Antitumor Effect Of New Multiple Antigen Peptide Based On T Cell Epitopes Of Human Telomerase Reverse Transcriptase

Background and Objectives:Despite multiple approaches for prevention and therapy, cancer remains a major causeof death in the world. Most approaches, such as radiotherapy or chemotherapy, killmalignant cells but also affect normal cells, which results in side effects that limit the use ofthese treatments. With the rapid increase in knowledge of the immune system and itsregulation, immunologic approaches have been explored to target and eliminate cancer cells.Dendritic cell (DC)-based immunotherapy, which has the advantages of inducing strongimmune responses, few side-effects and wide applicability, is becoming a promisingtherapy for malignant tumors. The use of tumor-associated antigen (TAA) loaded DCs is anideal tumor immunotherapy. However, the expression of most TAAs is restricted to a fewtumor types. In addition, the appearance of antigen-loss mutations in tumor cells in the faceof immune pressure is well described. To circumvent these issues, a new class of TAAs,termed “universal tumor antigens,” has been proposed.Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase,is highly expressed in over85%of cancer cells to maintain the immortality of the cancercells but is not active in most normal somatic cells. Therefore, hTERT may be an idealcandidate for a universal TAA. Meanwhile, unlike most other TAAs, the expression ofhTERT in tumor cells is associated with tumor growth, development and invasion.Targeting hTERT may also minimize immune escape because the inhibition of telomeraseactivity in hTERT-positive tumor cells leads to telomere shorting and tumor cell death byapoptosis. Our previous study indicated that a hTERT recombinant adenovirus vaccine anda peptide-nucleotide dual vaccine could also induce hTERT-specific CTLs in vivo and invitro. These results indicate that hTERT could be a universal TAA used in tumor immunotherapy.When compared with the vector-based vaccine that uses an adenovirus or lentivirus asa vector, peptide-based vaccines are simple, safe, stable and economical, as well as easy toproduce and modify. However, there are unique disadvantages, which includeHLA-restriction, low immunogenicity and a low level of memory responses. Branchedpeptides, such as multiple antigen peptides (MAPs), which consist of concentrated epitopesand possess an immunologically silent lysine core, were invented in1988by Tam, and theyhave been explored for uses in serodiagnosis, vaccines against bacteria, viruses andparasites, and as inhibitors of pathogenic infections[24]. MAPs may be considered for in thedevelopment of new strategies for antitumor immunotherapy targeting hTERT-positivetumor cells.In this study, we designed three tetrameric MAP vaccines based on HLA-A2-restrictedCTL epitopes of hTERT [hTERT-540(ILAKFLHWL), hTERT-865(RLVDDFLLV) andhTERT-572Y (YLFFYRKSV)]. The killing effects of CTLs induced by these MAP vaccinesand their corresponding linear peptides, as well as recombinant adenovirus-hTERT vectors,were studied using a standard4h51Cr-release assay. These findings will provide evidencesfor a potential clinical application of hTERT MAP vaccines in immunotherapy for cancers.Methods:three tetrameric MAP vaccines based on HLA-A2-restricted CTL epitopes of hTERT[hTERT-540(ILAKFLHWL), hTERT-865(RLVDDFLLV) and hTERT-572Y(YLFFYRKSV)] and their corresponding linear peptides were synthesized by the ShanghaiQiangyao Biotechnology Ltd. Co.(Shanghai, China). The molecular weights of the peptideswere validated by mass spectrometry (API2000, PE). Lyophilized peptides were dissolvedin dimethylsulfoxide (DMSO, Sigma) and stored at-20°C. One nonapeptide from the HIVsequence [HIVpol (476-484)(ILLEP VHGV)], which served as a negative control, wassynthesized by the Beijing Scilight Biotechnology Ltd. Co.(Beijing, China).The DCs from peripheral blood mononuclear cells (PBMCs) were generated using aprocedure described previously. These mature DCs were identified by flow cytometry. ThehTERT-specific CTLs were induced by mature DC pulsed with hTERT MAP vaccines andtheir crresponding linear peptides. The killing effect of these CTLs to various tumor cells[human colon carcinoma cells SW480(hTERT~+, HLA-A2~+), human gastric carcinoma cells KATO-III (hTERT~+, HLA-A2~+), human osteogenic sarcoma cells U2OS (hTERT-,HLA-A2~+), U2OS/hTERT (hTERT~+, HLA-A2~+), human breast cancer cell line MCF-7(hTERT~+, HLA-A2~+), and human hepatocellular carcinoma cells HepG2(hTERT~+,HLA-A2-), HepG2/HLA-A2cells (hTERT~+, HLA-A2~+)] were tested by a standard51Crreleasing assay. The lysis of CTLs on autologous lymphocytes and DCs with lowtelomerase activity is to test the possible side effects. ELISPOT was used to test the numberof IFN-γ producing effectors.DCs from C57BL/6-Tg (HLA-A2~+) mouse bone marrow (mDCs) were also generatedas described previously. The mature mDC was identified by flowcytometry. C57BL/6transgenic mice that were12weeks old were immunized once aweek by subcutaneous injection in the back with2×106mDCs-pulsed with the abovepeptides for three times. And then, the spleens of the mice were removed and thesplenocytes were harvested as effectors.Statistical analysis was performed using the Student’s t-test. The difference wasconsidered statistically significant when the P＜0.05. All statistical analyses wereconducted with SPSS17.0software.ResultsPeptides from human telomerase reverse transcriptase were produced by solid-phasepeptide synthesis (SPPS). The peptides were purified using reverse-phase high-performance liquid chromatography (RP-HPLC). The purity of the peptides was confirmedby High-performance liquid chromatography (HPLC). The molecules weight wasdetermined by high-performance liquid chromatography and mass spectrographic analysis(LC/MS). The purity of these peptides was reached more than95%.The experiments demonstrated that the hTERT MAP vaccines could trigger muchstronger hTERT-specific CTL responses than their corresponding linear peptides againstKATO-III, SW480, and MCF-7cells expressing hTERT and HLA-A2. On the other hand,the activated CTLs could neither kill the hTERT-negative tumor cells, such as U2OS cells,nor kill HLA-A2negative cells, such as HepG2cells. But to U2OS/hTERT cells transfectedwith full-length cDNA of hTERT and to HepG2/HLA-A2cells transfected with full-lengthcDNA of HLA-A2, the hTERT specific CTL responses was re-occurred. ELISPOT assaydemonstrated that MAP vaccines could increase the number of IFN-γ-producing effectors cells compared to their corresponding linear peptides. In ex vivo study, after deleting theCD4+T lymphocyte, the IFN-γ-producing effector cells were significantly decreased bothin MAP vaccines and their corresponding linear peptides vaccined mice. No obvioushTERT-specific CTL responses were found to autologous lymphocytes and DCs.Conclusions:In this study, we demonstrated that DCs-pulsed with MAPs based on hTERTHLA-A2-restricted CTL epitopes could trigger stronger antitumor immune response thantheir corresponding traditional linear peptides in vitro and ex vivo. This vaccine format,either as a single agent or in combination, may offer great promise for cancer treatments.