Study Of Tumor Immunity Induced By Human MUC1 VNTR DNA Vaccine

Tumor DNA vaccination is a novel strategy for the immunotherapy oftumors, which targets the tumor associated antigen (TAA). It is constructed bya specific gene fragment encoding TAA and a mammalian expression vector.The expression TAA in the host cells can be up-taken and presented to immunesystem by APC, which results in the activation of T cells, and consequentlyelicits TAA specific antibody and CTL responses to tumor cells. Therefore, itis the key point for prevention and treatment of cancer to select an idealantigen coding gene and an effective immunal strategy for elicit anti-tumorresponses.Mucinl (MUC1) is a tumor associated antigen (TAA), which existmainly in breast, pancreas, ovary and other epithelial tissues and apparatus. Inadenocarcinomas, there is a highly and altered expression in MUC1, whichmake it be a targets in immunotherapy. MUC1 is a large glycoprotein, belonging to type 1 transmembrane protein composed of the extracellular, transmembrane and intracellular domains, containing a variable number(20-125) of tandem repeats (VNTR) of a 20 amino acid sequence in its extracellular domain. On the apical surface of the normal ductal epithelia,VNTRs are heavily glycosylated at threonine and serine residues, up to 70%carbohydrate by weight, while malignant cells contain underglycosylatedVNTR domains, and overexpressed in 90% of all adenocarcinomas includingbreast, lung, pancreas, prostate, stomach, colon, and ovary. Due to such alteredglycosylation, new epitopes appear on the cell surface that are absent innormal tissues. These VNTR-derived epitopes will be attractive targets inimmunotherapy. Peptide epitopes, containing the PDTR fragment from VNTRof MUC1 have been found to be immunodominant in T-cell and B-cellresponses. Therefore targeting MUC1 for cancer immunotherapy can exploitthe difference between cancer and normal cells, and eliminating the cancerouscells while leaving the normal mammary cells unharmed.There are currently many active immunotherapies targeting MUC1 underinvestigation, especially some of the protein vaccines have been in the clinicaltrial. Most of the work has focused on the use of a synthetic peptide, either byitself or conjugated to a carrier protein. However, peptide vaccine may havesome disadvantages that protein-based vaccines are heat sensitive and requirelow temperature conditions during manufacturing and storage to maintainefficacy. Whereas DNA vaccine is simple, stable and inexpensive, and providea safe and efficient alternative. The chemical nature of DNA contributes to theconvenience in manufacturing, storage, and administration of DNA vaccines.DNA vaccine provides prolonged antigen expression, leading to amplificationof immune response and induces memory responses against a weaklyimmunogenic tumor-associated antigens (TAA). DNA vaccination representsan exceptionally potent strategy, which can activate both cellular and humoralimmune response as the encoded antigen is processed through bothendogenous and exogenous pathways and its peptide epitopes generated by proteolysis in antigen processing cell (APC) are presented by majorhistocompatibility complexes (MHC) classâ… andâ…¡.Although DNA vaccination alone can elicit potent humoral and cellularresponses to many antigens, it appears that for certain antigens, the immuneresponse generated by DNA vaccination may be sub-optimal for protection.To augment immune responses to vaccines, particularly when the vaccine istargeting weak antigens or self-antigens, such as MUC1, adjuvants are oftenrequired. Cytokines such as IL-2, IL-12, IL-18, and granulocyte-macrophagecolony-stimulating factor (GMCSF) are particularly strong choices to enhanceT helper 1 (Th1) cellular immune responses induced by DNA vaccinesexpressing a variety of antigens. Interleukin-2 (IL-2) is perhaps the mostextensively studied of all cytokine adjuvants. IL-2 has long been known to bea B- and T-cell growth factor and is capable of enhancing immune responseswhen administered in conjunction with a particular antigen.In this paper, we constructed recombinant expressing vectors encodinghuman multiple repeats of human MUC1, and established a tumor cell line andtumor-bearing model stably expressing MUC1 VNTR, and studied thepossibility in inducing cellular and humoral immune responses and anti-tumoractivities of the DNA vaccine. It was our hope that MUC1 VNTR DNAvaccine could be utilized for immunization strategies and induce protectiveimmunity against MUC1-expressing tumors and enhanced by the cytokineplasmid.In the first set of studies described in the dissertation, we constructed theeukaryotic expression vectors encoding 2 and 33 tandem repeats of humanMUC1 (VR1012-2m and VR1012-33m). A pair of primers consisting theenzyme sites of Salâ… and Xhoâ… respectively were designed and 1 repeat ofMUC1 was synthesized by PCR. The PCR product was cloned into the vector pGEM-T Easy. The ligations were then performed using two enzymes ofSalâ… and Xhoâ… which have different recognition sequences but producecompatible cohesive ends to construct 2 and 33 MUC1VNTRs (2m and 33m).The constructed gene was subcloned into the eukaryotic expression vectorVR1012. The sequencing result and restrictive enzyme digestion analysisshowed that the recombinant VR1012-2m and VR1012-33m were constructedsuccessfully.In the second set of studies, we established a tumor-bearing mouse modelstably expressing enhanced green fluorescent protein (EGFP) labeled 33m. Aeukaryotic vectors expressing 33m, pEGFP-33m, was constructed by cloning33m gene into pEGFP and 33m stably expressing murine Lewis lungcarcinoma (LLC) cell line (MUC1+LLC) was established byLipofectamine-mediated transfection of pEGFP-33m into LLC cells. Positivecell clones were selected by G418 and the EGFP expression in transfectedcells was observed under a fluorescent microscope. 33m expression in MUC1+LLC cells was confirmed by means of Western Blotting. A syngenic grafttumor model was generated by subcutaneous injection of MUC1+LLC cellsinto C57/BL6 mice and tumor size increased rapidly with time and cellnumber dependent manner. 33m mRNA expression in the forming tumor wasconfirmed by RT-PCR detection.In a third set of experiments, we examined the immune responses inBALB/c mice induced by DNA immunization of MUC1 VNTR gene usingdifferent immunization protocols, including different number of tandemrepeats, different number of immunizations, and co-injection of murineinterleukin-2 (IL-2) expression plasmid. BALB/c mice were immunized witheither DNA encoding 2 or 33 repeats of human MUC1 VNTR (VR-2m andVR-33m) alone or co-administered with VR-33m and a plasmid encoding murine interleukin-2 (VR-IL2). Anti-MUC1 VNTR antibody detection,Cytotoxic T lymphocyte (CTL) and T lymphocyte proliferation assay werecarried out to examine the immune response of the vaccine candidates. Theresults show that immunization with VR-2m, VR-33m or VR-33m/VR-IL2can induce both MUC1 VNTR-spcific cellular and humoral response, andthese effects can be enhanced by increasing the number of tandem repeats, thenumber of immunizations, and co-administration of the cytokine plasmidobviously. Furthermore, the specific CTL and proliferative T-cell responses ofVR-33m immunized mice are enhanced significantly by co-injection withVR-IL2.In our last set of experiments, we explore the possibility to develop anefficient anti-tumor vaccine strategy using the specific anti-tumor immunityinduced by MUC1 VNTR DNA vaccine combining the adjuvant effect of aplasmid expressing murine interleukin-2 (IL-2). In prophylactic test, C57BL/6mice immunized with VR empty vector, VR-IL2 and VR-33m alone orcoimmunized VR-33m with VR-IL2 were challenged subcutaneously withMUC1-expressing murine Lewis lung carcinoma (MUC1+LLC) cells. Tumorgrowth and mice survival were monitored to evaluate the anti-tumor activity.The results showed that VR-33m could significantly inhibit the volume andweight of the implanted MUC1+LLC tumors, but there was no statisticaldifference in tumor incidence and survival between the VR-33m alone and VRcontrol group. Vaccination with VR-33m/VR-IL2 could induce both tumorsuppression and a prolonged survival significantly. In therapeuticimmunization, C57BL/6 mice were inoculated with MUC1+LLC cells andthen immunized with VR-33m/VR-IL2. The data showed that co-injection ofVR-33m with VR-IL2 resulted in a significant protection against the challengeof a MUC1+tumor cell line and increasing survival compared with the vector control group.In our approach, though VR-33m immunization can significantly inhibitthe volume and weight of the implanted MUC1+LLC cell tumors, statisticallysignificant improved protection from latency was not achieved. Thisdemonstrates that the presence of MUC1-specific antibody and CTL inducedby VR-33m may be sub-optimal for translating into a clinical response asmeasured by time of tumor onset and survival. The co-administration ofVR-33m with VR-IL2 can reduce the formative rate and inhibit the growth ofthe subcutaneous translated tumor significantly, and prolong the life span andpromote the survival rate of the mice both before and after tumor challenge.In conclusion, we show that MUC1 VNTR DNA vaccination succeeds ininducing both humoral and cellular immune responses against MUC1 VNTR,and these effects can be enhanced obviously by IL-2 plasmid. IL-2 can act asan effective adjuvant for MUC1 through co-administration with theantigen-expressing plasmid. VR-33m/VR-IL2 DNA vaccination of mice iseffective in preventing and treating an established transplated tumor. AVR-33m/VR-IL2 DNA vaccine may offer important clinical benefit to patientswith MUC1+tumors.