H-2Kb–Restricted CTL Epitopes from Mouse Heparanase Elicit an Antitumor Immune Response In vivo

Abstract

The identification of CTL epitopes from tumor antigens is very important for the development of peptide-based, cancerspecific immunotherapy. Heparanase is broadly expressed in various advanced tumors and can serve as a universal tumorassociated antigen. Although several epitopes of heparanase antigen are known in humans, the corresponding knowledge in mice is still rather limited. The present study was designed to predict and identify the CTL epitopes in the mouse heparanase protein. For this purpose, H-2K b -restricted CTL epitopes were identified by using the following four-step procedure: (a) a computer-based epitope prediction from the amino acid sequence of mouse heparanase, (b) a peptidebinding assay to determine the affinity of the predicted epitopes with the H-2K b molecule, (c) the testing of the induction of CTLs toward various carcinoma cells expressing heparanase antigens and H-2K b , and (d) the induction of immunoprotection and immunotherapy in vivo. The results showed that, of the tested peptides, effectors induced by peptides of mouse heparanase at residue positions 398 to 405 (LSLLFKKL; mHpa398) and 519 to 526 (FSYGFFVI; mHpa519) lysed three kinds of carcinoma cells expressing both heparanase and H-2K b (B16 melanoma cells, EL-4 lymphoma cells, and Lewis lung cancer cells). In vivo experiments indicated that mHpa398 and mHpa519 peptides offered the possibility of not only immunizing against tumors but also treating tumorbearing hosts successfully. Our results suggest that the mHpa398 and mHpa519 peptides are novel H-2K b -restricted CTL epitopes capable of inducing heparanase-specific CTLs in vitro and in vivo. These epitopes may serve as valuable tools for the preclinical evaluation of vaccination strategies. [Cancer Res 2008;68(5):1529-37]

Tapasin modification on the intracellular epitope HBcAg18–27 enhances HBV-specific CTL immune response and inhibits hepatitis B virus replication in vivo

“…Serum HBsAg and HBV DNA were monitored at day 7 after the second and third immunization. The inhibition rate of serum HBsAg or viral DNA in HBV transgenic mice of CTPHBcAg 18-27 -Tapasin group was higher than CTP-HBcAg [18][19][20][21][22][23][24][25][26][27] or HBcAg 18-27 -Tapasin or IFN-a (Figures 4a and b). Serum HBsAg level from the mice immunized with CTP-HBcAg 18-27 -Tapasin decreased markedly as compared with the mice immunized with CTP-HBcAg 18-27 , HBcAg 18-27 -Tapasin, or IFN-a (*Po0.05).…”

“…Phorbol 12-myristate 13-acetate (PMA), ionomycin, monensin, phytohemagglutinin (PHA), and concanavalin A (ConA) were obtained from Sigma (USA). Soluble fusion proteins CTP-HBcAg 18-27 -Tapasin, CTP-HBcAg [18][19][20][21][22][23][24][25][26][27] , and HBcAg 18-27 -Tapasin were purified and had undetectable endotoxin level according to our former experiments (Figures 1a and b). 19 EL-4 cells (H-2K b ; from ATCC) were cultured in Dulbecco's modified Eagle's medium containing 10% FBS at 37 1C under a humidified condition of 5% CO 2 and 100 U/ml penicillin.…”

“…25 EL-4 cells were seeded at a density of 5 Â 10 4 cells per well in 96-well plates. Effector cells were incubated with EL-4 at different effector and target (E/T) ratios (5:1, 10:1 or 20:1) at 37 1C under 5% CO 2 for 4 h. The specific CTL activity was measured using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison WI, USA) for lactate dehydrogenase (LDH) release according to the manufacturer's instructions.…”

“…12,13 The low surface expression of MHC I molecules resulted in impaired positive and negative thymic T-cell selection, resulting in a reduction of CD8 þ T-cell number in the thymus and periphery, and reduced antigen presentation to T cells, resulting in poor in vivo CTL response to influenza virus. 14,15 HBcAg 18-27 epitope (HBcAg [18][19][20][21][22][23][24][25][26][27] ) is a widely accepted as a CTL epitope to stimulate CTL response to hepatitis B core antigen (HBcAg). 16,17 However, HBV-specific CTL responses induced by exogenous antigens such as HBV-encoded antigens are usually weak, mainly because of the prohibition of intracellular delivery of antigens and peptides by the lipophilic nature and selective permeability of biological membranes.…”

Downregulation of heparanase by RNA interference inhibits invasion and tumorigenesis of hepatocellular cancer cells in vitro and in vivo

“…Because heparanase plays a role in cell invasion through the degradation of HSPG in the ECM and BM (18,19), we tested the effect of heparanase RNAi on the invasive ability of HepG2 cells using the Matrigel invasion assay. As shown in Fig.…”