Construction Of Recombinant BCG Secreting IL-2 And IFN-α 2a And Immunotherapeutic Effect On Mice With Bladder Cancer

BACKGROUD Bladder cancer is the predominant disease in male genitourinary system. Up to eighty percent of all bladder cancers are diagnosed as superficial bladder cancers(SBC) and Up to seventy percent of patients with SBC have some recurrence within five years of treatment. Once positively diagnosed and biopsied, many SBC can be surgically removed by a procedure known as a transurethral resection of bladder tumor(TURBT) and follow-up intravesical instillation therapy. One of the most potent immunotherapies presently used is the application of Bacillus Calmette Guerin (BCG) to treate carcinoma in situ (CIS) and prevent recurrences of SBC. Intravesical BCG therapy is considered the most successful immunotherapy to date. Not only is it superior to intravesical chemotherapy with regard to the SBC recurrence rate, but also it acts beneficially on the progression rate of this tumor. Despite its successful use, two major problems exist: a significant proportion of patients do not respond to BCG therapy and side-effects are common. These limit its use in clinical practice.Intravesical instillation of BCG causes a non-specific activation of the local cellular immune response. This response is T-lymphocyte dependent and is modulated by T helper 1 (Th1) like cytokines, primarily interleukin-2 (IL-2), interferon gamma (IFN- γ) and tumor necrosis factor alpha (TNF- α ). Therefore, current studies aim at developing recombinant BCG (rBCG) secreting Th1-like cytokines to further improve the effectiveness of the therapy. Recombinant BCG strains might be potentially useful agents to improve this type of immunotherapy.It is well known that IL-2 not only enhance T cell and NK cell activities to induce IFN- γ in the local cellular immune response but also activate macrophages to induce TNF- α and other cytotoxic molecules such as free radicals in the local cellular immune, the antitumor effects of IFN- α 2a are believed to be primarily direct. IFN-α 2a not only inhibits tumor proliferation and induce differentiation but also enhances the expression of tumor-associated antigens and MHC molecules and expand the activity of NK cells. IL-2 and IFN- α 2a have been demonstrated to have a pivotal, cooperative role in metastatic renal cell carcinoma recently. The combination ofrBCG secreting IL-2 with rBCG secreting IFN- a 2a are hopeful to improve the therapeutic outcome of blaader cancer in essence.OBJECTIVE The aim of our present study was to construct recombinant BCG secreting cytokine and investigate the specific IL-2 and IFN- a 2a express high, steadily in rBCG vaccine respectively during its development, differentiation and maturation. To detect and analyze the specific antitumor activity, immunogenicity, immunotherapeutic function and Immunological protection of rBCG vaccine both in vitro and in vivo. The synergistic effect of rBCGIL-2 and rBCGIFN- a 2a on the therapy for bladder cancer has been investigated. This study will provide the experimental evidences for IL-2 and IFN- a 2a and BCG Immunotherapy for bladder cancer.METHODS (1) Construction and Identification of Recombinant Shuttle plasmid. BCG Ag85B signal sequence was amplified from the genome of BCG by using polymerase chain reaction (PCR) and cloned in E. coli-BCG shuttle-vector pMV261 to get pMS. The cDNA fragment encoding human IL-2 and IFN- a 2a were amplified from the genome of human IL-2 and IFN- a 2a by using PCR and inserted into the shuttle expression vector pMV261 respectively. The recombinant plamid pMSIL-2 and pMSIFN- a 2a were identified by restriction endonuclease digestion, PCR amplification and nucleotide sequencing respectively. (2) construction of recombinant BCG secreting cytokine and immunogenicity from BALB/c mice have been studied. BCG was transformed with pMSIL-2 and pMSIFN- a 2a by eletroporation, and designated as rBCGIL-2 and rBCGIFN- a 2a respectively. The DNA and protein expressions of cytokine gene in rBCG were determined by PCR and Western blotting respectively. Mimicing the standard BCG instillation schedule, BALB/c mice were inoculated intravenously through the lateral tail vein 5x10 colony forming units (CFU) of rBCG strain comparing with lxlO6CFU of BCG and PBS weekly for 6 weeks. MTT assay was used to confirm the effect of rBCG on induction of macrophage activity and proliferation of T lymphocyte, the change of CD4+ and CD8+ subset were examined by flow cytometry, Thl cytokines (IL-2, IFN- y , TNF- a and Th2 cytokines (IL-4) in the culture supernatant of mice spleen lymphocyte were detected by an enzyme-linked immunosorbent assay (ELISA) after 2, 4 and 6 successive weekly BCG instillations. The difference between the combined immunotherapy group and single immunotherapy group was alsoinvestigated. (3) The effect of rBCG on suppression of Mice bladder cell BTT in vitro and in vivo. CCK-8 assay was used to testify the effect of rBCG on proliferation of BTT cell and EJ cell line in vitro. The immunotherapeutic effect of rBCG on mice with bladder cancer was assessed and immunological protection induced by rBCG was studied by the challenge of tumor cell injection followed the inoculation. The ability of inducing Thl cytokines in mice spleen lymphocyte was also detected.RESULTS (1) By partial nucleotide sequencing, the cDNA was consistent with the reported IL-2 and IFN- a 2a in the GeneBank, and then were inserted into the shuttle expression vector pMV261 to construct recombinant plasmid pMSIL-2 and pMSIFN- a 2a respectively. rBCGIL-2 and rBCGIFN- a 2a were successfully transformed into BCG by eletroporation and were capable of synthesizing and secreting cytokineIL-2 and IFN- a 2a respectively. During 6 weekly BCG strain and rBCG inoculation a time dependent induction of the T lymphocyte proliferation, CD subset, macrophage activity and cytokine (including IL-2, IL-4, TNF-aandlFN-y) was observed. At the different time points, there was significant difference between the combined rBCG therapy group and single rBCG therapy group. (2) rBCG significantly inhibited the growth and proliferation of BTT cell and EJ cell line in vitro. In the mice model with pre-established subcutaneous BTT bladder cancer, vaccination with rBCG could inhibit tumor growth, but the immunization of the combined rBCG inhibited the tumor growth most significantly when compared with single rBCG. The survival time of the mice treated with combined rBCG was also greatly extended. (3) Histological examination showed that the most obvious tumor necrosis and infiltration of inflammatory cells was present inside and around the tumors of the tumor-bearing mice immunized with combined rBCG, and the inflammatory cells infiltration was also observed in mice immunized with single rBCG and BCG. (4) The single rBCG immunized mice exhibited resistance to tumor challenge, but the inhibition of tumor growth could be observed more significantly in mice after vaccination with combined rBCG when compared with single rBCG. (5) Immunization of mice with single rBCG could increase the production of IL-2 and interferon-y, but the Thl lymphocyte from mice immunized with combined rBCG secreted a more high level of IL-2 and IFN-y.CONCLUSIONS (1) rBCGIL-2 and rBCGIFN- a 2a were constructed successfully...