Massenspektrometrische Analyse der HLA-Ligandome des Nierenzellkarzinoms und des benignen Nierengewebes

Peptide vaccination is a promising immunotherapeutic approach for the treatment of malignancies. In this project, the unique opportunity to analyze HLA ligandomes of samples from tumor and adjacent benign tissue of renal cell carcinoma (RCC) patients by mass spectrometry was given. This allowed for the establishment of a novel approach of antigen definition by comparative profiling of malignant and benign HLA ligandomes. Analyses were performed for HLA class I and II of tumor and benign tissue sample pairs in a cohort of 33 RCC patients. Altogether, the acquired dataset includes 35.543 unique HLA class I ligand identifications derived from 11.279 unique source proteins and 8.344 unique HLA class II ligand identifications derived from 2.199 unique source proteins. Comparative analysis revealed a new class of tumor antigens based on tumor-exclusive representation of HLA ligand source proteins, termed ligandome-derived tumor-associated antigens (LiTAAs). A selection of LiTAA-derived HLA ligands (ligandome-derived tumor-associated peptides, LiTAPs) were tested for immunogenicity in high-throughput priming experiments using artificial antigen-presenting cells (aAPCs). Immunogenic LiTAPs will eventually provide a ‘warehouse’ of suitable vaccination peptides and allow for a personalized vaccination approach based on individual HLA typing and actual target expression on tumor cells. Furthermore, analysis of healthy (benign) tissue HLA ligandomes is a step towards a comprehensive map of the healthy human immunopeptidome, an important prerequisite for the definition of tumor-specific immunotherapy targets. In a second project, naturally presented HLA ligands derived from mycobacterium tuberculosis (Mtb) were identified by infection experiments. HLA ligands derived from Mtb antigens encoded in a viral vector could be identified, whereas analysis of macrophages infected with live Mtb did not reveal any new Mtb-derived HLA ligands. Such HLA ligands may provide the basis for the design of more efficient subunit vaccines against tuberculosis as well as development of enhanced infection screening assays.Die Peptidimpfung ist ein vielversprechender immuntherapeutischer Ansatz für die Behandlung maligner Erkrankungen. In diesem Projekt bestand die einmalige Möglichkeit, die HLA-Ligandome von Proben aus Tumor und benignem Gewebe am Nierenzellkarzinom erkrankter Patienten massenspektrometrisch zu analysieren. So konnte ein neuer Ansatz zur Antigendefinition durch vergleichende Analyse maligner und benigner HLA-Ligandome entwickelt werden. Die Analysen wurden für HLA Klasse I und II in Probenpaaren von Tumor und benignem Gewebe einer Kohorte von 33 Nierenzellkarzinom-Patienten durchgeführt. Insgesamt umfasst der erhaltene Datensatz 35.543 identifizierte unterschiedliche HLA Klasse I-Liganden die 11.279 verschiedenen Quellproteinen entstammen, sowie 8.344 unterschiedliche HLA Klasse II-Liganden, die 2.199 verschiedenen Quellproteinen entstammen. Die vergleichende Analyse offenbarte eine neue Klasse von Tumorantigenen, basierend auf einer Tumor-exklusiven Repräsentation der Quellproteine der HLA-Liganden, genannt Ligandom-abgeleitete Tumor-assoziierte Antigene (LiTAAs). Eine Auswahl von LiTAA-entstammenden HLA-Liganden (Ligandom-abgeleitete Tumor-assoziierte Peptide, LiTAPs) wurden auf ihre Immunogenität in hochdurchsatz-priming Experimenten mit künstlichen antigenpräsentierenden Zellen (aAPCs) untersucht. Immunogene LiTAPs werden letztendlich eine ‚Lagerhalle‘ mit geeigneten Impfpeptiden bilden und eine personalisierte Impfung basierend auf HLA-Typisierung und tatsächlicher Expression der Zielstrukturen auf Tumorzellen ermöglichen. Weiterhin ist die Analyse gesunder (benigner) HLA-Ligandome ein Schritt Richtung einer umfassenden Karte des gesunden humanen Immunpeptidoms, eine wichtige Voraussetzung für die Definition tumorspezifischer immuntherapeutischer Zielstrukturen. In einem zweiten Projekt wurden natürlich präsentierte HLA-Liganden aus Mycobacterium tuberculosis (Mtb) in Infektionsexperimenten identifiziert. HLA-Liganden aus in einem viralen Vektor codierten Mtb-Antigenen konnten identifiziert werden, während die Analyse mit lebenden Mtb infizierter Makrophagen keine neuen HLA-Liganden aus Mtb ergab. Solche HLA-Liganden können sowohl die Basis für das Design effizienterer subunit-Impfungen gegen Tuberkulose als auch für die Entwicklung verbesserter Infektions-Überprüfungen bilden

Influence of ATLG serum levels on CD3/CD19-depleted hematopoietic grafts and on immune recovery in pediatric haplo-HSCT

Anti-T lymphocyte globulin (ATLG) significantly reduces the risk of engraftment failure in allogeneic hematopoietic stem cell transplant (HSCT) but hampers posttransplant immune reconstitution. We hypothesized that in patients receiving haploidentical CD3/CD19-depleted grafts, these double-edged effects could be better balanced by attaining high ATLG serum concentrations before transplant but as low as possible on the day of transplant. Therefore, we moved the start of ATLG application to day -12 and determined serum concentrations of T-cell-specific ATLG in pediatric patients treated with 3 established dosing regimens (15, 30, or 60 mg/kg). Corresponding mean T-cell-specific ATLG serum concentrations at day 0 were 1.14, 2.99, or 12.10 μg/mL, respectively. Higher ATLG doses correlated with higher peak levels at days -8 and -7 and reduced graft rejection, whereas lower ATLG doses correlated with significantly faster posttransplant recovery of T and natural killer cells. The rate of graft-versus-host disease remained low, independent of ATLG doses. Moreover, in vitro assays showed that ATLG concentrations of 2.0 μg/mL and lower only slightly reduced the activity of natural killer cells, and therefore, the function of such effector cells might be preserved in the grafts. Pharmacokinetic analysis, compatible with linear first-order kinetics, revealed similar half-life values, independent of ATLG doses. Hence, the day on which a desired ATLG serum level is reached can be calculated before HSCT. Our retrospective study demonstrates the relevance of dosing and time of administration of ATLG on engraftment and immune recovery in ex vivo CD3/CD19-depleted haploidentical HSCT

Recurrent SARS-CoV-2 Infection and Impaired Immunologic Response in a Pediatric Oncologic Patient While Treated With Radiochemotherapy

Immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in pediatric patients with malignant disease may be affected by tumor therapy. Here, we report the case of a child with rhabdomyosarcoma and recurrent SARS-CoV-2 infection. Immunologic responses, analyzed by T-cell activity and anti-viral IgG levels, were impaired and not durable as a result of intensive radiochemotherapy

Case Report: Targeting of individual somatic tumor mutations by multipeptide vaccination tailored for HLA class I and II presentation induces strong CD4 and CD8 T-cell responses in a patient with metastatic castration sensitive prostate cancer.

Localized prostate cancer is curable, but metastatic castration sensitive prostate cancer has a low 5-year survival rate, while broad treatment options are lacking. Here we present an mCSPC patient under remission receiving individualized neoantigen-derived peptide vaccination as recurrence prophylaxis in the setting of an individual treatment attempt. The patient was initially analyzed for somatic tumor mutations and then consecutively treated with two different peptide vaccines over a period of 33 months. The first vaccine contained predicted HLA class I binding peptides only whereas the second vaccine contained both predicted HLA class I and II binding peptides. Intracellular cytokine staining after 12 day in-vitro expansion measuring four T-cell activation markers (IFNg, TNF-α, IL-2, CD154) was used to determine vaccine-induced T-cell responses. While the first vaccine induced only one robust CD4+ T-cell response after 21 vaccinations, co-vaccination of HLA class I and II peptides induced multiple strong and durable CD4+ and CD8+ T-cell responses already after sixth vaccinations. The vaccine-induced immune responses were robust and polyfunctional. PSA remained undetectable for 51 months. The results presented here implicate that neoantigen-targeting vaccines might be considered for those cancer subtypes where therapeutic options are limited. Furthermore, our findings suggest that both HLA class I and II restricted peptides should be considered for future peptide vaccination trials

Case Report: Long-Term Survival of a Patient with Cerebral Metastasized Ovarian Carcinoma Treated with a Personalized Peptide Vaccine and Anti-PD-1 Therapy

Ovarian cancer is one of the most common cancers among women and the most lethal malignancy of all gynecological cancers. Surgery is promising in the early stages; however, most patients are first diagnosed in the advanced stages, where treatment options are limited. Here, we present a 49-year-old patient who was first diagnosed with stage III ovarian cancer. After the tumor progressed several times under guideline therapies with no more treatment options available at that time, the patient received a fully individualized neoantigen-derived peptide vaccine in the setting of an individual healing attempt. The tumor was analyzed for somatic mutations via whole exome sequencing and potential neoepitopes were vaccinated over a period of 50 months. During vaccination, the patient additionally received anti-PD-1 therapy to prevent further disease progression. Vaccine-induced T-cell responses were detected using intracellular cytokine staining. After eleven days of in vitro expansion, four T-cell activation markers (namely IFN-ɣ, TNF-α, IL-2, and CD154) were measured. The proliferation capacity of neoantigen-specific T-cells was determined using a CFSE proliferation assay. Immune monitoring revealed a very strong CD4+ T-cell response against one of the vaccinated peptides. The vaccine-induced T-cells simultaneously expressed CD154, TNF, IL-2, and IFN-ɣ and showed a strong proliferation capacity upon neoantigen stimulation. Next-generation sequencing, as well as immunohistochemical analysis, revealed a loss of Beta-2 microglobulin (B2M), which is essential for MHC class I presentation. The results presented here implicate that the application of neoantigen-derived peptide vaccines might be considered for those cancer stages, where promising therapeutic options are lacking. Furthermore, we provide more data that endorse the intensive investigation of B2M loss as a tumor escape mechanism in clinical trials using anti-cancer vaccines together with immune-checkpoint inhibitors