Construction And Expression Of AntiCD3×antiCD19 Bispecific Antibody And Its Disulphide Stabilized Configuration And Analyzing Their Biological Activities

Background and Objective B cell leukemias and malignant lymphomas represent a heterogeneous group of hematological malignancies occurring in blood, lymph nodes and bone marrow, which frequently disseminate throughout the body. The most common forms of non-Hodgkin's lymphoma (NHL) are derived from the B cell lineage. Although NHL can be treated with reasonable success at early and intermediate stages, the results of conventional chemotherapy and radiation in advanced stages remain disappointing. This holds particularly true for the prevalent low-grade lymphomas such as chronic lymphatic leukemia and mantle cell. Although immunotherapy with antibodies was successfully applied in low grade and high grade B-NHL, for their specialities to target of antigen, it must be improved especially for some poorly responding subtypes, such as B-CLL. Bispecific antibodies (bsAbs) are a promising variant of these therapies. BsAbs are subtype of genetic engineering antibodies which are dimers, one chain comprising a VH domain from antibody A and a VL domain from antibody B, connected by a short peptide linker. So they possess of dual binding specificity, which one binding tumor antigens or tumor-associated antigens on malignant cells and the other binding activating receptors on immune effectors, recruiting the latter to the tumor cells. In terms of B-NHL, antibodies against B-lymphocyte dfferentiation antigens are playing an increasing role. Among them CD 19 holds an eminent place as a functional receptor molecule on B lymphocytes. With the important exception of stem cells, it is expressed by virtually all developmental stages of B-cell lineage, including malignant tumor derived from B lymphocytes. CD3 are expressed on the T lymphocytes and NK cells. In this experiment, I have constructed the antiCD3 X antiCD19 bsAbs vector of which poducts are capable of redirecting cytotoxic T lymphocytes or NK cells towards malignant cells that express tumour-associated antigens such as CD 19. To improve bsAbs stability I have introduced cysteine residues into the V domains of CD3 to promote the disulphide crosslinking of the dimer. Then expressed these two vector in E.coli 16C9, purified by 6 X His-tag affinity chromatography and analyzed their biological activities in vitro and in vivo.Methods Utilizing overlap PCR and PCR to construct the antiCD3 X antiCD19 expressing vector pAYZCD3CD19. Then introduced cysteine residues into the VL-100 and VH-44 domains of CD3 and built the expressing vector pAYZdsCD3CD19 of disulphide crosslinking of antiCD3 X antiCD19 also with overlap PCR and PCR. Transformed these two vectors into E.coli 16C9 for expressing, respectively and purified the Diabody or ds-Diabody by 6×His-tag affinity chromatography and confirmed the products by 12%SDS-PAGE and western-blot. Antigen binding activity of the Diabody or ds-Diabody to Jurkat cells which are CD3 positive or Raji cells which are CD 19 positive were analyzed by indirect immunological fluorescent assays or competitive immunological fluorescent assays with FACS. Diabody or ds-Diabody were incubated at 37℃for various times in PBS containing 0.2%(w/v) human serum albumin (HSA) at a concentration of 20μg/mL. Comparing the remaining binding activity with Jurkat or Raji cells (1×106 cells) which were determined by flow cytometry.A non-radioactive cytotoxicity assay was performed to evaluate the efficacy of the Diabody or ds-Diabody in mediating T-cell cytotoxicity by fluorescent dyes Calcein-AM. Human peripheral blood lymphocytes (PBL) were isolated using Ficoll-Hypaque density-gradient centrifugation from heparinized blood of healthy volunteers and depleted of monocytes by adherence to plastic flasks. Then T cells, CD4+and CD8+T cells were selected by FACS. In vitro cytotoxity test was performed by using Raji cells as targets and T cells, CD4+and CD8+T cells as effectors firstly. The cytotoxity of effectors mediated by Diabody or ds-Diabody in different concentration and E/T ratio was compared to PBS, antiCD3scFv+antiCD19scFv and antiCD3/antiPgp. Additionally CD 19 negtive cells k562 was set up as unrelated target. The secretion of IL-2 and IL-4 by activated T cells in groups of E/T 25:1, concentration of antibody 500ng/ml were analyzed by ELISA. Also releasing of perform and granzyme A, expressing the membrane molecular CD25 and CD69 on activated T cells of the same group, and apotosis of these T cells were analyzed by qPCR or FACS respectively. Female BALB/C nude mice were inoculated subcutaneously with Raji cells to establish the xenograft model. Mice were treated by intravenous injections of three different doses of ds-Diabody or Diabody or PBS, antiCD3scFv+antiCD19scFv and antiCD3/antiPgp combined with pre-activated PBL in PBS via the tail vein, every week for 3 weeks. Observing toxity of the antibody and evaluating the inhibition of xenograft.The expression of CD80 or CD86 on Nalm6 cell which were stimulated by Ara-C in different concentration for 72h were evaluated by FACS. Furthermore, the inhibition of growth of Nalm6 cells by Ara-C in different concentration was analyzed by MTT. Determing the concentration of Ara-C which can stimulate the CD80 or CD86 increasing while having no effect on growth. Then combing Ara-C at the concentration and Diabody or ds-Diabody to do non-radioactive cytotoxicity assay samely with Raji cells. Result the DNA sequence of the expression vector containing antiCD3×antiCD 19 diabody or disulphide stabilized diabody were correct. The yield of purified diabody or ds-diabody was up to 5mg/L. There were two bands of Diabody at molecular weight 26kD and 28kD indicated by SDS-PAGE and one band at 28kD by western-blot because the diabody was separated into two segments in SDS-PAGE. While there was only one band at 45kD of ds-Diabody in the non-reducing SDS-PAGE and two in the reduing conditions which were located at 26kD and 28kD respectively. The Diabody and ds-Diabody can specifically bind to CD 19+Raji cells and CD3+Jurkat cells while competitively inhibit binding of HIT3a and HIT 19a to such cells. The ds-Diabody was much more stable than Diabody evaluated by flow cytometry when incubatied in PBS containing 0.2%(w/v) HSA at 37℃for prolonged periods of time.Diabody and ds-Diabody increased the cytotoxity of T cells in vitro apparently than the contrast groups (p<0.05).So the secretion of IL-2 and IL-4, the release of perform and Granzyme A, CD25 and CD69 expressed on activated T cells were increased much more than the other groups (p<0.05). While apotosis of activated T cells mediated by Diabody or ds-Diabody reduced greatly. Furthermore, the Diabody and ds-Diabody showed improved tumor inhibiting activities (p<0.05) and ds-Diabody had a two-fold improved antitumor activity over the diabody in nude mice bearing Raji xenografts.Ara-C in final concentration of 0.25μmol/L which was incapable of inhibiting the growth of Nalm6 cells increased the expression of CD80 (B7.1) on Nalm6 cells but no effect on CD86 (B7.2). The cytotoxity of T cells mediated by Diabody and ds-Diabody associated with Ara-C raised apparently than the Diabody and ds-Diabody alone (p< 0.05). The result was the same in the secretion of IL-2 and IL-4, the release of perforin and Granzyme A, CD25 and CD69 expressed on the activated T cells. Apotosis of T cells activated by Diabody or ds-Diabody with Nalm6(Ara-C) were more reduced than Nalm6 without Ara-C (p<0.05).Conclusion Successfully constructed the expression vector of antiCD3×antiCD19 diabody and its disulphide stabilized configuration, acquired the purified diabody in high level yield with high specific affinity, Compared with the diabody, the ds-Diabody obtained was more stable in human serum at 37℃, without loss of affinity or cytotoxicity activity in vitro. Furthermore, ds-Diabody improved antitumor activity over the diabody in nude mice bearing Raji xenografts with a two-fold. Ara-C in final concentration of 0.25μmol/L which was incapable of inhibiting the growth of Nalm6 cells increased the expression of CD80 (B7.1) on Nalm6 cells. The cytotoxity of T cells mediated by Diabody or ds-Diabody associated with Ara-C was increased 20%than by Diabody or ds-Diabody alone.