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Identification of HLA-A2.1 Restricted Epitopes in Human Heparanase and Induction of a Specific Anti-tumor Immune Response

Author: ChenZuo
Tutor: YangShiMing；WanZuo
School: Third Military Medical University
Course: Internal Medicine
Keywords: Heparanase Dendritic cells Epitope HLA-A2.1 restriction Tumor vaccine T lymphocyte cytokine
CLC: R392
Type: Master's thesis
Year: 2007
Downloads: 93
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Abstract

[Background and purpose of biological treatment Following surgery, radiotherapy and chemotherapy after clinical treatment of malignant tumors of the fourth treatment mode. With the in-depth study of the anti-tumor immune mechanisms, people have come to realize induced in vivo cytotoxic T lymphocyte (Cytotic T Lymphocyte, CTL) play the role of anti-tumor is not the entire tumor antigen molecules, but combined with the MHC molecules short peptide CTL epitopes (epitopes), looking for specific tumor antigens and their corresponding CTL epitopes become the key to cancer immunotherapy. Dendritic cells (Dendritic cells, DC) as the body of the most powerful antigen-presenting cells, the immune response process initiating and regulator. The DC load CTL epitope vaccine for its immunogenicity and side effects, ease of application and other advantages to become a hot topic of current anti-cancer therapy research. Heparanase (Heparanase, Hpa) is a newly discovered tumor metastasis-related genes in lymphocytes and bone marrow and immune tissue, only low expression in normal tissues, but widespread in almost all metastatic malignant cells, and the level of expression and tumor metastasis, TMN staging poor clinical outcome indicators significantly related heparanase in advanced cancer treatment has become a good target. Heparanase whether as a tumor-associated antigen for tumor immunotherapy? Our past research showed that the the heparanase length gene load DC can be induced in vitro heparinase specific CTL, heparinase positive and MHC matches gastric cancer cell killing effect heparinase positive MHC does not match the gastric cancer cell killing effect, suggesting that heparanase amino acids in the full-length sequence must exist epitopes can induce specific CTL responses . The purpose of this study is to find exists in the full-length sequence of heparanase can effectively stimulate the body's anti-tumor effect of CTL epitopes. In view of the HLA-A2.1 HLA-A2.1-restricted CTL epitope Chinese population, the most common HLA class I molecules type, the positive rate of up to 50%, so the identification of tumor metastasis-associated antigen Hpa will the Chinese population heparanase expression-positive malignancies provide the basis for immunotherapy. Spatial conformation [method] 1, ultramafic sequence and combination of quantitative motif heparanase HLA-A2.1-restricted CTL epitope prediction and analog epitope peptide and MHC molecules using molecular simulation technology to analyze the combination of parameters in order to further improve the accuracy of the forecasts, and then select from the results in the high score of 5 heparanase epitope peptide synthesis, purification, molecular weight identification using T ₂ cell characteristics carried affinity synthetic candidate peptides with HLA-A2.1 molecules; using the standard ^{51 Cr release assay to detect heparanase-specific CTL induced above heparanase epitope peptide load DC on KATO-Ⅲ gastric immune cell killing effect, which filter out effectively activate CTL responses heparanase epitopes. 2, by density gradient centrifugation to separate and get human peripheral blood mononuclear cells using rhGM-CSF, rhIL-4 and rhTNF-α-induced amplification of human mature dendritic cells, and were identified from the morphology, cell surface CD molecules ; that the screening of candidate peptide load DC induce specific CTL, using the standard ^{51 Cr release assay heparanase-specific CTL KATO-Ⅲ gastric cancer cells (Hpa ^{, HLA-A2.1 ^{), U-2OS osteosarcoma cells (Hpa ^{, HLA-A2.1 ^{), SW480 colon cancer cells ( Hpa ^{, HLA-A2.1 ^{), MCF-7 breast cancer cells (the Hpa ^{- , HLA-A2.1-^{), HepG2 hepatoma cells (Hpa ^{, HLA-A2.1 ^{- ), and transfection of the full-length gene of heparanase MCF-7/Hpa breast cancer cells (Hpa ^{, HLA-A2.1 ^{) and transfected with the gene HLA-A2.1 HepG2/HLA-A2.1 hepatoma cells (Hpa ^{, HLA-A2.1 ^{) immune destruction effect; HLA-A2.1 monoclonal antibody to HLA-A2.1 loci closed above the target cells, or CD8 monoclonal antibody closed effector cells in CD8 locus, and then heparanase epitope-specific CTL killing KATO-III cancer cells, to study screening heparinase epitope is affected by the source of HLA-A2.1 restriction of CTL effector cells; using standard ^{51 Cr release experimental study heparanase-specific CTL immune cytotoxic activity of autologous lymphocytes, to determine its possible side effects; IFN-γ ELISPOT to detect heparanase specific effector cells release. 3, after the above screening the heparinase candidate epitope the load C57BL/6-Tg (HLA-A2.1) 1Enge / J mouse bone marrow-derived DC (mDC), mice immunized three times, spleen lymphocytes as effector cells ^{51 Cr release assay heparanase-specific CTL KATO-III Osteosarcoma cell line U2OS was, SW480, MCF-7, MCF-7/Hpa, HepG2, HepG2/HLA-A2.1 cells and autologous lymphocytes and the mDC killing effect; ELISPOT to detect heparanase-specific effector cell IFN-γ release. [Results] 1, ultra-motif and quantitative motif method jointly predicted five score highest heparanase epitope peptide: Hpa (525-533) (PAFSYSFFV), Hpa (353-361) (PLLSDTFAA), Hpa ( 277-285) (KMLKSFLKA), Hpa (400-408) (PLPDYWLSL), Hpa (405-413) (WLSLLFKKL); the T ₂ cell affinity analysis found five predicted polypeptide can T ₂ effective combination of cells; using standard ^{51 the heparinase-specific CTL induced by Cr release assay above heparanase epitope peptide load DC KATO-Ⅲ gastric cancer cells The immune destruction effects, the results show that, of Hpa (525-533), Hpa (277-285), of Hpa (405-413) CTL induced gastric cancer cells KATO-Ⅲ has a significant killing effect, while of Hpa (353-361) Hpa (400-408) CTL induced KATO-Ⅲ gastric cancer cell killing effects of negative peptide, suggesting of Hpa (525-533), Hpa (277-285), Hpa (405-413) heparanase specific antigen epitope. 2, HLA-A2.1-positive healthy volunteers in peripheral blood mononuclear cells (PBMC), separated by density gradient centrifugation with recombinant human cytokines GM-CSF, IL-4, and TNF-α induced by amplification, Morphological observation and the use of flow cytometry to identify cell surface CD molecules, were successfully prepared PBMC derived mature DC; using the standard ^{51 Cr release assay Hpa (525-533), Hpa (277 -285) of Hpa (405-413) heparanase-specific CTL epitopes load DC-induced immune destruction of the different sources tumor cells effect, results showed that the heparanase-specific CTL heparanase-positive and HLA- A2.1-positive KATO-Ⅲ, U2OS and SW480 cell killing effect of heparanase negative but HLA-A2.1-positive MCF-7 cells and heparanase positive HLA-A2.1 negative HepG2 cells are not killing effect the heparanase full-length gene transfected MCF-7 cells (MCF-7/Hpa) and transfected with full-length gene HLA-A2.1 HepG2 cells (HepG2/HLA of -A2.1) to regenerate the immune destruction effects suggest that CTL responses induced by heparanase epitopes a heparanase specific and limited by HLA-A2.1; single HLA-A2.1 and CD8 molecules anti closed and then the target cell surface sites and the effect of the HLA-A2.1 CD cell surface molecules, respectively destruction experiment results show that no significant heparanase-specific CTL closed the HLA-A2.1 KATO-Ⅲ cells killing effect, closed CD8 effector cells KATO-Ⅲ cells had no significant killing effect, and further suggest that this killing effect is the limit of the HLA-A2.1 CTL effector cells from CD8-positive T lymphocytes; using standard ^{51 Cr release experimental examination detected above heparanase epitopes induced heparanase-specific CTL on autologous lymphocyte immune killing effect, results showed that the heparanase-specific CTL against autologous lymphocytes no significant destruction effect, suggesting that the security of heparanase peptide vaccine (Table 1); heparanase-specific effector cells detected by ELISPOT IFN-γ secretion capacity, heparanase epitope potential for IFN-γ effector cells. secretion. 3, the enzyme with the above heparin epitope peptide load C57BL/6-Tg (HLA-A2.1) 1Enge / J mouse bone marrow-derived DC (mDC), and then the immunized mice three times, spleen lymphocytes were used as effector cells to the different sources of a variety of tumor cells as target cells, with ^{51 Cr release assay killing effect, experimental results show that the epitope specificity of CTL heparinase heparanase-positive and HLA- 2 positive KATO-Ⅲ, U2OS and SW480 cell killing effect of heparanase negative but HLA-A2.1-positive MCF-7 cells and heparanase-positive but negative for HLA-A2.1 HepG2 cells not killing effect, but infected MCF-7 breast cancer cells in the of heparanase full-length gene recombinant adenovirus (rAd-Hpa) (MCF-7/rAd-Hpa) and transfected with HLA-A2.1 HepG2/HLA-A2.1 cells regenerate significant killing effect, there is no obvious autologous lymphocytes and mDC killing effects (Table 1). ELISPOT to detect the heparinase antigen the table potential energy effective promotion of a heparanase specific effector cells to secrete IFN-γ. [Conclusion] 1, using ultra motif and quantitative motif method and screened by cultured cells from the entire length of the amino acid sequence of heparinase 3 HLA-A2.1-restricted CTL epitopes, i.e. HPA (525 - 533) (PAFSYSFFV), Hpa (277-285) (KMLKSFLKA), Hpa (405-413) (WLSLLFKKL), above heparanase epitopes German scholars reported. From in vitro and animal experiments confirmed heparanase epitopes of Hpa (525-533) of Hpa (277-285), Hpa (405-413) can induce heparanase-specific CTL heparanase positive and HLA-A2.1 match the tumor cells with a strong immune killing activity, does not have a killing effect on heparanase negative or HLA-A2.1 negative tumor cells, but transfected with heparinase or HLA -A2.1 tumor cell killing effect, suggesting that the CTL response induced by heparanase epitopes heparanase specific and limited by HLA-A2.1 heparanase-specific CTL comes mainly from CD8-positive T lymphocytes . From in vitro and animal experiments confirmed heparanase-specific CTL autologous lymphocytes and / or DC no significant immune cytotoxic activity, suggesting that heparanase peptide vaccine clinical application security. 4, from the second aspect of the in vitro and in animal experiments confirmed heparanase epitope HPA (525-533), the HPA (277-285), HPA (405-413) of DC can promote IFN-γ releasing effector cells. These studies have shown that human heparanase-specific antigen epitopes [Hpa (525-533), Hpa (277-285), Hpa (405-413)] is not only to stimulate the body's specific anti-tumor effect, but also induced a virtuous cycle of non-specific anti-tumor, this heparanase peptide vaccine with broad-spectrum, high efficiency, specific security advantages for heparanase epitope peptide vaccine clinical application provides a theoretical basis.}}}}}}}}}}}}}}}}}}}}}}

Identification of HLA-A2.1 Restricted Epitopes in Human Heparanase and Induction of a Specific Anti-tumor Immune Response

Abstract

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