Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

BACKGROUND:Class II MHC molecules (MHC II) are cell surface receptors displaying short protein fragments for the surveillance by CD4+ T cells. Antigens therefore have to be loaded onto this receptor in order to induce productive immune responses. On the cell surface, most MHC II molecules are either occupied by ligands or their binding cleft has been blocked by the acquisition of a non-receptive state. Direct loading with antigens, as required during peptide vaccinations, is therefore hindered. PRINCIPAL FINDINGS:Here we show, that the in vivo response of CD4+ T cells can be improved, when the antigens are administered together with 'MHC-loading enhancer' (MLE). MLE are small catalytic compounds able to open up the MHC binding site by triggering ligand-release and stabilizing the receptive state. Their enhancing effect on the immune response was demonstrated here with an antigen from the influenza virus and tumour associated antigens (TAA) derived from the NY-ESO-1 protein. The application of these antigens in combination with adamantane ethanol (AdEtOH), an MLE compound active on human HLA-DR molecules, significantly increased the frequency of antigen-specific CD4+ T cells in mice transgenic for the human MHC II molecule. Notably, the effect was evident only with the MLE-susceptible HLA-DR molecule and not with murine MHC II molecules non-susceptible for the catalytic effect of the MLE. CONCLUSION:MLE can specifically increase the potency of a vaccine by facilitating the efficient transfer of the antigen onto the MHC molecule. They may therefore open a new way to improve vaccination efficacy and tumour-immunotherapy

Anchor Side Chains of Short Peptide Fragments Trigger Ligand-Exchange of Class II MHC Molecules

Class II MHC molecules display peptides on the cell surface for the surveillance by CD4+ T cells. To ensure that these ligands accurately reflect the content of the intracellular MHC loading compartment, a complex processing pathway has evolved that delivers only stable peptide/MHC complexes to the surface. As additional safeguard, MHC molecules quickly acquire a ‘non-receptive’ state once they have lost their ligand. Here we show now that amino acid side chains of short peptides can bypass these safety mechanisms by triggering the reversible ligand-exchange. The catalytic activity of dipeptides such as Tyr-Arg was stereo-specific and could be enhanced by modifications addressing the conserved H-bond network near the P1 pocket of the MHC molecule. It affected both antigen-loading and ligand-release and strictly correlated with reported anchor preferences of P1, the specific target site for the catalytic side chain of the dipeptide. The effect was evident also in CD4+ T cell assays, where the allele-selective influence of the dipeptides translated into increased sensitivities of the antigen-specific immune response. Molecular dynamic calculations support the hypothesis that occupation of P1 prevents the ‘closure’ of the empty peptide binding site into the non-receptive state. During antigen-processing and -presentation P1 may therefore function as important “sensor” for peptide-load. While it regulates maturation and trafficking of the complex, on the cell surface, short protein fragments present in blood or lymph could utilize this mechanism to alter the ligand composition on antigen presenting cells in a catalytic way

Characterisation of an h-ABL specific CD4+ T cell response and the implication of AdEtOH as a catalyst of peptide loading

1\. EINLEITUNG 1 1.1. Onkogene und Tumorantigene 1 1.2. Die Rolle des Immunsystems bei der Krebsentstehung 2 1.2.1. „Immunosurveillance“ 3 1.3 Formen der Leukämie und deren Entstehung 6 1.3.1. Chronische myeloische (CML) und chronische lymphatische Leukämie (CLL) 6 1.3.2. Akute myeloische (AML) und akute lymphatische Leukämie (ALL) 7 1.4. Das Fusionsprotein BCR-ABL 8 1.4.1. Entstehung des Philadelphia Chromosoms und krankheits-bildende Faktoren 8 1.4.2. BCR-ABL-Variationen und deren Assoziation mit der Ausprägung der Leukämie 9 1.4.3. Auswirkung des Fusionsproteins BCR-ABL auf die Regulation der Zelle 10 1.5. T-Zell-vermittelte Immunität 12 1.5.1. Effektorfunktion und Effektormoleküle 14 1.5.2. Toleranzinduktion als Kontrollelement der Immunreaktion 15 1.6. Tumorimmunologische Therapieansätze 18 1.6.1. Behandlungsformen und therapeutische Ansätze für die CML 19 1.6.2. Generelle Tumor-Behandlungsstrategien 21 1.6.3. Die Rolle der CD4+-T-Zellen für eine Tumorbehandlung 23 1.7. Zielsetzung der Arbeit 28 2\. MATERIAL UND METHODEN 31 2.1. Material 31 2.1.1. Zelllinien 31 2.1.2. Puffer und Chemikalien 33 2.1.3. Peptide und biologisches Material 36 2.1.4. Monoklonale Antikörper 38 2.1.5. Geräte 39 2.1.6. Software 39 2.2. Methoden 40 2.2.1. Polymerase-Ketten- Reaktion (PCR) 40 2.2.2. Agarose Gelelektrophorese, DNA Aufreinigung 42 2.2.3. Restriktionsspaltung, Ligation, Transformation und Plasmidaufarbeitung 43 2.2.4. Prokaryotische Expression und Aufreinigung von Proteinen 43 2.2.5. Kultivierung von Zellen und Zellpräparation 47 2.2.6. Durchflusszytometrie (FACS-Analyse) 51 2.2.7. Zelloberflächlicher MHC-Beladungsassay 52 2.2.8. Tumorinduktion /CML-Mausmodell 53 2.2.9. Induktion von primären Immunantworten 54 2.2.10. ELISPOT-Assay 55 2.2.11. Proliferationsassay 56 2.2.12. Transfektion und retrovirale Infektion von Zellen 57 3.ERGEBNISSE 60 3.1. Charakterisierung des humanen Peptides ABL908-922 60 3.1.1. Identifizierung der Immunogenität verschiedener ABL- Epitope 60 3.1.2. Die Prozessierbarkeit des hABL908-922 Epitopes im ABL-Protein 65 3.1.3. BCR-ABL-transduzierte Zellen präsentieren hABL908-922 70 3.2. Untersuchung der hABL-spezifischen CD4+-T -Zell-antwort im BCR-ABL+-Tumormodell 74 3.2.1. Modulation der Immunantwort hABL908-922 durch Inhibition der regulatorischen T-Zell-Population 79 3.3. AdEtOH, ein allelspezifischer Katalysator 83 3.3.1. Die katalytische Aktivität des AdEtOH 83 3.3.2. Die katalytische Aktivität des AdEtOH ist allelspezifisch 84 3.3.3. Anwendung des AdEtOH als Katalysator für verschiedene HLA-DR1 restringierte Peptide 86 3.3.4. Modulation CD4+ spezifischer T-Zellantworten in vitro durch den Einsatz von AdEtOH 89 3.3.5. Modulation CD4+-spezifischer T-Zellantworten in vivo durch den Einsatz von AdEtOH 92 3.3.6. Die Verstärkung vom Tumorantigenen durch AdEtOH 94 4.DISKUSSION 96 4.1. Die Charakterisierung der hABL908-922-spezifischen CD4+-Immunantwort 96 4.2. AdEtOH ist ein effektiver Katalysator für die Peptid-Beladung von MHC-Klasse-II-Varianten 103 5\. LITERATURVERZEICHNIS 106 5.1. Eigene Publikationen 106 5.2. Publikationen 106 6\. ZUSAMMENFASSUNG 116 7\. SUMMARY 118 8\. ABKÜRZUNGEN 120 9.DANKSAGUNG 122CD4+-T-Lymphozyten übernehmen als Helfer-Zellen eine entscheidende Aufgabe in der Induktion und Koordination produktiver Immunreaktionen. Als Suppressorzellen können CD4+-T-Zellen destruktive inflammatorische Prozesse, welche zu Autoimmunreaktionen führen, unterbinden. Daneben können diese Zellen aber auch tumorspezifische Immunantworten supprimieren und zur Progression des Tumorwachstums beitragen. Dagegen kann eine gezielte Aktivierung spezifischer CD4+-Effektorzellen (Th1, Th2, Th17) zu einer effektiven anti-tumoralen Immunantwort führen. In diesem Kontext befassen sich viele Gruppen mit der Identifizierung tumorspezifischer Epitope, welche therapeutisch eingesetzt werden könnten. Bei der Identifizierung BCR-ABL-relevanter Antigene wurde der Fokus größtenteils auf CD8+-Epitope aus der Breakpoint-Region des Fusionsproteins gelegt. Eine Reihe von Studien zeigen jedoch, dass die Aktivierung sowohl von CD8+T-Zellen als auch von CD4+-T-Zellen von großer Bedeutung ist. In dieser Arbeit wurde ein neues Antigen vorgestellt, welches der ABL-Kinase entstammt, die in BCR-ABL transformierten Zellen überexprimiert ist. Das CD4+-Antigen hABL908-922 ist HLA-DR1-restringiert und kann im humanisierten Mausmodell als Xenoantigen eine starke CD4+-spezifische Immunreaktion induzieren. Die hABL908-922-spezifischen T-Zellen können aber auch ex vivo in Lymphknoten und Milz BCR-ABL-transplantierter Mäuse detektiert werden. Gewisse anti-tumorale Effekte durch eine Vakzinierung mit hABL908-922 lassen sich erst nach Depletion von CD25+-T-Zellen feststellen. Da aber hABL908-922 eine reine CD4+-spezifische Immunreaktion induziert, ist das Epitop, trotz des Mangels einer vollständigen Tumorabstoßung, durchaus ein interessantes ABL-Epitop. In Anlehnung an therapeutische Zwecke ist ein Vakzinierungsansatz mit autoreaktiven Antigenen, wie es für hABL908-922 im Menschen der Fall wäre, generell kritisch zu betrachten, da mögliche Autoimmunreaktionen nicht auszuschließen sind. Dennoch kann die Identifizierung solcher Antigene für mögliche Modifikationen bezüglich spezifischer T-Zell-Rezeptoren relevant werden, da für die Abwehr nicht- viraler Tumore die Induktion der Autoimmunität ein wichtiger Faktor ist. Ein weiterer Teil der hier vorgelegten Arbeit beschäftigte sich mit der Untersuchung der niedermolekularen chemischen Verbindung 2-(1-adamantyl)ethanol (AdEtOH). AdEtOH konnte als allelspezifischer Katalysator der Peptid-Beladung identifiziert werden. Als Katalysator kann AdEtOH sowohl Antigen-spezifische CD4+-Immunreaktionen in vitro als auch in vivo verstärken. Die Ergebnisse zeigen, dass die allelspezifische katalytische Aktivität des AdEtOH mit der Ausprägung der Aminosäure Glycin an der Position beta 86 in der P1-Tasche des MHC-Klasse-II-Moleküls korreliert. Eine allelspezifische Verstärkung der Immunreaktion kann auch bei Zugabe von AdEtOH als Additive zum Adjuvans erreicht werden.CD4+ T cells play a crucial role in induction and coordination of immune reaction leading to tumor regression and better survival. However CD4+ T cells, namely regulatory T cells control immune response and have suppressive function to prevent autoimmune reactions. Hence such cells could also suppress specific immune responses against tumor antigens. Several studies could show, that the activation of tumor specific T cells and the depletion of regulatory T cells leads to an overall better clinical outcome. Current immunotherapies are based on CD8+ T cells recognizing antigens from the breakpoint region, which are actually tumor-specific antigens. However there are only some candidates for immune based therapeutic application. It could demonstrate that the vaccination with tumor-specific antigens induce specific CD8+ T cell response. Nevertheless up to now there is no appreciable clinical success. However recent publications show that CD4+ T cells play also a key role in induction of effective anti-tumor response. Therefore the combination of both activation of CD8+ and CD4+ T cells could increase and ameliorate tumor regression. For that reason there is a need to discover tumor-specific and tumor-associated antigens in terms of CD4 T cells. In this context we investigated here the immunogenecity of our newly identified ABL-derived CD4+ T cell epitope. This epitope is located within the ABL-region, which itself is over expressed in BCR-ABL expressing cells. Due to the construction of the BCR-ABL fusion protein, the ABL-component is basically equal in all Ph+ leukemias. Here we show that the epitope ABL908-922, which is restricted to HLA-DR1, is able to induce strong immune reaction in HLA-DR1tg mice. Moreover we could demonstrate that this peptide is processed and presented by HLA-DR1 expressing dendritic cells, which were previously infected with BCR-ABL protein. In addition hABL-specific CD4+ T cells can be activated by transplantation of BCR-ABL+ tumor cells. Furthermore the effectiveness of hABL908-922 specific CD4 T cells was investigated in a BCR-ABL+ tumor model. With immunization of hABL908-922 we could not detect any tumor reduction or delay. But the inclusion of Treg depletion within the vaccination protocol promote the activation of hABL908-922 specific CD4 T cells, which results in certain tumor delay. Additional to the analysis of ABL-antigens, we investigated the role of MHC loading enhancer (MLE), which are small organic compounds. Recently we published that MLEs like pCP are capable to enhance peptide loading at the surface of Antigen presenting cells. Here we could characterize an organic molecule, AdEtOH which catalyzes in an allele-specific manner. The allele-specificity is correlated with the expression of Glycin at the position 86 in the P1-pocket. Moreover we used AdEtOH to investigate the effect in vivo. We could show that the enhancement of AdEtOH as adjuvant additive is reflected in an increased specific immune response in vivo.. However the role of such MLE for treatment is not clear and have to be explored in further studies

TNIK signaling imprints CD8+ T cell memory formation early after priming.

Co-stimulatory signals, cytokines and transcription factors regulate the balance between effector and memory cell differentiation during T cell activation. Here, we analyse the role of the TRAF2-/NCK-interacting kinase (TNIK), a signaling molecule downstream of the tumor necrosis factor superfamily receptors such as CD27, in the regulation of CD8+ T cell fate during acute infection with lymphocytic choriomeningitis virus. Priming of CD8+ T cells induces a TNIK-dependent nuclear translocation of β-catenin with consecutive Wnt pathway activation. TNIK-deficiency during T cell activation results in enhanced differentiation towards effector cells, glycolysis and apoptosis. TNIK signaling enriches for memory precursors by favouring symmetric over asymmetric cell division. This enlarges the pool of memory CD8+ T cells and increases their capacity to expand after re-infection in serial re-transplantation experiments. These findings reveal that TNIK is an important regulator of effector and memory T cell differentiation and induces a population of stem cell-like memory T cells

Small organic compounds enhance antigen loading of class II major histocompatibility complex proteins by targeting the polymorphic P1 pocket

MHC molecules are a key element of the cellular immune response. Encoded by the major histocompatibility complex (MHC) they are a family of highly polymorphic peptide receptors presenting peptide antigens for the surveillance by T cells. We have shown that certain organic compounds can amplify immune responses by catalyzing the peptide-loading of human class II MHC molecules HLA-DR. Here we show now that they achieve this by interacting with a defined binding site of the HLA-DR peptide receptor. Screening of a compound library revealed a set of adamantane derivatives that strongly accelerated the peptide-loading rate. The effect was evident only for an allelic subset and strictly correlated with the presence of glycine at the dimorphic position ss86 of the HLA-DR molecule. The residue forms the floor of the conserved pocket P1, located in the peptide binding site of MHC molecule. Apparently, transient occupation of this pocket by the organic compound stabilizes the peptide-receptive conformation permitting rapid antigen loading. This interaction appeared restricted to the larger ss86G-pocket and allowed striking enhancements of T cell responses for antigens presented by these 'adamantyl-susceptible' MHC molecules. As catalysts of antigen-loading, compounds targeting P1 may be useful molecular tools to amplify the immune response. The observation, however, that the ligand repertoire can be affected through polymorphic sites form the outside may also imply that environmental factors could induce allergic or autoimmune reactions in an allele-selective manner

2680 Argx-110 Targeting CD70, in Combination with Azacitidine, Shows Favorable Safety Profile and Promising Anti-Leukemia Activity in Newly Diagnosed AML Patients in an Ongoing Phase 1/2 Clinical Trial

Outcomes in elderly patients with acute myeloid leukemia (AML) are still adverse, as the majority does not qualify for intensive therapy or allogenic stem cell transplantation (ASCT). DNA hypomethylating agents (HMAs) induce remissions and prolong survival in a fraction of these patients. However, overall prognosis remains dismal and all patients progress due to therapy-resistant leukemia stem cells (LSCs). We recently demonstrated that HMAs upregulate the expression of CD70 on primary human AML LSCs, potentially contributing to HMA resistance and that blocking the cell-autonomous CD70/CD27 signaling inhibits proliferation and myeloid differentiation of LSCs and contributes to HMA resistance. Consequently, combining HMA treatment with a blocking αCD70 monoclonal antibody potently reduced colony formation of AML LSCs in vitro and effectively eliminated human AML LCSs in xenograft experiments. Based on these results, we initiated an open-label, non-controlled, non-randomized Phase 1/2 trial combining the HMA azacitidine (AZA) with ARGX-110, a human monoclonal antibody targeting CD70, in newly diagnosed AML patients unfit for intensive chemotherapy (ARGX-110-1601, NCT03030612)