4-1bbl / Anti-cd20 Fusion Protein To Enhance The Levels Of Cd3 Anti-/ Anti-cd20 Bispecific Antibody Cytotoxicity Mechanism

The three main drawbacks in conventional chemotherapy and radiotherapy of cancer are the lack of specificity of the currently available therapeutic agents, multidrug resistance of tumor cells and tumor micrometastases. So, antibodies represent a unique class of new therapeutics owing to their high specificity for a defined antigen. Recent clinical success with antibody-based therapeutics has led to an upsurge in the development of these agents. Bispecific antibodies, with one binding arm to surface markers on tumor cells and the other to molecules on effector cells, represent an alternative approach to the conventional antibody-based cancer therapeuticsCell-mediated responses play a central role in antitumor immunity. The aim of tumor immunotherapy has been to generate long-lasting functionally active CD8+ T cells specific for the tumor cells. In order to activate a naive T cell, two signals are thought to be requisite.The primary signal or signal one occurs through the TCR:MHC:Ag (antigen) complex while the second signal or signal two is provided through costimulation. If costimulatory signal was not served to activated T lymphocyte, T cell may lead to activation induced cell death (AICD) or annergy which finally impair antitumor immunity. Tumor cells often expressed low level of costimulatory molecules to provide insufficient signals to antigen presenting cells or lymphocytes, hence to escape immunosurveilence. Such kind of tumors usually manifestated poor therapeutic outcomes and prognosis.Therefore, costimulatory pathway in T cell activation and proliferation plays an important role. CD28/B7 pathway is one of the most importment costimulatory pathways that promote the naive T cell activation. However, a number of molecules have been identified which function to further enhance and extend the activation of T cells. These include the more recently described 4-1BB/4-1BB ligand (4-1BBL) molecules that regulate T cell activation by means of increasing cell proliferation, prolonging cell survival, even increase the intracellular storage of perforin and granule enzyme. All of these characters provide a new target in cancer immunotherapy.The CD20 antigen is an attractive target for specific treatment of B-cell lymphoma as it is expressed in pre-B cells, mature B cells and more than 95% of B-cell lymphoma. To specifically target B-cell lymphoma via the TAA CD20, we before engineered a bispecific diabody recognizing the CD3-TCR complex, as well as CD20 (anti-CD3/anti-CD20 diabody) and constructed the human extracellular domain of 4-1BBL(ex4-1BBL). They can be used not only to redirect preactivated cytotoxic T cells toward the tumor, but, moreover, are able to stimulate resting or even anergic T cells if sufficient costimulatory signaling (for example, via the 4-1BB/4-1BBL pathway) is provided. Artificial signaling via the CD3 antigen mimicks the physiological antigen-specific activation of T lymphocytes by MHC-bound antigen. On the other hand. The simultaneous use of CD3 and 4-1BB monoclonal antibodies may, thus, substitute for the T-cell stimulatory capacity of professional antigen-presenting cells. However, to avoid a systemic T-cell activation that may result in undesirable clinical side effects, the CD3/4-1BB signaling has to be localized strictly to the tumor site. To provide tumor-specific 4-1BB costimulation, here we constructed and produced a recombinant human 4-1BB ligand (4-1BBL) /anti-CD20 fusion protein and examined its antitumor activity, alone and in combination with an anti-CD3/anti-CD20 bispecific diabody. The 4-1BBL/anti-CD20 fusion protein retained both the costimulatory activity of 4-1BBL on T cells and the tumor-targeting ability of CD20 antibody on B cells. The fusion protein bound as efficiently to 4-1BB- and CD20-positive cells as its respective parental antibodies, and was capable of cross-linking human T lymphocytes and CD20-positive tumor cells. We continued to identify that combination treatment with the 4-1BBL/anti-CD20 fusion protein and the anti-CD3/anti-CD20 diabody led to significantly increased T cell cytotoxicity to human B lymphoma cells in vitro and drastically more potent tumor inhibitory activity in vivo in xenografted B-cell lymphoma in SCID mice. Mechanistic studies revealed that the combination treatment remarkably inhibited apoptosis of human peripheral blood lymphocytes, accompanied by up-regulation of Bcl-xL and Bf1-1, perforin and granzyme B mRNA and increased IL-2 production.Our results demonstrated that 4-1BBL/CD20, as a targeted immunoadjuvant, can modulate the PBL activation and enhance the outcomes of PBL-based antitumor biotherapy. The combined administration of 4-1BBL/CD20 and diabody could strongly potentiate the antitumor activity of the diabody, thus may have significant clinical application in the treatment of human CD20-positive B cell malignancies.