Cytotoxic T lymphocytes (CTL) are major effector cells in the adaptive immune response against intracellular pathogens and cancers, killing targets with high precision. Precision is achieved through the specificity of the clonally expressed T cell receptor (TCR). TCRs recognise a specific peptide chain loaded into a major-histocompatability complex, triggering signalling, inducing the CTL to attach and kill target cells. Key stages in this attack are the initial conjugation followed by polarisation and docking of the centrosome to the junction of the two cells, the immune synapse (IS). This focuses secretion of the cytolytic components, perforin and granzyme, from modified lysosomes to kill the target cell. My PhD has utilised amino acid substitutions in the target peptide to alter its signal strength and shown this alters the subsequent killing efficiency of a target population. I developed new imaging and analysis techniques to investigate the effect of TCR signal strength at each step of the killing process. I show the first step, conjugation, is reduced for a percentage of cells with dwell times decreasing as TCR signal strength decreased. The next key step of centrosome polarisation and docking at the IS was also impaired for an increasing proportion of cells as TCR signalling reduced. Impaired centrosome docking reduced efficient granule recruitment to the IS, necessary for target killing. Centrosome docking was linked with the TCR-induced intracellular calcium flux, the duration of which increases with the strength of TCR signalling. This demonstrates how the process of CTL killing can be fine-tuned by the quality of antigen.

A peptide nucleic acid (PNA) encoded 1296 member peptide library was synthesised and incubated with a variety of cell types. Library members entering cells were extracted, hybridised onto DNA microarrays and the peptide identity was determined via deconvolution. Global consensus analysis highlighted the tetrapepide, Glu-Llp- Glu-Glu (Llp is 6-hexamine-N-aminoacetic acid), a surprise in view of the basic residues typically observed in cell penetrating peptides. When evaluated, Glu-Llp- Glu-Glu revealed cellular uptake comparable to a known basic peptide (tetraLlp). In depth delineation via clustering analysis allowed assessment of differential cellular uptake, with the identified peptides showing clear cellular specificity. This was verified by peptide synthesis and cellular uptake analysis by flow-cytometry, and in all cases an endocytic uptake mechanism was confirmed. This approach establishes a strategy for the identification of short peptides as tools for selective delivery into specific cell types. The incubation of a 10,000 member PNA-encoded peptide library with D54 and HEK293T transfected with CCR6 cells followed by microarray analysis allowed detailed information on the interaction between peptide-ligands and cell surface receptors to be extracted. This allowed the identification of new ligands for integrins and G-protein coupled receptors and offers a novel approach to ligand discovery allowing the comparative analysis of different cell types for the identification of differences in surface-receptor ligands and/or receptor expression between various cell types. In addition, this work included the development of a novel method for the indirect amplification of a PNA library by amplification of a complementary DNA library hybridised to the PNA. The generation of 10,000 defined pieces of DNA would have a myriad of applications, not least in the area of defined or directed sequencing and synthetic biology, but also in applications associated with encoding and tagging. By this approach DNA microarrays were used to allow the linear amplification of immobilised DNA sequences on an array followed by PCR amplification. Arrays of increasing sophistication (1; 10; 3875; 10,000 defined oligonucleotides) were used to validate the process, with sequences verified by selective hybridisation to a complementary DNA microarray with DNA sequencing demonstrating error rates of ca ≈ 0.2%. This technique offers an economical and efficient way of producing hundreds to thousands of specific DNA primers, while the DNA-arrays can be used as “factories” allowing specific DNA oligonucleotide pools to be generated with or without masking. This study also demonstrated a significant variance observed between the sequence frequencies found via Solexa sequencing compared to microarray analysis.

Cancer is a leading cause of morbidity and mortality in the developed world. While much has been learned about these diseases in the last few decades, one of the main barriers to widespread advancement is the heterogeneity of cancer biology. A growing body of evidence supports the idea that certain protein receptors are overexpressed on the surface of tumor cells as compared to normal tissues. These extracellular biomarkers provide a unique opportunity to selectively target the tumor with both imaging and therapeutic modalities. The research in this dissertation focuses on targeting proteins on the tumor cell surface with peptidomimetic ligands. Following a description of various extracellular receptors, chapter one discusses targeting ligands designed to specifically and selectively bind these receptors. It reviews recent literature on targeted alpha-particle therapy and ends with an explanation of the advantages of peptide ligands. Three distinct approaches to imaging and therapeutic modalities are then discussed in subsequent chapters. First, a peptide ligand was designed to target radionuclides to malignant melanoma cells in an effort to develop companion radiotherapeutics and diagnostic imaging agents. The second research project describes the synthesis of a novel antagonist peptide ligand with conjugated near infrared dye, and its utility for real-time intraoperative guidance during pancreatic adenocarcinoma resection. Finally, the last chapter describes how the relatively new field of immunomodulatory effectors may be enhanced by their derivatization with peptide targeting ligands.

Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

In dieser Arbeit wurde die Induktion spezifischer, immunologischer T-Zelltoleranz als therapeutische Strategie bei Autoimmunerkrankungen im Mausmodell untersucht. Da davon ausgegangen werden muss, dass viele der Autoimmunkrankheiten durch T-Zellen vermittelt sind, ist die Induktion spezifischer T-Zelltoleranz eine besonders interessante Therapiestrategie. Spezifische T-Zelltoleranz kann mittels Injektion des entsprechenden Peptidantigens induziert werden. Insgesamt sind zur Induktion einer solchen Toleranz, zumindest beim Menschen, relativ hohe Dosen an Peptidantigen notwendig. Die Produktion dieser Peptidantigene ist teuer. Bei unvorsichtiger Gabe kann es zur Anaphylaxie kommen. Es sollte also von Vorteil sein, die zu applizierende Menge an Peptid möglichst gering, dabei aber effizient zu halten. Vermutlich werden Antigene in Form von Peptiden schnell von unspezifischen Peptidasen und Proteasen in nicht-immunogene Fragmente gespalten und ausgeschieden, was wiederum eine hohe Dosierung erforderlich macht. Im Anfang der vorliegenden Arbeit stand die Hypothese, dass eine Stabilisierung des zu applizierenden Antigens zum Schutz vor Fragmentierung (und damit Wirkungsverlust) eine geeignete Methode sein könnte, Toleranzinduktion effektiver bzw. kostengünstiger zu gestalten. Bezüglich einer Stabilisierung von Peptiden zeigte sich, dass Peptide, welche aus rechtsdrehenden (D-)Aminosäuren zusammengesetzt sind, nur verzögert durch Proteasen/Peptidasen abgebaut werden. Wir setzten deshalb in dieser Arbeit D-Aninosäuren-substituierte Peptid-Varianten des Ovalbumin323-339-Peptidepitops (OVA323-339) ein und betrachteten die Wirkung dieser Peptide in vitro sowie in vivo auf spezifische DO11.10 T-Zellen. Basierend auf dem Peptidantigen OVA323-339, wurde zunächst ein minimales Epitop definiert, welches bei etwa gleicher Potenz um 6 Aminosäuren verkürzt werden konnte. Anschließend wurde eine Substitutionsanalyse durchgeführt, in der die ursprüngliche Aminosäuresequenz durch Austausch einiger L-Aminosäuren mittels D-Aminosäuren verändert wurde. Diese neu synthetisierten Peptide wurden zunächst auf ihre Fähigkeit überprüft, die OVA323-339 spezifischen DO11.10 T-Zellen in vitro zu aktivieren. Parallel konnte gezeigt werden, dass diese synthetisierten Peptidepitope in vitro eine deutlich verlängerte Serumhalbwertszeit aufwiesen. Im Weiteren wurde versucht, durch systemische Injektion von 300µg D-Peptid-Varianten in BABLB/c Mäusen T-Zelltoleranz zu induzieren. Die ex vivo restimulierten Lymphknoten-Zellen dieser Mäuse präsentierten je nach appliziertem Peptid eine reduzierte Proliferationsbereitschaft und IL-2 Sekretion. Die hier induzierte Toleranz konnte bis zu 60 Tagen post injectionem sowohl für das OVA323-339 als auch für einige der eingesetzten D-Peptide nachgewiesen werden. Auch nach Reduktion der Peptiddosis auf nur 100µg/Maus, waren die verkürzten und D-Aminosäuren-substituierten Peptide immer noch in der Lage sicher Toleranz zu induzieren. Die induzierte Toleranz durch D-Peptide war dabei der durch das Ausgangspeptid OVA323-339 induzierten Toleranz vergleichbar stark. Mit der Hilfe eines Transfermodells in unmanipulierte Mäuse, wurde das Verhalten der spezifischen T-Zellpopulation in vivo beobachtet. Durch den Transfer konnten in den Empfängermäusen (Balb/c) definierte T-Zellpopulationen bekannter Größe erzeugt werden. Mit dem Antikörper KJ1-26.1, der spezifisch den DO11.10-T-Zellrezeptor erkennt, konnten die transferierten Zellen in Geweben der Empfängermaus per FACS-Analyse nachgewiesen und deren Verhalten ex vivo studiert werden. Die intravenöse Injektion der serumstabilisierten Peptidanaloge führte in den transferierten Mäusen je nach Peptid zu einer funktionellen Nichtreaktivität (Anergie) als auch zur Deletion der für das Ausgangs(L-)Peptid spezifischen DO11.10 T-Zellen. In den oben genannten Versuchen ergaben sich Hinweise dafür, dass die D-Peptide ebenso effektiv sind wie das wesentlich längere Ausgangspeptid OVA323-339. Zukünftige Experimente werden weitere Aufschlüsse über einen möglichen Vorteil des Einsatzes von D-Peptiden in der Toleranzinduktion erbringen. / Induction of antigen-specific peripheral T cell tolerance in autoimmune diseases is an interesting therapeutically strategy. It can be induced by systemic injection of high-dose antigen. Investigations in induction of peripheral T cell tolerance in autoimmune mouse models revealed promising results. But it was also shown that the induced T cell tolerance spontaneously reverses after a period of time. This is probably due to a short in vivo half-life of the administrated peptide antigens. Since durable tolerance is required for this strategy to be of therapeutic value the administrated antigen-dose has to be of a very high and has to be injected repeatedly, and therefore bears an increased risk of anaphylactic reactions or exacerbation of the autoimmune disease. Because of these restrictions and also the high costs of peptide-production and purification, it is not surprising that this therapy didn t really find its way in to the clinical practice. The discovery that Peptides assembled partly or totally from D-amino acids are much more stable to proteolysis then natural L-peptides and therefore show an increased stability, lead to a wide interest of pharmacologists and immunologists. In former investigations it was shown that D-peptides used as vaccines elicited high levels of neutralizing antibodies so that there is no doubt about their immunogenic potency in vivo. It is also known that a single T cell receptor recognizes a wide range of peptide analogues that closely mimic the natural antigen. These observations led to our hypothesis, that the induction of peripheral T cell tolerance by systemic administration of D-Peptide substituted antigen variants should be possible and could be much more effective than the induction by the wild-type L-peptide. To verify our hypothesis we have chosen the well known OVA323-339 antigen which is recognized by T cells through the presentation in the I-Ad context. In a first step we performed a truncation analysis of OVA323-339 to identify a minimal epitope in it. We were able to demonstrate that the sequence OVA327-337 is as well potent as the original and 6 amino acids longer OVA323-339 sequence. The potency of new defined epitopes was tested by stimulating the OVA323-339 -specific DO11.10 T cells in vitro. In a stepwise performed substitution analysis we attempted to insert some D-amino acids in this novel peptide epitope. The DO11.10 cells only tolerated a few D-amino acid substitutions into the original sequence with the effect of now showing reduced proliferation. Performing an analysis of their half-life in vitro we identified two peptides as interesting candidates for the in vivo tolerance induction experiments. In the in vivo part of this work we induced peripheral tolerance by injecting the novel peptides into BALB/c mice. To monitor the behaviour of the tolerated T cells we also performed adoptive transfer experiments by transferring DO11.10 cells into naive BALB/c mice. With the help of the KJ26-1 antibody which specifically recognizes the DO11.10 T cell receptor it became possible to detect the transferred T cells ex vivo. Our results demonstrate that induction of peripheral T cell tolerance through injection of D-peptides is possible and long lasting (up to 60 days). Even with a dose reduction we found a stable T cell tolerance under ex vivo restimulation with the original peptide. Summarizing we were able to show that D-peptides are at least as effective as the natural occurring L-peptides inducing tolerance. Much more, the transfer experiments revealed that the kind of induced T cell tolerance (i.e. anergy and/or deletion through activation induced cell death) is antigen dependent and probably differs due to the agonistic potency of the given antigen.

Les récepteurs couplés aux protéines G constituent la plus grande famille de protéines membranaires et interviennent dans de nombreux processus physiologiques. La compréhension de l’interaction ligand-récepteur d’un point de vue mécanistique mais également thérapeutique est cruciale. Appartenant à la famille des récepteurs couplés aux protéines G, les récepteurs de la vasopressine et de l’ocytocine ont été choisis comme modèle d’étude. Ces hormones jouent un rôle important dans la modulation de l’attachement et de l’affect chez les mammifères. Afin d’accélérer la découverte de ligands ocytocinergiques et d’explorer les mécanismes fondamentaux de leurs interactions, nous avons conçu les premiers ligands fluorescents non peptidiques des récepteurs de la vasopressine V1a et de l’ocytocine. Ces ligands ont été utilisés pour développer des tests de liaisons par TR-FRET et démontrer la dimérisation des récepteurs de la vasopressine V1a et V2 sur cellules. Des études autour de petites plates-formes dérivées d’aza-dicétopipérazine ont permis d’accéder à un nouvel antagoniste non peptidique du récepteur de l’ocytocine. L’optimisation de dérivés benzodiazépines ocytocinergiques par des études de relations structure-activité a permis d’identifier les meilleurs agonistes non peptidiques du récepteur de l’ocytocine à ce jour. Une étude in vivo chez la souris et chez le singe est amorcée pour apporter dans un futur, une solution thérapeutique aux problèmes d’interaction sociale en général et d’autisme en particulier. / G protein coupled receptors are the largest membrane protein family and play an important role in a large number ofphysiological processes. The comprehension of the ligand-receptor interaction from a mechanistic point of view but alsofor therapeutic use is crucial. Belonging to the G protein coupled receptors, the oxytocin and vasopressin receptors havebeen used as a model system. These two hormones play an important role in the modulation of attachment and affectin mammals. To accelerate the discovery of new ligands for oxytocin and vasopressin receptors and to explore thefundamental role of their interactions, we designed the first non-peptide fluorescent ligands for oxytocin and vasopressin V1a receptors. These ligands have been used to develop new binding tests based on TR-FRET technology and to prove the V1a and V2 receptor dimerisation. In parallel, we developed a new non-peptide oxytocin antagonist around an aza-diketopiperazine platform. . Optimization of benzodiazepine derivatives enables us to identify the best non peptideoxytocin agonists to date. In vivo studies in mice and monkeys are initiated to bring in the future a therapeuticsolution to social interaction problems in general and autism in particular