Anti-cancer vaccination - immunomonitoring of a clinical phase I/II study in prostate cancer patients vaccinated with a RhoC-derived synthetic peptide

Die Vakzinierung mit Peptiden, die von tumorassoziierten oder -spezifischen Antigenen entstammen, welche eine Art der Immuntherapie darstellt, verfolgt das Ziel, eine spezifische, gegen den Tumor gerichtete Immunantwort zu induzieren, oder eine vorhandene Immunantwort zu verstärken. Das Ziel der Arbeit war, zu untersuchen, ob solch eine Aktivierung des Immunsystems im Rahmen einer klinischen Phase I/II Peptid-Vakzinierungs-Studie bei Prostatakarzinom (PCa)- Patienten vorliegt. Die Patienten wurden wiederholt mit einem der RhoC Zielstruktur entstammenden synthetischen langen Peptid (SLP, 20 Aminosäuren), welches potentiell CD8 und CD4 Zellen aktivieren kann, zusammen mit Montanide ISA-51 vakziniert. Eine Vakzine-spezifische Immunantwort konnte in 86% der Patienten nachgewiesen werden, welche mindestens noch zehn Monate nach der letzten Vakzinierung nachweisbar war. Die Vakzin-spezifischen Zellen waren überwiegend multifunktionale CD4 Zellen, die einen Effektor-Gedächtnis-T-Zellen Phänotypen aufwiesen. Insgesamt konnten drei promiskuitiv präsentierte humane Leukozytenantigen (HLA)-Klasse II Peptide nachgewiesen werden. Die aktivierten CD4 Zellen zeigten auch nach wiederholten Vakzinierungen keinen Phänotypen der Exhaustion. Eine zusätzliche Vakzin-spezifische CD8 Zell Antwort, die durch HLA-B*27:05 restringiert war, konnte bei einem Patienten nachgewiesen werden. Die Untersuchung zeigt, dass das RhoC-entstammende Vakzin-Peptid immunogen ist und eine langanhaltende Immunantwort in einer Vielzahl von Patienten hervorruft. Der zweite Teil der Arbeit befasst sich mit den technischen Aspekten die zur Durchführung der hier präsentierten klinischen Studie wichtig sind. Zuerst werden in einem Review die gebräuchlichsten Methoden zum Nachweis von Antigen-spezifischen T Zellen in der experimentellen Immuntherapie, neben einer neu etablierten Methode zur Identifizierung von Antigen-spezifischen CD8 Zellen, erläutert. Methoden, die mitunter beschrieben werden, werden in der Folge für den Nachweis von Antigen-spezifischen CD8 und CD4 Zellen mit Hilfe von SLPs eingesetzt, dessen Protokolloptimierung danach beschrieben wird. Wir zeigen, dass der Einsatz von Poly-ICLC (Hiltonol®), ein Toll-ähnlicher Rezeptor 3 Agonist, sowie eine erhöhte SLP Konzentration führte zu einem verbesserten Nachweis von Peptid-spezifischen Zellen. Das optimierte Protokoll findet sein Einsatzgebiet nicht nur im Monitoring von klinischen Studien, sondern zum Beispiel auch in der Identifizierung von Epitopen.Vaccination with peptides derived from tumor-associated or -specific antigens is a form of immunotherapy that specifically induces or reactivates T cell immune response to fight cancer. The aim of the present work was to assess the immune responses in prostate cancer (PCa) patients upon peptide vaccination in a clinical phase I/II study. PCa patients were repeatedly vaccinated with a RhoC-derived synthetic long peptide (SLP, 20 amino acids), which potentially activates CD8 and CD4 cells, emulsified in Montanide ISA-51. An immune response was detected in 86% of the patients after vaccination, which lasts at least ten months after the last vaccination. Vaccine-specific T cells were mainly poly-functional CD4 cells of the effector memory T cell phenotype. In total, three promiscuously presented human leucocyte antigen (HLA)-class II peptides were identified. No exhausted T cell phenotype was observed after multiple vaccinations. For one patient, a vaccine-directed CD8 cell response restricted by HLA-B*27:05 was detected in addition to the CD4 cell response. In conclusion, the RhoC-derived peptide is immunogenic and induces a long-lasting immune response in the majority of patients. The second part of the thesis deals with technical aspects that are important for the presented clinical study. First, the common methods for the monitoring of clinical studies are described in a review, besides a newly established method for the identification of antigen-specific CD8 T cells. Methods that are described in this review are used for the identification of antigen-specific CD8 and CD4 cells using SLPs, which protocol optimization is described afterwards. We showed that the addition of Poly-ICLC (Hiltonol®), a toll-like receptor 3 agonist, together with an increased SLP concentration, lead to an optimized identification of peptide-specific cells. The optimized protocol could be used for the monitoring of clinical studies, as well as for the identification of epitopes, for example

Adhering to adhesion : assessing integrin conformation to monitor T cells

Monitoring T cells is of major importance for the development of immunotherapies. Recent sophisticated assays can address particular aspects of the anti-tumor T-cell repertoire or support very large-scale immune screening for biomarker discovery. Robust methods for the routine assessment of the quantity and quality of antigen-specific T cells remain, however, essential. This review discusses selected methods that are commonly used for T-cell monitoring and summarizes the advantages and limitations of these assays. We also present a new functional assay, which specifically detects activated beta(2) integrins within a very short time following CD8(+) T-cell stimulation. Because of its unique and favorable characteristics, this assay could be useful for implementation into our T-cell monitoring toolbox

CAF-immune cell crosstalk and its impact in immunotherapy

Tumour cells do not exist as isolated entities. Instead, they are surrounded by a variety of cells and extracellular matrix, which form the tumour microenvironment (TME). The interaction between cancer cells and their microenvironment is increasingly acknowledged as essential in dictating the outcome of the patients. The TME includes everything that surrounds tumour cells and is often highjacked by the latter to promote their growth, invasion, and immune escape. Immune cells and cancer-associated fibroblasts (CAFs) are essential components of the TME, and there is increasing evidence that their interaction constitutes a major player not only for tumour progression but also for therapy response.Recent work in the field of immuno-oncology resulted in the development of novel therapies that aim at activating immune cells against cancer cells to eliminate them. Despite their unprecedented success, the lack of response from a large portion of patients highlights the need for further progress and improvement. To achieve its ultimate goal, the interaction between cancer cells and the TME needs to be studied in-depth to allow the targeting of mechanisms that are involved in resistance or refractoriness to therapy. Moreover, predictive and prognostic biomarkers for patient stratification are still missing. In this review, we focus on and highlight the complexity of CAFs within the TME and how their interaction, particularly with immune cells, can contribute to treatment failure. We further discuss how this crosstalk can be further dissected and which strategies are currently used to target them

Sleep enhances numbers and function of monocytes and improves bacterial infection outcome in mice.

Sleep strongly impacts both humoral and cellular immunity; however, its acute effects on the innate immune defense against pathogens are unclear. Here, we elucidated in mice whether sleep affects the numbers and functions of innate immune cells and their defense against systemic bacterial infection. Sleep significantly increased numbers of classical monocytes in blood and spleen of mice that were allowed to sleep for six hours at the beginning of the normal resting phase compared to mice kept awake for the same time. The sleep-induced effect on classical monocytes was neither caused by alterations in corticosterone nor myelopoiesis, bone marrow egress or death of monocytes and did only partially involve Gαi-protein coupled receptors like chemokine receptor 2 (CCR2), but not the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) or lymphocyte function-associated antigen 1 (LFA-1). Notably, sleep suppressed the expression of the clock gene Arntl in splenic monocytes and the sleep-induced increase in circulating classical monocytes was abrogated in Arntl-deficient animals, indicating that sleep is a prerequisite for clock-gene driven rhythmic trafficking of classical monocytes. Sleep also enhanced the production of reactive oxygen species by monocytes and neutrophils. Moreover, sleep profoundly reduced bacterial load in blood and spleen of mice that were allowed to sleep before systemic bacterial infection and consequently increased survival upon infection. These data provide the first evidence that sleep enhances numbers and function of innate immune cells and therewith strengthens early defense against bacterial pathogens

Simultaneous Identification of Functional Antigen-Specific CD8+ and CD4+ Cells after In Vitro Expansion Using Elongated Peptides

Elongated peptides (EPs), containing possibly one or multiple epitope/s, are increasingly used for the screening of antigen-specific CD8+ and CD4+ cell responses. Here, we present an in vitro protocol that allows the amplification of antigen-specific cells and the subsequent functional analysis of both T cell types using EPs. Known viral-derived epitopes were elongated to 20 mer EPs on the N-, C-, and both termini for HLA class I binders, or on the N- and C- termini for HLA class II binders. With EP stimulation only, the percentage of responding CD8+ T cells was dependent on the elongation site of the EP, whereas CD4+ T cell responses were completely lost in 22% of the tests performed ex vivo. A short-term amplification step plus the addition of a TLR3 agonist (Poly-ICLC) together with an increased EP concentration improved markedly the detection of CD8+ and CD4+ T cell reactivities

Vaccination against RhoC induces long-lasting immune responses in patients with prostate cancer: results from a phase I/II clinical trial

Background Peptide-based vaccination is a rational option for immunotherapy of prostate cancer. In this first-in-man phase I/II study, we assessed the safety, tolerability and immunological impact of a synthetic long peptide vaccine targeting Ras homolog gene family member C (RhoC) in patients with prostate cancer. RhoC is a small GTPase overexpressed in advanced solid cancers, metastases and cancer stem cells.Methods Twenty-two patients who had previously undergone radical prostatectomy received subcutaneous injections of 0.1 mg of a single RhoC-derived 20mer peptide emulsified in Montanide ISA-51 every 2 weeks for the first six times, then five times every 4 weeks for a total treatment time of 30 weeks. The drug safety and vaccine-specific immune responses were assessed during treatment and thereafter within a 13-month follow-up period. Serum level of prostate-specific antigen was measured up to 26 months postvaccination.Results Most patients (18 of 21 evaluable) developed a strong CD4 T cell response against the vaccine, which lasted at least 10 months following the last vaccination. Three promiscuouslypresented HLA-class II epitopes were identified. Vaccine-specific CD4 T cells were polyfunctional and effector memory T cells that stably expressed PD-1 (CD279) and OX-40 (CD134), but not LAG-3 (CD223). One CD8 T cell response was detected in addition. The vaccine was well tolerated and no treatment-related adverse events of grade ≥3 were observed.Conclusion Targeting of RhoC induced a potent and long-lasting T cell immunity in the majority of the patients. The study demonstrates an excellent safety and tolerability profile. Vaccination against RhoC could potentially delay or prevent tumor recurrence and metastasis formation.Trial registration number NCT03199872