Targeting a cancer-specific LYPD3 glycoform for tumor therapy

Introduction: One of the most drastic changes in cancer is the altered glycosylation of proteins and lipids, giving rise to truncated O-glycans like the Thomsen Friedenreich (TF) or Thomsen nouvelle (Tn) antigen, which are almost absent on normal cells. Combined protein-carbohydrate epitopes comprising these specific glycans are ideal candidates for potent targeted therapies given their excellent tumor specificity and broad cancer expression.Methods and results: We have generated GT-002, a monoclonal antibody specifically targeting the epithelial glycoprotein LYPD3 only in the presence of a TF glycosylation. It does not cross-react with non-glycosylated LYPD3 or TF on other glycoproteins in ELISA and flow cytometry. GT-002 binds to various tumor cell lines and stains tumor tissues of different cancer indications including squamous cell carcinoma of the head and neck. The remarkable tumor specificity was confirmed in an immunohistochemistry study on a normal human tissue panel including several LYPD3-positive organs, where GT-002 elicited almost completely abolished normal tissue binding. Consequently, we observed markedly reduced binding of GT-002 to normal human tissues compared to Lupartumab, a conventional anti-LYPD3 antibody previously in clinical development as antibody-drug conjugate (BAY1129980). Neuraminidase treatment of healthy tissues, resulting in cleavage of sialic acid residues, re-established binding of GT-002 comparable to Lupartumab, showing that the GT-002 epitope is masked by sialic acid in normal cells.Discussion: We believe that GT-002 is a promising candidate for development of antibody-drug- and radio-conjugates as well as bispecific molecules and chimeric antigen receptor therapeutics and highlights the powerful potential of antibodies against combined protein-carbohydrate epitopes to reduce on-target/off-tumor cytotoxicity

Development and characterization of miscellaneous novel bi-specific antibodies based on PankoMab-GEX™ for improvement of tumor therapy

T-Zellen besitzen zahlreiche wertvolle Eigenschaften für die Tumortherapie. Mit bis zu 80% bilden die verschiedenen T-Zell Subpopulationen den Hauptteil der Lymphozyten im peripheren Blut und zeichnen sich zusätzlich durch eine starke Proliferation in Folge einer Aktivierung und einer hohen Zytotoxizität aus. Eine Möglichkeit T-Zellen für die Tumortherapie gezielt zu nutzen, stellen T-Zell-rekrutierende bispezifische Antikörper dar, wobei die therapeutische Anwendung häufig von hohen Nebenwirkungen aufgrund von Zytokinsekretion der T-Zellen, aber auch aufgrund der unzureichenden Selektivität der Expression des Zielantigens begleitet wird. So zeichnen sich die meisten der aktuell verwendeten Tumorantigene nur durch eine erhöhte Expression auf Tumorzellen aus, werden aber auch auf zahlreichen Normal- geweben exprimiert. Um die Anwendbarkeit von T-Zell-rekrutierenden Antikörpern weiter zu verbessern, sind daher Tumorantigene von höchstem Interesse, die keine Expression auf Normalgeweben zeigen. Ein solches Antigen ist TA-MUC1, das nur auf Tumorzellen für Antikörper zugänglich ist. Das Ziel der Arbeit bestand in der Produktion und Charakterisierung von verschiedenen, gegen TA- MUC1 gerichteten, T-Zell-rekrutierenden Antikörpern, um zu untersuchen, ob TA- MUC1 ein geeignetes Antigen für die Rekrutierung von T-Zellen darstellt. Als Grundlage für die bispezifischen Antikörper wurden zunächst verschiedene gegen CD3 gerichtete monoklonale Antikörper hinsichtlich ihrer T-Zell-Bindung und -Aktivierung untersucht und die vielversprechendsten Kandidaten für die Expression als bispezifische Antikörper ausgewählt. Dabei konnten auch die bispezifischen Antikörper mit hohen Produktivitäten in GlycoExpress® herstellt und durch die Etablierung geeigneter Aufreinigungsschritte erfolgreich isoliert werden. Funktionell wurden zunächst drei Konstrukte verglichen, bei denen verschiedene anti-CD3 scFv an die Cκ-Domäne eines IgG1 fusioniert wurden. Trotz vergleichbarer Bindung zeigten die scFv-Domänen Unterschiede in der vermittelten T-Zell-Aktivierung und induzierten Zytokinfreisetzung in unterschiedlichem Maße, wobei scFvOkt3.2 sich durch eine verringerte Tumorzell-unabhängige Zytokinfreisetzung und T-Zell-Aktivierung auszeichnete. Um einen Einfluss des gewählten Formates zu untersuchen, wurden anschließend zwei Konstrukte detaillierter funktionell analysiert, bei denen scFvOkt3.2 an die Cκ- oder an die CH3-Domäne fusioniert wurde. Dabei führte die Fusion an die CH3-Domäne zu den besten funktionellen Ergebnissen. So vermittelte dieses Konstrukt eine im Vergleich verbesserte CD3-Bindung, was zu einer starken Aktivierung und Proliferation von T-Zellen sowie zu einer starken zytotoxischen Wirkung gegen TA-MUC1-positive Tumorzellen führte. Überraschend war jedoch das Ergebnis, dass trotz der verbesserten CD3-Bindung eine verbesserte Tumorzell-unabhängige Aktivierung und Costimulation durch das Cκ- Konstrukt vermittelt wurde. Zusammenfassend konnte in dieser Arbeit die Eignung von TA-MUC1 als vielversprechendes Tumorantigen für T-Zell- rekrutierenden Antikörper gezeigt werden. Die untersuchten PM-CD3-Konstrukte vermittelten in vitro alle wichtigen Funktions-weisen, wie Aktivierung und Proliferation von T-Zellen sowie Zytotoxizität. Für eine klinische Anwendung sind allerdings noch weiterführende sicherheitsrelevante Untersuchungen nötig.T cells possess several valuable properties for tumor therapy. With up to 80% different T cell subsets account for the majority of the lymphocyte population within the peripheral blood, which proliferate strongly and are highly cytotoxic after activation. One opportunity to use T cells for tumor therapy is the development of T cell recruiting bi-specific antibodies. However, the therapeutic application of those antibodies is often accompanied by severe adverse events that mainly depend on cytokine release by T cells as well as the non-selective expression of targeted tumor antigen. Most tumor antigens in clinical use are characterized by upregulated expression on tumor cells but show broad expression on normal healthy tissue as well. To reduce effects on healthy cells antigens that are exclusively expressed on tumor cells are of highest interest. One such promising tumor antigen is TA-MUC1. During this study different T cell recruiting bi-specific antibodies that target TA-MUC1 were produced and characterized to analyze if TA-MUC1 is a suitable antigen for T cell recruitment. Four different anti-CD3 antibodies were analyzed regarding T cell binding and activation to choose the most promising candidates for expression as bi-specific antibodies. As a first step three constructs with three different anti-CD3 scFv’s fused to the Cκ domain of an IgG1 were compared in functional assays. Despite comparable CD3 binding the scFv’s showed clear differences in T cell activation and cytokine release. The antibody construct containing scFvOkt3.2 induced less target cell independent cytokine release and T cell activation compared to the other constructs. Following the analysis of the different scFv’s two constructs, scFvOkt3.2 fused to the Cκ or CH3 domain, were characterized in more detail to analyze a potential influence of the chosen construct format. The subsequent functional tests showed best results for the CH3 fusion. This construct mediated improved CD3 binding leading to enhanced T cell activation and proliferation as well as to increased cytotoxicity against TA-MUC1 positive tumor cells. Surprisingly stronger target cell independent activation and costimulation was mediated by the Cκ construct. In summary this study shows that TA-MUC1 is a valuable tumor antigen for T cell recruiting bi-specific antibodies. The analyzed PM-CD3 constructs are able to induce in vitro T cell activation and proliferation as well as T cell dependent cytotoxicity. However, for clinical application additional assays regarding the safety profile are necessary

CAR-Ts redirected against the Thomsen-Friedenreich antigen CD176 mediate specific elimination of malignant cells from leukemia and solid tumors

This research was in part funded by: "From CARs to TRUCKs: Induction of a concerted anti-tumor immune response by engineered T cells" (Deutsche Krebshilfe/German Cancer Aid-Priority Program in Translational Oncology #111975), "The Thomsen-Friedenreich antigen CD176: New target of chimeric antigen receptor (CAR)-modified immune cells in adoptive cancer immunotherapy" (Deutsche Kinderkrebsstiftung, Projekt DKS 2020.17), and Glycotope GmbH. AcknowledgmentsIntroduction: Chimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "on-target/off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts. Methods: Using the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2 generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8 T cells upon co-cultivation with CD176 tumor cell lines as models for CD176 blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity. Results: Specific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-κB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176 cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-γ, granzyme B and perforin), mediated cytotoxicity against CD176 cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176 control cells, their response proved to be target-specific. Discussion: Genetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity