Rational On Anti-tumor Mechanism Of Anti-CD20 Antibody/Flt3l Bispecific Fusion Protein

Object:CD20 is a pan-B cell marker expressed from pre-B cells until B cells are differentiated into plasma cells. It is present on >90% of B-cell lymphomas but not in stem cells, pro-B cells, normal plasma cells, or other normal tissues and is neither shed nor internalized after antibody binding, making it an ideal target for passive immunotherapy of B-cell lymphoma. Rituximab (Rituxan, IDEC-C2B8) is the first therapeutic monoclonal antibody (mAb) approved for the treatment of relapsed/refractory low-grade or follicular B-cell non–Hodgkin's lymphomas. Multiple mechanisms have been proposed for the action of Rituximab in the depletion of B cells including its ability to mediate complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, and to induce cell apoptosis. Despite the effectiveness of Rituximab, only 48% of patients respond to the treatment and complete responses are <10%. In addition, a significant number of patients have progressive disease during antibody therapy. There is an urgent need to develop more effective anti-CD20 molecules to further improve the efficacy of antibody therapy for B-cell lymphoma.Flt3 ligand (FL) is a member of a small family of growth factors that stimulate the proliferation of hematopoietic cells by binding to and activating distinct tyrosine kinase receptors. FL plays a major role in activating the immune system via its ability to stimulate the production of both dendritic and natural killer cells. Since both DCs and NK cells are key elements in induction of host immune responses, it is reasonable that systemic administration of Flt3L could induce antitumor immune responses that protect against tumor challenges and mediate the regression of established tumors. Administration of either soluble FL protein or tumor cells transfected with FL gene and expressing FL molecules on cell surfaces only cause transient tumor regression in immunogenic tumor models. These treated animals usually developed recurrent tumors after the termination of treatment. Recently, a promising alternative approach has been reported in which local administration of FL attracted NK cell and DCs into tumor tissues and caused the regression of subcutaneously growing tumors in combination with low dose radiotherapy. An ideal therapy for treatment of cancers should be effective both in destroying tumor cells and in the induction of antitumor immunity. To further determine this hypothesis, we developed two genetically engineered bifunctional fusion proteins (BiFPs), designated CD20scFvCH/FlexCL and FlexFc-CD20scFv. Methods:CD20scFvCH/FlexCL and FlexFc-CD20scFv were contructed. The in vitro and in vivo antitumor activity of BiFPs were examined and compared with the parental CD20 antibody and Flt3L.Results:The BiFPs were shown to be effective in mediating CDC and ADCC. The effect of CD20scFvCH/FlexCL was a little bit lower than that of parental CD20 antibody but significantly superior to that of FlexFc-CD20scFv. Both BiFPs could stimulate the bone marrow and promote the bone marrow hematopoiesis. In vivo study showed that BiFPs concentrated in CD20+ tumor tissue. Results from immunotherapeutic studies further revealed that CD20scFvCH/FlexCL was far more effective in inhibiting the tumor development of mice bearing CD20+ lymphomas than parental chimeric CD20 mAbs in combination FL. The mice developed a long-lasting anti-tumor immunity following CD20scFvCH/FlexCL treatment. The effect might be mediated through CD3+CD8+ T cell subpopulation. These results suggest that BiFP CD20scFvCH/FlexCL might be a promising therapeutic agent for the treatment of B-cell lymphoma.Conclusion:Provided herein is a chimeric protein that permits the simultaneous eradication of tumor cells and the stimulation of an effective anti-tumor immune response. Specifically, the chimeric protein comprises two components. The first component is a biologically active fragment of Flt3. Flt3 is a potent chemotactic molecule and activator for DCs and other anti-tumor effectors such as NK cells. The second component is a tumoricidal agent-anti-CD20 antibody that incude cell death through CDC. Thus, the chimeric protein reduces tumor burden by directly inducing the apoptosis of tumor cells while also targeting and activating DCs, and other antitumor effectors, e.g., NK cells, to infiltrate the tumor tissues. Tumor antigens released by the dying tumor cells then can be processed and presented by Flt3-activated DCs. The DCs then effectively serve as antigen-presenting cells for a specific anti-tumor immune response. Therefore, this chimeric protein simultaneously effects direct and indirect tumor cell elimination while eliciting an effective active immune response against the tumor cells that prevents the recurrence of tumor growth.