Anti-cd3 / Anti-pgp The Diabody Transformation And Activity

A major issue in the treatment of cancer in terms of a poor response or relapse is the development of multidrug resistance(MDR) by the tumor cells. Pgp,encoded by the MDR1 gene,is a 170 kDa transporter consisting of 1280 amino acids that is located in the plasma membrane and is responsible for cancer resistance to multiple chemotherapeutic agents.The level of Pgp expression is an adverse prognostic factor for complete remission and survival in malignant diseases.Pgp may therefore act as a potential therapeutic target for cancer intervention.Immunotherapy with bispecific antibodies(BiAbs) is a very promising approach for targeting to tumors.T cells play a pivotal role acting against tumors by directly eliminating the tumor cells through the formation of cytotoxic T cell-tumor cell synapses.However,the complexity of T cell recognition offers a variety of strategies for tumor cells to evade specific T cell recognition.Anti-CD3/anti-TAA(tumor associated antigen) BiAbs can possess two specificities,one directed at the T cell and the other at the cancer cell.This enables them to serve as mediators between T cells and cancer cells,bypassing the conventional T cell recognition process. Bispecific diabodies are the smallest BiAbs with about one-third the size of IgG.Bispecific diabodies are dimers,one chain comprising a V_H domain from antibody A and a V_L domain from antibody B,connected by a short peptide linker,and vice versa.The linker is too short to allow pairing between domains of the same chain,thus driving the pairing between complementary domains on different chains and forming two antigen binding sites that point away from each other.In addition the relatively small size of diabodies(55 kDa) facilitates penetration into solid tumors as compared to larger whole antibodies.Diabodies lack Fc domains thus eliminating the undesirable side-effects they have in immunotherapy.Further,diabodies can be readily produced by secretion from bacteria at a yield of up to 1g/L,thus possessing a considerable potential for application in a clinical setting.A critical and important factor contributing to the therapeutic effect of recombinant antibodies is stability.The two chains of diabodies are associated non-covalently and are therefore capable of dissociation.By introducing a disulphide bond into the recombinant Fv fragment,between two conserved framework residues,a significant improvement in the antibody stability is achieved.The disulphide bond locks the two peptide chains covalently while retaining full or even improved antigen binding activity.The disulphide bond stabilized Fv fragment or BsAbs may thus have the same,or even higher antitumor activity,compared to its non-stabilized counterpart.In our previous study it was suggested that an anti-Pgp/anti-CD3 diabody might be an effective agent in the treatment of MDR tumors. However,the diabody contains an Etag peptide,which can lead to immunogenicity,the poor stability of the diabody limits the antitumor response and reduces its effectiveness in cancer therapy.The required dose of anti-Pgp/anti-CD3 diabody was relatively high and a rapid tumor relapse occurred only one week after therapy.In this study,we generated an anti-Pgp/anti-CD3 diabody without Etag by the technology of gene egineering,which retain the full binding activity. It was reported that ARA-C can induce CD80 or CD86 expression in tumor cells.We used diabody in combination with ARA-C to inhibit MDR xenografts, and achieve the better therapeutic effect.During the observation window, no tumor was detected.To enhance the stability,We introduced cysteine residues into the CD3(dsCD3-Diabody) or Pgp(dsPgp-Diabody) V-domain to covalently lock the two chains together.The dsPpg-diabody failed to form disulphide bond properly.The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly.Compared with the parent diabody,the dsCD3-Diabody obtained was more stable in human serum at 37℃,without loss of affinity or cytotoxicity activity in vitro. Furthermore,the dsCD3-Diabody showed improved tumor localization and a two-fold improved antitumor activity over the parent diabody in nude mice bearing Pgp-overexpressing K562/A02 xenografts.