pressure assay they were 100, 30, 9, and 7%, respectively.

Active-site studies of neurohypophyseal hormones: synthesis and pharmacological properties of [5-(N4,N4-dimethylasparagine)]oxytocin.

Synthesis and biological properties of [5-(N4,N4-dimethylasparagine)]oxytocin are reported. In this analogue, the hydrogens of the primary carboxamide moiety in the side chain of the asparagine residue in position 5 of the posterior pituitary hormone oxytocin have been replaced by two methyl groups. The protected nonapeptide intermediate was prepared by a stepwise procedure using solution techniques. The analogue possesses 4.60 +/- 0.03 units/mg (mean +/- SEM) uterotonic activity on the isolated rat uterine horn and 9.14 +/- 0.03 units/mg of avian vasodepressor activity. Moreover, it displays an identical intrinsic activity in the in vitro rat uterotonic assay as oxytocin, when tested in the presence of either 0.5 mM Ca2+ (standard assay conditions) or at reduced levels of Ca2+ (0.3, 0.15, and 0.05 mM). This result is significant in view of the proposed biologically active model of oxytocin, in which the side chain of the 5 position residue was assigned to contain an "active element" responsible for the intrinsic activity of the hormone when bound to the uterine receptor.

Oxytocin and lysine-vasopressin with N5,N5-dialkylglutamine in the 4 position: effect of introducing sterically hindered groups into the hydrophilic cluster of neurohypophyseal hormones.

The synthesis and pharmacological potencies of oxytocin and lysine-vasopressin analogues are reported in which the N5-amide of their glutaminyl residues are dialkylated. These analogues have been studied as an ongoing exploration of the biological effects on the natural hormones of substituting individually one of the amino acid residues, which has a hydrophilic side chain and which are thought to be part of the hydrophilic surface of the hormones. [4-(N5,N5-Dimethylglutamine)]oxytocin (17), [4-(N5,N5-di-n-propylglutamine)]oxytocin (18), and [4-(N5,N5-dimethylglutamine)] lysine-vasopressin (19) were synthesized by clasical solution techniques. Potencies in the in vitro rat uterotonic, avian vasodepressor, rat pressor, and rat antidiuretic assays were determined and are as follows, respectively: for compound 17 3.01 +/- 0.14 units/mg, 4.55 +/- 0.03 units/mg, tachyphylaxis and tachyphylaxis; for compound 18 less than 0.1, less than 0.1, less than 0.05, and less thad 1.88 +/- 0.04 units/mg. The potencies in all cases are significantly less than those of the parent hormone. The results are discussed in terms of the proposed biologically active conformations of the hormones at the uterotonic receptor and at the antidiuretic receptor.

Effect of changing the COOH-terminal amide group present in the hydrophilic cluster of oxytocin to dimethylamide.

Oxytocinoic acid dimethylamide was synthesized by stepwise solution techniques as part of an ongoing evaluation of the effects on the biological activity of oxytocin caused by individually changing the groups that comprises the hydrophilic surface of the hormone to more hydrophobic and more bulky groups. The analogue exhibited approximately 3% of the potency of oxytocin in the in vitro uterotonic assay. In addition, it possesses potencies of less than 0.07, less than 0.01, and 0.096 unit/mg in the avian vasodepressor, rat pressor, and rat antidiuretic assays, respectively. In the in vitro uterotonic assay, oxytocinoic acid dimethylamide showed a reduced affinity for the oxytocin receptor, a nonparallel dose-response relationship, and most importantly a reduced intrinsic activity as compared to oxytocin. The results suggest that the replacement of the protons of the primary carboxyamide of Gly9-NH2 of oxytocin by methyl groups displaces the active elements from the orientation for obtaining maximal intrinsic activity in the isolated rat uterus.

Importance of the N-terminal domain of the type II angiotensin antagonist sarmesin for receptor blockade.

Analogues of the competitive angiotensin antagonist [Sar1,Tyr(ME)4]angiotensin II (sarmesin) with modifications at the N-terminus have been prepared by the solid-phase method and purified by reversed-phase HPLC. Substitution of the Sar1 residue of sarmesin with N,N-dimethyl-Gly, N-ethyl-Gly, aminoisobutyric, (methylamino)isobutyric, aminocaproic, and oxamic acids gave analogues that had the following respective antagonist activities (pA2) in the rat isolated uterus assay: less than 6, 6.9, 5.5, 6.0, less than 6, and 5.3. The additional substitution of Ile for Phe at the C-terminus of the latter four peptides gave pA2 values of 7.1, 5.1, less than 5, and 5. Substitution of the Arg2 residue of sarmesin with Nle or Sar abolished antagonist activity. These data emphasize the stringent and discriminating structural requirements in the N-terminal domain of sarmesin that endow this analogue with its antagonist properties and suggest the presence of defined steric constraints in this region of the molecule during receptor blockade.

Conformationally restricted C-terminal peptides of substance P. Synthesis, mass spectral analysis and pharmacological properties.

Four cyclic analogues of the C-terminal hepta- or hexapeptide of substance P were prepared by the solution method. The cyclizations were obtained by substituting with cysteine the residues normally present in positions 5 or 6 or 11 of substance P and by subsequent disulfide bond formation. The final products were identified by ordinary analytical procedures and advanced mass spectroscopy. The biological activities were determined on three bioassays: the guinea pig ileum, the guinea pig trachea and the rabbit mesenteric vein. Results obtained with these assays indicate that all peptides with a disulfide bridgehead in position 11 are inactive and that a cycle between positions 5 and 6 already strongly reduces the biological activity. The acyclic precursors containing thiol protection groups display weak biological activities. These results further underline the importance of the side chain in position 11 of substance P and suggest that optimal biological activities may require a linear peptide sequence.

Changes in residues 1 and 4 of angiotensin II affect differently its agonist and tachyphylactic properties.

Two series of angiotensin II analogues with modifications at positions 1 or 4 of the peptide chain were studied with respect to their tachyphylactic properties and to the kinetics of relaxation of the guinea-pig ileum after a contractile response to maximally effective concentrations. Tachyphylaxis was measured by the decrease in response amplitude after three successive treatments ("tachyphylactic index") and the relaxation rate was evaluated by the time taken for the tonus to reach half of its value at the moment of agonist washout ("half relaxation time"). A correlation between tachyphylactic index and half relaxation time was found for the series of position 1 analogues, but not for the position 4 analogues. For the two series, the half relaxation times of the tachyphylactic analogues decreased from the first to the third of a series of successive treatments. Bulky substituents at position 1, which did not greatly affect the agonist activity, suppressed the tachyphylactic property. The results provide evidence that the agonist and tachyphylactic properties of angiotensin II are due to its interaction, respectively, with an agonist site and a "tachyphylaxis" site on the receptor and that the structural requirements for binding to the two sites are different.

Ethanol effects on striatal dopamine receptor-coupled adenylate cyclase and on striatal opiate receptors.

The perturbation of neuronal cell membranes by ethanol may result in specific functional changes through modification of the activity of various membrane-bound proteins. In mouse striatum, adenylate cyclase, a membrane-bound enzyme, is coupled to dopamine, as well as to opiate, receptors. Ethanol stimulates striatal adenylate cyclase activity by modifying the regulatory protein ("G-protein")-adenylate cyclase interaction to produce an increased amount of activated enzyme. This action is additive with the effects of dopamine on adenylate cyclase. Ethanol also modifies striatal opiate receptor-effector coupling processes. In the presence of ethanol, opiate receptor affinity is altered, and this alteration is modified by GTP, suggesting that ethanol influences the interaction of the opiate receptor complex with the G-protein. Our results suggest that ethanol can affect receptor-effector coupling, including the binding of opiate agonists to their receptors, through its membrane-disordering capacity, and that particular systems may react in a relatively specific manner with ethanol.

Carotid atherosclerosis in type 2 diabetes mellitus: potential role of endothelin-1, lipoperoxides, and prostacyclin.

Factors were studied that may initiate macroangiopathy or enhance or aggravate its pathogenesis in patients with type 2 diabetes mellitus. A total of 151 diabetics were compared with healthy controls (n=50); all patients and subjects were normotensive and without renal failure. Plasma endothelin-1 and free radical levels were measured. In addition, plasma prostacyclin levels were assessed by assaying its stable, spontaneous, breakdown product 6-keto-prostaglandin-F1a. Diabetics were divided into three groups: those with clinically evident macroangiopathy and those with early or without atherosclerosis (as determined by the carotid intima-media thickness. Plasma endothelin-1 levels were increased in all diabetics with atherosclerosis. Plasma free radical levels were increased in diabetics with macroangiopathy when compared with control subjects. The plasma levels of 6-keto-prostaglandin-F1a were slightly, but significantly, decreased in the diabetics with macroangiopathy when compared with control subjects. The carotid intima-media thickness was significantly greater in diabetics without macroangiopathy when compared with the controls. Furthermore, the intima-media thickness increased significantly in this group of diabetics but not in the controls over a 30-month follow-up period. Several factors may contribute to atherogenesis in diabetics. These include increased plasma endothelin-1 and free radical levels as well as a deficiency of prostacyclin. These factors may become targets for intervention as well as markers of disease progression.

Sulphonamide-based bombesin prodrug analogues for glutathione transferase, useful in targeted cancer chemotherapy.

Glutathione transferases (GSTs) are enzymes involved in cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of toxic compounds and xenobiotics, including certain chemotherapeutic drugs. The encountered chemotherapeutic resistant of tumour cells, thus, has been associated with the increase of total GST expression. GSTs, in addition to GSH-conjugating activity, exhibit sulphonamidase activity, catalyzing the GSH-mediated hydrolysis of sulphonamide bonds. Such reactions are of interest as potential tumour-directed prodrug activation strategies. In the present work we report the design and synthesis of novel chimaeric sulphonamide derivatives of bombesin, able to be activated by the model human isoenzyme GSTA1-1 (hGSTA1-1). These derivatives bear a peptidyl-moiety (analogues of bombesin peptide: R-[Lue(13)]-bombesin, R-[Phe(13)]-bombesin and R-[Ser(3),Arg(10),Phe(13)]-bombesin, where R=C(6)H(5)SO(2)NH-) as molecular recognition element for targeting the drug selectively to tumour cells. The released S-alkyl-glutathione, after hGSTA1-1-mediated cleavage of the sulphonamide bond, provides an inhibitor of varied strength against GSTs from different sources. These prodrugs are envisaged as a plausible means to sensitize drug-resistant tumours that overexpress GSTs.

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide.

A novel liposomal formulation was developed for the encapsulation of the oligopeptide leuprolide (GlpHisTrpSerTyr-D-LeuLeuArgProNHEt), a potent analogue of gonadotropin releasing hormone used in the treatment of advanced prostate cancer, endometriosis and precocious puberty. Leuprolide was synthesized using solid phase methodology on a {3-[(ethyl-Fmoc-amino)-methyl]-1-indol-1-yl}-acetyl AM resin and Fmoc/tBu chemistry. The new liposomal formulation, called 'liposomes in liposomes' is composed of egg phosphatidylcholine:dipalmitoylphosphatidylglycerol in a molar ratio of 98.91:1.09 (internal liposomes) and egg phosphatidylcholine:dipalmitoylphosphatidylglycerol:cholesterol in a molar ratio of 68.71:0.76:30.53 (external liposomes). It offers high encapsulation efficiency (73.8% for leuprolide); it can provide new delivery characteristics and it may have possible advantages in future applications regarding the encapsulation and delivery of bioactive peptides to target tissues. Furthermore, the physicochemical characteristics (size distribution and zeta-potential) of the liposomal formulations and the thermal effects on leuprolide in model lipidic bilayers composed of dipalmitoylphosphatidylcholine were studied using differential scanning calorimetry. Finally, the dynamic effects of leuprolide in an egg phosphatidylcholine/cholesterol system were examined using solid state 13C MAS NMR spectroscopy.

A phase I study of the optimized cryptic peptide TERT(572y) in patients with advanced malignancies.

OBJECTIVE: It was the aim of this study to evaluate the safety of the optimized cryptic peptide TERT(572Y) in pretreated patients with advanced cancer. METHODS: Nineteen patients with progressive and chemotherapy-refractory tumors received escalated doses (2-6 mg) of 2 subcutaneous injections of the optimized TERT(572Y) peptide followed by 4 subcutaneous injections of the native TERT(572) peptide every 3 weeks. Both TERT peptides were coinjected with adjuvant Montanide ISA51. Toxicity was evaluated every 3 weeks and peptide-specific CD8+ cells were detected by flow cytometry using TERT(572Y) tetramers. RESULTS: Fourteen out of 19 patients completed the vaccination program. No grade III/IV toxicity was observed. Grade I anemia was observed in 4 patients and local skin reaction at the injection site in 11 patients. Other nonhematologic toxicities were mild, and no late toxicity was observed after a median postvaccination follow-up period of 10.7 months. There was no dose-limiting toxicity. Peripheral blood TERT(572Y)-specific CD8+ lymphocytes were detected in 13 out of 14 evaluable patients after 2 injections with the optimized TERT(572Y) peptide. There was no complete or partial response, but 4 patients (21%) with persistent TERT(572Y)-specific CD8+ experienced stable disease for a median of 10.5 months. CONCLUSION: TERT(572Y) peptide vaccine is well tolerated and effective in eliciting specific TERT(572Y) CD8+ lymphocytes in pretreated cancer patients, demonstrating that cryptic peptides could be used in cancer immunotherapy.

Synthesis of histidine2-angiotensin II analogues.

The synthesis of histidine2-angiotensin II analogues, namely H-Asp-alpha-(im-Bzl)His-Val-Tyr-Ile-(im-Bzl)His-Pro-Phe-OH and H-Asp-beta-(im-Bzl)His-Val-Tyr-Ile-(im-Bzl)His-Pro-Phe-OH, are described. Also a new route leading to the synthesis of alpha-benzyl-L-aspartate, using N-trityl-L-aspartate di-benzyl ester as the starting material, is reported.

Synthesis of [1-Aib]-angiotensin II, an analogue with higher potency than [1-Asn,5-Val]-angiotensin II.

[1-Aib]-angiotensin II was synthesized by Merrifield's solid-phase procedure. The analogue, tested on rabbit aorta strips, showed intrinsic activity alpha E = 1, and when tested on rat blood pressure it gave a pD2 of 8.06; a 3.2 +/- 1.3-fold higher potency than the Ciba-Hypertensin standard.

Oxytocin analogs with oxygen-containing side chains in position 3.

We have synthesized three oxytocin analogs containing an oxygen atom in the amino acid side chain in position 3 to determine the influences of increased side chain length and of hydrophilicity on the potencies and specificities of the resulting analogs. These three analogs: [3-O-methylhomoserine] oxytocin, [3-O-ethylserine] oxytocin, and [3-O-methylthreonine] oxytocin - have the following activities in U/mg: 490, 208, 265, milk ejection; 125, 129, 63, uterus in vivo; 0.2, 16, 0.03, antidiuretic; and 0.1, 0.5, 0.1, pressor. The results show that a longer side chain, [3-O-methylhomoserine] and [3-O-ethylserine] vs. [3-O-methylthreonine], tends to increase all activities. Moving the hydrophilic oxygen farther away from the peptide backbone, on the other hand, decreases vasopressin-like activities but increases or has no effect on oxytocin-like activities.

Synthesis and pharmacological properties of [1-N4-dimethyl-asparagine]-angiotensin II.

[1-N4-Dimethyl-asparagine]-angiotensin II was synthesized by Merrifield's solid-phase procedure. The analogue gave rat blood pressure about 70% relative potency to Hypertension (Ciba). Rabbit aorta strips gave intrinsic activity alpha E = 1, a PD2 of 6.92 +/- 0.09 and an affinity relative to [Asn1]-angiotensin II of 6.5%.

Plasma levels of endothelin and early carotid atherosclerosis in diabetic patients.

An increased thickness of the carotid artery wall is thought to be a sign of early atherosclerosis. Since plasma endothelin concentrations were released from vascular endothelial cells, we have investigated the possible relationship between endothelin 1 (ET-1) and arterial wall thickness. Ninety-eight patients with Type 2 diabetes without evidence of macroangiopathy, hypertension, proteinuria or proliferative retinopathy, and 50 non-diabetic subjects were studied. After an overnight fast, blood was taken for ET-1, glucose, HbA1c, lipids, insulin and C-peptide. Arterial wall thickness was measured as the mean of the maximum intimal-medial thickness (IMT) in 16 carotid segments by B-mode ultrasound. ET-1 levels were significantly elevated in diabetic patients with IMT>1100 microm, 8.3 pmol/l (5.2-12.9) compared with control subjects, 7.6 pmol/l (5.0-11.0), p<0.01 and with diabetic subjects with IMT<500 microm, 7.43 pmol/l (4.8-11.1), p<0.01. The diabetic (IMT>1100 microm) study group had also significantly higher levels of insulin, 102.8 +/- 46.4 pmol/l vs control subjects, 77.5 +/- 32.4 pmol/l, p<0.01. In diabetic subjects, no correlation was found between ET-1 and IMT with glucose, HbA1c, lipids, age or duration of diabetes, respectively. We conclude that ET-1 levels are elevated in Type 2 diabetic patients with increased IMT. Thus providing further support for the role of endothelin in atherosclerosis.