Search

Herein, we describe the generation of potent NK cell engagers (NKCEs) based on single domain antibodies (sdAbs) specific for NKp46 harboring the humanized Fab version of Cetuximab for tumor targeting. After immunization of camelids, a plethora of different VHH domains were retrieved by yeast surface display. Upon reformatting into Fc effector‐silenced NKCEs targeting NKp46 and EGFR in a strictly monovalent fashion, the resulting bispecific antibodies elicited potent NK cell‐mediated killing of EGFR‐overexpressing tumor cells with potencies (EC₅₀killing) in the picomolar range. This was further augmented via co‐engagement of Fcγ receptor IIIa (FcγRIIIa). Importantly, NKp46‐specific sdAbs enabled the construction of various NKCE formats with different geometries and valencies which displayed favorable biophysical and biochemical properties without further optimization. By this means, killing capacities were further improved significantly. Hence, NKp46‐specific sdAbs are versatile building blocks for the construction of different NKCE formats.

BACKGROUND: Acute myeloid leukemia (AML) is a fatal clonal hematopoietic malignancy, which results from the accumulation of several genetic aberrations in myeloid progenitor cells, with a worldwide 5-year survival prognosis of about 30%. Therefore, the development of more effective therapeutics with novel mode of action is urgently demanded. One common mutated gene in the AML is the DNA-methyltransferase DNMT3A whose function in the development and maintenance of AML is still unclear. To specifically target "undruggable" oncogenes, we initially invented an RNAi-based targeted therapy option that uses the internalization capacity of a colorectal cancer specific anti-EGFR-antibody bound to cationic protamine and the anionic siRNA. Here, we present a new experimental platform technology of molecular oncogene targeting in AML. METHODS: Our AML-targeting system consists of an internalizing anti-CD33-antibody-protamine conjugate, which together with anionic molecules such as siRNA or ibrutinib-Cy3.5 and cationic free protamine spontaneously assembles into vesicular nanocarriers in aqueous solution. These nanocarriers were analyzed concerning their physical properties and relevant characteristics in vitro in cell lines and in vivo in xenograft tumor models and patient-derived xenograft leukemia models with the aim to prepare them for translation into clinical application. RESULTS: The nanocarriers formed depend on a balanced electrostatic combination of the positively charged cationic protamine-conjugated anti-CD33 antibody, unbound cationic protamine and the anionic cargo. This nanocarrier transports its cargo safely into the AML target cells and has therapeutic activity against AML in vitro and in vivo. siRNAs directed specifically against two common mutated genes in the AML, the DNA-methyltransferase DNMT3A and FLT3-ITD lead to a reduction of clonal growth in vitro in AML cell lines and inhibit tumor growth in vivo in xenotransplanted cell lines. Moreover, oncogene knockdown of DNMT3A leads to increased survival of mice carrying leukemia patient-derived xenografts. Furthermore, an anionic derivative of the approved Bruton's kinase (BTK) inhibitor ibrutinib, ibrutinib-Cy3.5, is also transported by this nanocarrier into AML cells and decreases colony formation. CONCLUSIONS: We report important results toward innovative personalized, targeted treatment options via electrostatic nanocarrier therapy in AML., Journal of Hematology & Oncology, 15 (1), ISSN:1756-8722

Blockade of the CD47-SIRPα axis improves lymphoma cell killing by myeloid effector cells, which is an important effector mechanism for CD20 antibodies in vivo. The approved CD20 antibodies rituximab, ofatumumab, and obinutuzumab are of human immunoglobulin G1 (IgG1) isotype. We investigated the impact of the variable regions of these 3 CD20 antibodies when expressed as human IgA2 isotype variants. All 3 IgA2 antibodies mediated antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cellular cytotoxicity (ADCC) by polymorphonuclear cells. Both effector mechanisms were significantly enhanced in the presence of a CD47-blocking antibody or by glutaminyl cyclase inhibition to interfere with CD47-SIRPα interactions. Interestingly, an IgA2 variant of obinutuzumab (OBI-IgA2) was consistently more potent than an IgA2 variant of rituximab (RTX-IgA2) or an IgA2 variant of ofatumumab (OFA-IgA2) in triggering ADCC. Furthermore, we observed more effective direct tumor cell killing by OBI-IgA2 compared with RTX-IgA2 and OFA-IgA2, which was caspase independent and required a functional cytoskeleton. IgA2 variants of all 3 antibodies triggered complement-dependent cytotoxicity, with OBI-IgA2 being less effective than RTX-IgA2 and OFA-IgA2. When we investigated the therapeutic efficacy of the CD20 IgA2 antibodies in different in vivo models, OBI-IgA2 was therapeutically more effective than RTX-IgA2 or OFA-IgA2. In vivo efficacy required the presence of a functional IgA receptor on effector cells and was independent of complement activation or direct lymphoma cell killing. These data characterize the functional activities of human IgA2 antibodies against CD20, which were affected by the selection of the respective variable regions. OBI-IgA2 proved particularly effective in vitro and in vivo, which may be relevant in the context of CD47-SIRPα blockade.

Monoclonal antibodies directed against the CD20 surface antigen on B cells are widely used in the therapy of B cell malignancies. Upon administration, the antibodies bind to CD20 expressing B cells and induce their depletion via cell- and complement-dependent cytotoxicity or by induction of direct cell killing. The three antibodies currently most often used in the clinic are Rituximab (RTX), Ofatumumab (OFA) and Obinutuzumab (OBI). Even though these antibodies are all of the human IgG1 subclass, they have previously been described to vary considerably in the effector functions involved in therapeutic B cell depletion, especially in regards to complement activation. Whereas OFA is known to strongly induce complement-dependent cytotoxicity, OBI is described to be far less efficient. In contrast, the role of complement in RTX-induced B cell depletion is still under debate. Some of this dissent might come from the use of different in vitro systems for characterization of antibody effector functions. We therefore set out to systematically compare antibody as well as C1q binding and complement-activation by RTX, OFA and OBI on human B cell lines that differ in expression levels of CD20 and complement-regulatory proteins as well as human primary B cells. Applying real-time interaction analysis, we show that the overall strength of C1q binding to live target cells coated with antibodies positively correlated with the degree of bivalent binding for the antibodies to CD20. Kinetic analysis revealed that C1q exhibits two binding modes with distinct affinities and binding stabilities, with exact numbers varying both between antibodies and cell lines. Furthermore, complement-dependent cell killing by RTX and OBI was highly cell-line dependent, whereas the superior complement-dependent cytotoxicity by OFA was independent of the target B cells. All three antibodies were able to initiate deposition of C3b on the B cell surface, although to varying extent. This suggests that complement activation occurs but might not necessarily lead to induction of complement-dependent cytotoxicity. This activation could, however, initiate complement-dependent phagocytosis as an alternative mechanism of therapeutic B cell depletion.

BACKGROUND: Mast cell and basophil activation by antigen crosslinking of FcεRI-bound IgE is central to allergy pathogenesis. We previously demonstrated global suppression of this process by rapid desensitization with anti-FcεRIα mAbs. OBJECTIVE: Determine whether use of monovalent (mv) anti-FcεRIα mAbs increases desensitization safety without loss of efficacy. METHODS: mv anti-human (hu) FcεRIα mAbs were produced with mouse-derived immunoglobulin V regions and huIgG1 or huIgG4 C regions, and used to suppress murine IgE-mediated anaphylaxis and food allergy. mAbs were administered as a single dose or as serially increasing doses to mice that express hu instead of mouse FcεRIα, mice that additionally have an allergy-promoting IL-4Rα mutation, and hu cord blood-reconstituted immunodeficient, hu cytokine-secreting, mice that have large numbers of activated hu mast cells. Anaphylaxis susceptibility was sometimes increased by treatment with IL-4 or a β-adrenergic receptor antagonist. RESULTS: mv anti-huFcεRIα mAbs have considerably less ability than divalent mAbs to induce anaphylaxis and deplete mast cell and basophil IgE, but still strongly suppress IgE-mediated disease. The mv mAbs can be safely administered as a single large dose to mice with typical susceptibility to anaphylaxis, while a rapid desensitization approach safely suppresses disease in mice with increased susceptibility. Our huIgG4 variant of mv anti-huFcεRIα mAb is safer than our huIgG1 variant, apparently because reduced interactions with FcγRs decrease ability to indirectly crosslink FcεRI. CONCLUSION: mv anti-FcεRIα mAbs more safely suppress IgE-mediated anaphylaxis and food allergy than divalent variants of the same mAbs. These mv mAbs may be useful for suppression of huIgE-mediated disease.

Antibodies against epidermal growth factor receptor (EGFR) are used in therapy for solid cancers such as head and neck cancer, colorectal and non-small lung carcinoma. Though complement-dependent cytotoxicity (CDC) is thought to be an important mechanism involved in immunotherapy, single unmodified EGFR IgG1 antibodies fail to trigger efficient CDC. Here, we generated a CDC-optimized variant of the EGFR antibody matuzumab (H425-wt) by introducing amino acid substitutions K326A/E333A (H425-mt). This antibody was used to investigate the impact of complement activation on the capacity of effector cells such as mononuclear cells (MNC) and polymorphonuclear cells (PMN) to exert antibody-dependent cell-mediated cytotoxicity (ADCC). H425-mt but not H425-wt significantly induced complement deposition, release of anaphylatoxins and CDC against distinct tumor cell lines, while no differences in ADCC by isolated MNC or PMN were detected. Notably, compared to H425-wt stronger cytotoxicity was induced by H425-mt in whole blood assays and in experiments using MNC or PMN combined with serum. While MNC-ADCC was not affected by C5 cleavage, PMN’s cytotoxic activity in the presence of serum strongly depended on C5 cleavage, suggesting a direct interaction between complement and PMN-ADCC. PMN, which strongly express C5a receptors, were stimulated with C5a resulting in an up-regulation of activated complement receptor 3 (CR3) and an enhanced CR3-dependent PMN-ADCC against tumor cells. In conclusion, complement-optimized EGFR antibodies may constitute a promising strategy to improve tumor cell killing by enhancing the interaction between humoral and cellular effector functions in antibody-based tumor therapy.

Die Verwendung monoklonaler Antikörper stellt eine wichtige und erfolgreiche Option für die Diagnostik und Behandlung von Krebserkrankungen dar. Die Entwicklung neuer gentechnologischer Verfahren erlaubte Anfang der 1990er Jahre die Generierung genetisch vollständig humaner Antikörper. Neue Screening- und Engineering-Technologien ermöglichten darüber hinaus die Schaffung einer neuen Antikörpergeneration in einer Vielfalt an Formaten mit verbesserten Eigenschaften bei gleichzeitig reduzierter Immunogenität. Einen vielversprechenden Ansatz bilden die Generierung und der Einsatz bispezifischer Antikörper, welche zwei verschiedene Tumor-assoziierte Antigene binden können oder Antigene auf der Oberfläche von Tumorzellen und Effektorzellen erkennen. Ziel dieser Arbeit war die Charakterisierung von humanen monoklonalen Antikörpern gegen das Tumor-assoziierte Antigen CEA und das Effektorzellantigen NKG2D in verschiedenen mono- und bispezifischen Formaten hinsichtlich der Antigenbindung, des Aggregationsverhaltens und ihrer Funktionalität. Ausgehend von scFv-Fragmenten, die mittels Phagendisplay und Panning aus einer Antikörpergenbibliothek selektiert wurden, konnten auf Grundlage eines Kassetten-basierten Vektorsystems erfolgreich Antikörper in den Formaten scFv-Fc, IgG, scFv-Fc-scFv und IgG-scFv z.T. mit ADCC-optimiertem Fc-Teil generiert und produziert werden. Die Antikörper wiesen in den verschiedenen Formaten eine spezifische Antigenbindung auf. Darüber hinaus konnte bei einem der CEA-spezifischen Antikörper der Einfluss von Aviditätseffekten auf die Antigenbindung nachgewiesen werden. Die Aggregationsneigung variierte in Abhängigkeit vom Format stark. Die geringste Aggregationsneigung wiesen Antikörper im IgG-Format auf, wohingegen die Tendenz zur Bildung von Dimeren am stärksten im scFv-Fc-scFv-Format ausgeprägt war. Im Zytotoxizitätsassay konnte die Funktionalität der verschiedenen mono- und bispezifischen Konstrukte sowie eine Verstärkung der Zelllyse durch den ADCC-optimierten Fc-Teil nachgewiesen werden. Die Ergebnisse dieser Arbeit deuten auf eine grundlegende Eignung der analysierten mono- und bispezifischen Antikörper für die Weiterentwicklung im Zusammenhang mit therapeutischen Zwecken hin., The use of monoclonal antibodies represents an important and effective option for the diagnosis and therapy of cancer. During the nineties of the last century, the development of new genetic engineering methods allowed the generation of antibodies from fully human genes. Furthermore, new screening and engineering methods enabled the creation of a new antibody generation with a variety of formats, improved characteristics with simultaneously reduced immunogenicity. The generation and application of bispecific antibodies, that recognise two tumor associated antigens or a tumor associated antigen and an effector antigen, comprises a promising appoach for therapeutics. The aim of this work was the characterization of human monoclonal antibodies against the tumor associated antigen CEA and the effector antigen NKG2D in different mono- and bispecific formats with regards to antigen specificity, aggregation tendency and functionality. Based on scFv fragments that were selected from antibody gene libraries using phage display and panning, and by using a cassette based vector system, several antibodies were genereated in differend fomats and produced sucsessfully. The different formats comprised scFv-Fc, IgG, scFv-Fc-scFv und IgG-scFv antibodies partially including an ADCC optimized Fc part. The generated antibodies bound specifically to their antigen and one CEA specific antibody showed avidity effects during antigen binding. The aggregation tendency of the antibodies varied greatly depending on the used format. IgG antibodies showed the lowest aggregation tendency while scFv-Fc-scFv antibodies were more prone to dimerization. Several generated mono- and bispecific antibodies were functional in a cytotoxicity assay and enhanced cell lysis due to presence of the ADCC optimized Fc part. Taken together, the results indicate that the analyzed mono- and bispecific antibodies may be suitable for further developments towards therapeutic use.

Untersuchungen zu direkten und indirekten Effektormechanismen des Anti-ICAM-1-Antikörpers MSH-TP15 beim Multiplem Myelom.

Es ist bekannt, dass CD4+CD25+ regulatorische T-Zellen (Tregs) im Blut und Pankreasgewebe von Patienten mit duktalem Pankreasadenokarzinom (PDAC) erhöht sind und ihr Vorkommen mit einer schlechten Prognose korreliert. Das Adhäsionsmolekül L1CAM wird vermehrt im PDAC exprimiert ist ebenfalls mit einer ungünstigen Prognose verbunden. Die chronische Pankreatitis, bei der L1CAM im Vergleich zu Normalgewebe ebenfalls erhöht ist, gilt als Risikofaktor für die Entwicklung eines PDAC. Daraus ergab sich die Frage, ob L1CAM eine Rolle bei der Anreicherung von Tregs im PDAC spielt. CD4+ T-Effektorzellen und Tregs mit dem hoch immunsuppressiven Phänotyp CD4+CD25+CD49d-CD127- wurden aus dem Blut gesunder Spender isoliert und mit Pankreasgangepithelzelllinien cokultiviert. Die Pankreatitis und benigne Pankreasgangepithelzellen wurden durch die Zelllinie H6c7, das PDAC dagegen durch die maligne Zelllinie Panc1 simuliert. Beide Zelllinien wurden jeweils mit niedriger und hoher Expression von L1CAM eingesetzt. Dann wurden die T-Zellen bezüglich Phänotyp, Proliferation und Migration analysiert. Zudem wurden Prävalenzen unterschiedlicher T-Zell-Populationen im Blut und Gewebe von Patienten mit chronischer Pankreatitis und PDAC sowie im Blut gesunder Spender bestimmt und mit klinischen Parametern korreliert. Die Ergebnisse zeigen, dass L1CAM die Bildung einer neuen Subpopulation von Tregs mit einem CD4+CD69+CD25- Phänotyp und immunsuppressiver Potenz fördert. Der Phänotyp von CD4+CD25+CD49d-CD127- Tregs blieb unter gleichen Bedingungen stabil, wobei deren Migration in Anwesenheit von L1CAM im Vergleich zu T-Effektorzellen deutlich erhöht war. Zudem war die Proliferation von T-Effektorzellen nach Cokultur mit den L1CAM-exprimierenden Zelllinien erniedrigt. Neben seiner bekannten Bedeutung in der Vermittlung von Chemoresistenz und Migration bzw Invasion von Tumorzellen konnte in dieser Arbeit somit eine neue Rolle von L1CAM in der Tumorgenese des PDAC aufgezeigt werden.