Tracking the progeny of adoptively transferred virus-specific T cells in patients posttransplant using TCR sequencing.

Adoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase 1/2 trial, we prophylactically administered multivirus-specific T-cell products to protect recipients of T-cell-depleted allogeneic stem cell grafts against viral reactivation. To establish treatment efficacy, it is important to determine the fate of the individual transferred T-cell populations. However, it is difficult to unequivocally distinguish progeny of the transferred T-cell products from recipient- or stem cell graft-derived T cells that survived T-cell depletion during conditioning or stem cell graft manipulation. Using messenger RNA sequencing of the T-cell receptor ß-chains of the individual virus-specific T-cell populations within these T-cell products, we were able to track the multiple clonal virus-specific subpopulations in peripheral blood and distinguish recipient- and stem cell graft-derived virus-specific T cells from the progeny of the infused T-cell products. We observed in vivo expansion of virus-specific T cells that were exclusively derived from the T-cell products with similar kinetics as the expansion of virus-specific T cells that could also be detected before the T-cell product infusion. In addition, we demonstrated persistence of virus-specific T cells derived from the T-cell products in most patients who did not show viral reactivation. This study demonstrates that virus-specific T cells from prophylactically infused multiantigen-specific T-cell products can expand in response to antigen encounter in vivo and even persist in the absence of early viral reactivation.

Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after allo-HSCT.

Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed prospectively the safety and efficacy of stem cell-donor- or third-party-donor-derived CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial. Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo major histocompatibility complex-Streptamer-isolated CMV epitope-specific donor T cells. Forty-four allo-HSCT patients receiving a T-cell-replete (D+ repl; n=28) or T-cell-depleted (D+ depl; n=16) graft from a CMV-seropositive donor were screened for CMV-specific T-cell immunity. Eight D+ depl recipients received adoptive T-cell therapy from their stem cell donor. CMV epitope-specific T cells were well supported and became detectable in all treated patients. Complete and partial virological response rates were 62.5% and 25%, respectively. Owing to longsome third-party donor (TPD) identification, only 8 of the 57 CMV patients transplanted from CMV-seronegative donors (D-) received antigen-specific T cells from partially human leukocyte antigen (HLA)-matched TPDs. In all but one, TPD-derived CMV-specific T cells remained undetectable. In summary, adoptive transfer correlated with functional virus-specific T-cell reconstitution in D+ depl patients. Suboptimal HLA match may counteract expansion of TPD-derived virus-specific T cells in D- patients.

Synthesis and biological evaluation of destruxin A and related analogs.

This report describes the development of an efficient solid-phase synthesis protocol and adaptation of reported solution phase procedures for the synthesis of the cyclic depsihexapeptide destruxin A and related analogs. The solid-phase method described is based on standard Fmoc peptide chemistry, including a new synthetic method for the assembly of the depsi bond-containing unit. In order to select analogs of destruxin A for synthesis and evaluation of insecticidal activity, the work of Hellberg et al., describing a set of Z-descriptors for amino acid side-chains comparing their physicochemical properties, was utilized. Destruxin A and 27 different analogs with structural variations in four residues were synthesized and insecticidal activity was evaluated via injections into tobacco budworm (Heliothis virescens) larvae. Several destruxin A analogs were found to be at least as potent as the native compound.

Sequential nucleophilic substitution on halogenated triazines, pyrimidines, and purines: a novel approach to cyclic peptidomimetics.

A novel concept for the synthesis of macrocyclic peptidomimetics which incorporate heteroaromatic units is reported. The method involves sequential SNAr reactions of orthogonally protected amino groups of peptides and other linear oligomers on halogenated heterocycles such as 2,4,6-trichloro[1,3,5]triazine, 2,4,6-trichloropyrimidine, 4,6-dichloro-5-nitropyrimidine, and 2,6,8-trichloro-7-methylpurine. The scope of this novel solid-phase approach was systematically evaluated by means of the SPOT-synthesis methodology on planar cellulose membranes. Besides the question of the accessibility of different ring sizes and the compatibility with protecting groups of commonly used amino acids, the applicability of the technique toward different halogenated heteroaromatics and peptidomimetics was studied. It was found that the procedure is well suited to assemble a wide variety of cyclic peptidomimetics differing in both size (11- to 37-membered rings) and chemical nature of the assembled backbones.

Spatially addressed synthesis of amino- and amino-oxy-substituted 1, 3,5-triazine arrays on polymeric membranes.

Effective spatially addressed parallel assembly of trisamino- and amino-oxy-1,3,5-triazines was achieved by applying the SPOT-synthesis technique on cellulose and polypropylene membranes. In addition to developing a suitable linker strategy and employing amines and phenolate ions as building blocks, a highly effective microwave-assisted nucleophilic substitution procedure at membrane-bound monochlorotriazines was developed. The 1,3, 5-triazines obtained could be cleaved in parallel from the solid support by TFA vapor to give compounds adsorbed on the membrane surface in a conserved spatially addressed format for analysis and screening. The reaction conditions developed were employed for the synthesis of 8000 cellulose-bound 1,3,5-triazines which were probed in parallel for binding to the anti-transforming growth factor-alpha monoclonal antibody Tab2 in order to identify epitope mimics.

Trichostatin A-mediated regulation of gene expression and protein kinase activities: reprogramming tumor cells for ribotoxic stress-induced apoptosis.

Recently we described a new signal transduction-based tumor therapeutic strategy involving first sensitization of tumor cells by trichostatin A (TSA), an inhibitor of histone deacetylation, and thereafter efficient apoptotic triggering by ribotoxic agents, which activate stress-activated protein kinases. In the present work we investigate the molecular basis of the sensitization step in this therapeutic model system and describe TSA-induced changes in mRNA and protein expression of several candidate genes identified previously by complex hybridization. Furthermore, activities of 15 different protein kinases were followed after TSA application, using a new filter-based technique (PhosphoSpots-Assay). The obtained data suggest that TSA induces pro-apoptotic genes like ID1, ID2, ID3, and down-regulates anti-apoptotic genes like Hsp27 and Bcl-xL, thereby shifting the cellular equilibrium from life to death. Furthermore, activities of calcium/calmodulin-dependent kinase II and protein kinase C, which have been assigned pro-apoptotic function in other systems, are induced.

Vascular endothelial growth factor (VEGF) receptor II-derived peptides inhibit VEGF.

Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration via tyrosine kinase receptors of the split kinase domain family. It mediates vascular growth and angiogenesis in the embryo but also in the adult in a variety of physiological and pathological conditions. The potential binding site of VEGF with its receptor was identified using cellulose-bound overlapping peptides of the extracytosolic part of the human vascular endothelial growth factor receptor II (VEGFR II). Thus, a peptide originating from the third globular domain of the VEGFR II comprising residues 247RTELNVGIDFNWEYP261 was revealed as contiguous sequence stretch, which bound 125I-VEGF165. A systematic replacement with L-amino acids within the peptide representing the putative VEGF-binding site on VEGFR II indicates Asp255 as the hydrophilic key residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding with an IC50 of 0.5 microM on extracellular VEGFR II fragments and 30 microM on human umbilical vein cells. VEGF165-stimulated autophosphorylation of VEGFR II as well as proliferation and migration of microvascular endothelial cells was inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal concentration of 3-10, 0.1, and 0.1 microM, respectively. We conclude that transduction of the VEGF165 signal can be interrupted with a peptide derived from the third Ig-like domain of VEGFR II by blockade of VEGF165 binding to its receptor.

Identification of peptides inhibiting enzyme I of the bacterial phosphotransferase system using combinatorial cellulose-bound peptide libraries.

The phosphoenolpyruvate(P-pyruvate)-dependent sugar phosphotransferase system (PTS) is a transport and signal-transduction system which is almost ubiquitous in bacteria but does not occur in eucaryotes. It catalyzes the uptake and phosphorylation of carbohydrates and is involved in signal transduction, e.g. catabolite repression, chemotaxis, and allosteric regulation of metabolic enzymes and transporters. EI (Enzyme I of the PTS) is the first and central component of the divergent PTS (P-pyruvate-dependent sugar phosphotransferase system) phosphorylation cascade. Using immobilized combinatorial peptide libraries and phosphorimaging, heptapeptides and octapeptides were identified which selectively inhibit EI in vitro. The IC50 of the best peptides is 30 microM which is close to the K(M) (6 microM) of EI for its natural substrate HPr (histidine containing phosphoryl carrier protein of the PTS). The affinity-selected peptides are better inhibitors than a peptide with the active-site sequence of HPr. The selected peptides contain several basic residues and one aromatic residue which do not occur in the active site of HPr. The large proportion of basic residues most likely reflects charge complementarity to the strongly acidic active-site pocket of EI. Guanidino groups might facilitate by complexation of the phosphoryl group the slow phosphorylation of the peptide.

Dissociation of the signalling and antiviral properties of SDF-1-derived small peptides.

BACKGROUND: The chemokine receptor CXCR4 (a receptor for the Cys-X-Cys class of chemokines) is a CD4-associated coreceptor for T-cell-tropic strains of human immunodeficiency virus 1 (HIV-1) and represents a target for antiviral therapy. Infection by T-tropic HIV-1 can be blocked by stromal-cell-derived factor-1 (SDF-1), the natural ligand of CXCR4. The broad variety of cells expressing CXCR4 and the perturbations observed in mice deficient for SDF-1 suggest that antiviral compounds antagonizing the signalling activity of CXCR4 might have severe side effects in vivo. Compounds that interfere selectively with HIV entry and not with SDF-1 signalling would therefore be useful. RESULTS: A series of peptides, each of 13 residues, spanning the whole SDF-1alpha sequence were tested for their ability to block HIV-1 infection. The antiviral and signalling properties of SDF-1 were retained by a peptide corresponding to its amino terminus. Removal of the first two residues resulted in an antiviral antagonist of the SDF-1-CXCR4 signalling pathway. We prepared 234 single-substitution analogues and identified one antiviral analogue that had drastically reduced agonistic or antagonistic properties. The antiviral peptides competed with the monoclonal antibody 12G5 for CXCR4 binding. Their antiviral activity seems to be due to receptor occupancy rather than induction of receptor endocytosis. CONCLUSIONS: The amino terminus of the SDF-1 chemokine is sufficient for signal transduction via CXCR4 and for inhibition of HIV-1 entry, but these activities could be dissociated in a peptide analogue. This peptide represents a lead molecule for the design of low molecular weight antiviral drugs.

Substrate specificity of the DnaK chaperone determined by screening cellulose-bound peptide libraries.

Hsp70 chaperones assist protein folding by ATP-dependent association with linear peptide segments of a large variety of folding intermediates. The molecular basis for this ability to differentiate between native and non-native conformers was investigated for the DnaK homolog of Escherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues. In the folded proteins these sites are mostly buried and in the majority found in beta-sheet elements. The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues. Acidic residues are excluded from the core and disfavored in flanking regions. The energetic contribution of all 20 amino acids for DnaK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.

Molecular cloning, DNA sequence and transcriptional analysis of the Rhodospirillum molischianum B800/850 light-harvesting genes.

The amino acid sequences of the B800/850 light-harvesting proteins from Rhodospirillum molischianum were determined by Edman degradation. On the basis of these amino acid sequences, two degenerated oligonucleotides were synthesized and used for PCR of genomic DNA. The resulting 150 bp DNA fragment was cloned, sequenced and used for subsequent Southern blot analysis of digested genomic DNA. A 2.3 kbp EcoRI fragment strongly hybridized to the probe and a size selected genomic library from genomic DNA was constructed. One clone scored positive during screening of the library with the PCR-fragment and subsequent DNA sequence analysis of the clone revealed the presence of three A-genes (A1A2A3) encoding alpha-polypeptides and of two B-genes (B1B2) encoding beta-polypeptides of the B800/850 complex. The arrangement of the different genes are B1A1, B2A2 and A3 where only B1 and B2 are preceded by typical Shine-Dalgarno sequences. In addition, typical nucleotide sequences for a rho-independent termination of transcription are located downstream of the genes A1 and A2. The deduced amino acid sequences revealed that the alpha-genes encoded for identical polypeptides, whereas the deduced beta-polypeptides differed in their amino acid sequence at four positions. Transcriptional operon analysis revealed that the genes A1B1 and A2B2 are both dicistronically transcribed, whereas the gene A3 is not.

Spectroscopic properties of the light-harvesting complexes from Rhodospirillum molischianum.

Spectroscopic properties, including low-temperature absorbance, linear and circular dichroism and site-selection fluorescence of the antenna complexes from Rhodospirillum molischianum have been determined. The unique 'LH1-like' character of the amino acid sequence from LH2 of this bacterium is reflected in the circular dichroism of the B850 band of this complex. The wavelength dependence of the polarization of the LH2 complex shows an unusual shape that is attributed to the octameric state of this complex. The complete amino acid sequence for the LH1 alpha-polypeptide and most of the beta-polypeptides are presented. These conform to the general features of other LH1 polypeptides. This result, in combination with spectroscopic data for LH1 imply that the organisation of the core in this bacterium is not much different from that in other purple non-sulphur bacteria.

Unexpected similarities of the B800-850 light-harvesting complex from Rhodospirillum molischianum to the B870 light-harvesting complexes from other purple photosynthetic bacteria.

The B800-850 light-harvesting complex (also called LH2) was isolated from photosynthetic membranes of Rhodospirillum molischianum DSM 119 using molecular sieve and ion-exchange chromatography. Its two bacteriochlorophyll a-binding polypeptides (alpha-subunit and beta-subunit) were purified with a reverse-phase HPLC system. The complete amino acid sequences of both subunits have been determined. The alpha- and beta-subunits consist of 56 and 45 amino acids, respectively, corresponding to molecular weights of 5939 and 5133. In contrast to the B800-850 complexes from other photosynthetic bacteria, the native B800-850 complex from Rs. molischianum is most likely an octamer of monomers with a stoichiometry of three bacteriochlorophyll a and 1.5 lycopenes per alpha,beta-subunit. Resonance Raman spectra provide evidence for a 5-coordinated Mg2+ in the BChl, and a carotenoid mainly in the all-trans configuration. A comparison between resonance Raman data from different photosynthetic bacteria indicates that the BChl a-binding site of the B800-850 complex from Rs. molischianum is more similar to the B870 complexes (also called LH1) than to the B800-850 complexes of other photosynthetic bacteria. Sequence similarities especially between the beta-subunits of the B800-850 complex of Rs. molischianum and the B870 and B800-850 complexes of other photosynthetic bacteria agree with this result and provide information on the mode of pigment binding in bacterial antenna complexes.