.4-1bbl Membrane Protein And Tetravalent Anti-cd3 / Anti-drug-resistant Tumor Effects Of Anti-pgp Bispecific Antibody

It is a promising application to treat P-glycoprotein(Pgp) -overexpressing multidrug resistance(MDR) tumors with anti-CD3/anti-Pgp diabody.As the antitumor effect relies on the cytotoxic T lymphocyte(CTL) derived from peripheral blood lymphocytes(PBL),therefore,strategy for promoting T cell activation can further enhance the antitumor efficacy of PBL.This study includes the following aspects:1.Promoting PBL activation with soluble human 4-1BBL4-1BBL/4-1BB signal,as a key costimulation pathway,can help to achieve optimal cellular immune response.The mechanisms involve in increasing cell proliferation, prolonging cell survival,even increasing the intracellular storage of perforin and granule enzyme.This study aimed to regulate the antitumor response with extracellular domain of human 4-1BBL(ex4-1BBL),a soluble protein produced by prokaryotic expression system.Immunohistochemistry analysis,PI staining and radio- immunoassay were used to determine the binding specificity of ex4-1BBL to 4-1BB receptor,reduction of Jurkat cell apoptosis and increased secretion of IL-2.Furthermore,we identified that ex4-1BBL could influence PBL to enhance proliferation(191.66%vs 146.67%,P＜0.05),increase IL-2 secretion(54.26±5.92 pg/ml vs 22.57±6.53 pg/ml,P＜0.05),reduce LDH release and improve cell survival status.The cytotoxicity of PBL regulated by ex4-1BBL combined with anti-CD3/anti-Pgp diabody or anti-CD3/anti-CD20 diabody was measured.Results showed increased cytotoxicity on their targtet cells(K562/A02 or Raji) at different E:T ratios.Further study in vivo was performed to observe the antitumor immunity of PBL against multidrug resistant K562/A02 xenografts in nude mice.Tumor growth was drmatically inhibited by the combined therapy of ex4-1BBL and anti-CD3/anti-Pgp,and no tumor relapse was observed again even 100 days after treatment initiation.Our results demonstrated that ex4-1BBL,as an immunoadjuvant,could upregulate the PBL activation and improve the outcomes of PBL-based antitumor biotherapy. Ex4-1BBL may become a new agent with extensive application for bioimmunotherapy.2.Enhanced efficacy of PBL by constructing tetravalent anti-CD3/anti-Pgp diabodyTetravalent diabody can obtain increased molecule weight and binding domains by reconstructing bifunctional antibody(also named bispecific antibody,BsAb),so it can prolong half life time,provide potent binding and extend the interactivation indirectly. Moreover,increased binding valency to CD3 molecules on T cell can promote T cell activation and benefit antitumor response.To increase therapeutic potential of bispecific antibodies,we constructed multiforms of tetravalent diabodies and expressed them in E.coli cells.To increase the yield of the tetravalent fusion protein,the conditions for protein expression,such as temperature and duration for expression, components of medium,even the expression vector,were optimized.The increased yield may provide enough protein for further study in the future.