Immunotherapy with the trifunctional anti-CD20 x anti-CD3 antibody FBTA05 (Lymphomun) in paediatric high-risk patients with recurrent CD20-positive B cell malignancies.

Children with B cell malignancies refractory to standard therapy are known to have a poor prognosis and very limited treatment options. Here, we report on the treatment and follow-up of ten patients diagnosed with relapsed or refractory mature B-cell Non Hodgkin Lymphoma (B-NHL), Burkitt leukaemia (B-AL) or pre B-acute lymphoblastic leukaemia (pre B-ALL). All children were treated with FBTA05 (now designated Lymphomun), an anti-CD3 x anti-CD20 trifunctional bispecific antibody (trAb) in compassionate use. Within individual treatment schedules, Lymphomun was applied (a) after allogeneic stem cell transplantation (allo-SCT, n = 6) to induce sustained long-term remission, or (b) stand alone prior to subsequent chemotherapy to eradicate residual disease before allo-SCT (n = 4). Nine of ten children displayed a clinical response: three stable diseases (SD), one partial remission (PR) and five induced or sustained complete remissions (CR). Five of these nine responders died during follow-up. The other patients still maintain CR with a current overall survival of 874-1424 days (median: 1150 days). In conclusion, despite the dismal clinical prognosis of children refractory to standard therapy, immunotherapy with Lymphomun resulted in a favourable clinical outcome in this cohort of refractory paediatric patients.

Fully human, HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells.

The Human Combinatorial Antibody Library (HuCAL) was screened for antibodies specific to human leukocyte antigen-DR (HLA-DR) that induce programmed death of lymphoma/leukemia cells expressing the target antigen. The active Fab fragments were affinity-matured, and engineered to IgG(4) antibodies of sub-nanomolar affinity. The antibodies exhibited potent in vitro tumoricidal activity on several lymphoma and leukemia cell lines and on chronic lymphocytic leukemia patient samples. They were also active in vivo in xenograft models of non-Hodgkin lymphoma. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms, and was selective to activated/tumor-transformed cells. Although the expression of HLA-DR on normal hematopoietic cells is a potential safety concern, the antibodies caused no long-lasting hematological toxicity in primates, in vivo. Such monoclonal antibodies offer the potential for a novel therapeutic approach to lymphoid malignancies.

Bi20 (fBTA05), a novel trifunctional bispecific antibody (anti-CD20 x anti-CD3), mediates efficient killing of B-cell lymphoma cells even with very low CD20 expression levels.

Trifunctional bispecific antibodies can efficiently mediate tumor cell killing by redirecting T cells and immune accessory cells to the tumor cell. Here, we describe the new trifunctional antibody, Bi20 (FBTA05, anti-CD20 x anti-CD3), that connects B cells and T cells via its variable regions and recruits FcgammaRI(+) accessory immune cells via its Fc region. Bi20 mediated efficient and specific lysis of B-cell lines and of B cells with low CD20 expression levels that were derived from CLL patients. Remarkably, T-cell activation and tumor cell killing occurred in an entirely autologous setting without additional effector cells in 5 of 8 samples. In comparison, rituximab, a chimeric monoclonal CD20 antibody, demonstrated a significantly lower B-cell eradication rate. Additionally, Bi20, but not rituximab, upregulated the activation markers CD25 and CD69 on both CD4(+) and CD8(+) T cells in the presence of accessory immune cells. CD14(+) accessory cells and the monocyte cell line THP-1 were activated via binding of the Fc region of Bi20, given that T cells were simultaneously engaged by the antibody. Bi20 induced a strong Th1 cytokine pattern characterized by high IFN-gamma and very low IL-4 secretion. In conclusion, Bi20 may offer new immunotherapeutic options for the treatment of B-cell lymphomas.

Rituximab and alemtuzumab induce a nonclassic, caspase-independent apoptotic pathway in B-lymphoid cell lines and in chronic lymphocytic leukemia cells.

The monoclonal antibodies (MoAbs) alemtuzumab (anti-CD52) and rituximab (anti-CD20) produce objective clinical responses in patients with chronic lymphocytic leukemia (CLL). However, their mechanisms of action are not fully understood. Therefore, we investigated the mechanisms of lymphoma and CLL cell killing by two anti-CD20 antibodies (rituximab, B1) and by alemtuzumab. All antibodies induced complement-independent cell death in B-lymphoid cell lines Raji, Ramos, and Mec-1. The efficiency of cell killing was increased by the addition of human complement in Raji but not Ramos cells. Both alemtuzumab and rituximab also killed freshly isolated CLL cells, with a much stronger response for alemtuzumab (from eight of eight patients) compared to rituximab (from two of six patients). Cell morphology and Western blot analyses revealed that the antibody-induced cell death lacked some typical features of apoptosis such as chromatin condensation or poly-ADP-ribose polymerase (PARP) cleavage. Taken together, the results suggest that the tumor killing activity of these MoAbs is not only mediated by complement-mediated cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC), but also by a nonclassic, caspase-independent apoptotic pathway.