Targeting the Glyco-Antigen CD75s with the Tetravalent, Fc-Engineered Antibody 'Ebu-141 Tetra' Induces Potent Killing of B Cell Lymphoma and Plasma Cell Tumors

While monoclonal antibodies (MoAb) are already well established for the treatment of B cell-derived malignancies and usually show a good safety profile, not all patients benefit and relapses may be a problem. In order to identify novel surface structures suitable for antibody-based therapies and to improve killing mechanisms, 'EBU-141 Tetra' was developed.

The parental MoAb EBU-141 is of mouse IgMk isotype, was generated in our laboratory and recognizes the glyco-antigen CD75s (previously CDw75), which are α-2,6-sialylated lactosamines on cell surface glycoproteins and glycolipids. CD75s was found on normal mature B cells and subsets of T cells, but is also expressed on certain lymphatic and solid tumors, e.g. mature B cell lymphoma, pancreatic and prostate cancer cells. EBU-141 specifically binds to CD75s on most B cell lymphoma, including Burkitt's lymphoma, FL, DLBCL, MCL, CLL, and interestingly plasma cell tumors. In addition, EBU-141 showed reactivity on a few cases of peripheral T-cell lymphoma, whereas classical Hodgkin lymphomas were consistently negative. Previously a chimeric IgG1k antibody, chEBU-141, was derived from EBU-141. Compared to the parental IgM antibody, chEBU-141 showed strongly reduced binding avidity, but was moderately effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) of mature B cell lymphoma and malignant plasma cells via recruitment of NK cells. However, chEBU-141 lacked the potent complement-dependent cytotoxicity (CDC) observed with the parental EBU-141 antibody. The aim of this study was to generate a tetravalent binding, Fc-engineered chEBU-141 IgG1 antibody with enhanced binding avidity for CD75s and potent effector functions for antibody-based therapy of mature B cell lymphomas and multiple myeloma.

Using the variable regions of EBU-141, the chimeric IgG1κ antibody with a protein-engineered Fc and tetravalent binding properties, named 'EBU-141 Tetra', was generated. This MoAb and relevant controls were produced by transient transfection of 293T cells and purified from cell culture supernatants by affinity chromatography. Direct anti-tumor effects and Fc-mediated modes of action were investigated in cell proliferation assays and chromium release experiments using lymphoma and myeloma cell lines. Peripheral blood mononuclear cells and serum of healthy donors were used as source of human effector cells and complement in the cytotoxicity experiments.

The 'EBU-141 Tetra' showed improved binding to CD75s on cell surface of mature B cell lymphoma as well as myeloma plasma cells compared to the bivalent binding chEBU-141 IgG1. The higher avidity for CD75s resulted in markedly improved ADCC activity of the 'EBU-141 Tetra' against Daudi Burkitt's lymphoma and U266 plasma cells with EC₅₀ values in the picomolar range and higher maximum lysis rates. In addition, the 'EBU-141 Tetra' regained CDC activity of the parental EBU-141 and demonstrated efficient killing of Burkitt's lymphoma and myeloma cell lines with human serum as complement source. Thus, recruitment of immune effector cells and activation of the complement system are the main modes of action of the novel, tetravalent, chimeric, Fc-engineered antibody 'EBU-141 Tetra' antibody.

Our findings further demonstrate that highly potent IgG-like antibodies against glycan-structures can be generated from mouse IgM antibodies and may open a new therapeutic window for therapy of patients with mature B cell lymphomas and multiple myeloma.