Yan-Fang Sui

Heat shock protein 70/MAGE-3 fusion protein vaccine can enhance cellular and humoral immune responses to MAGE-3 in vivo

MAGE-3, a member of melanoma antigen (MAGE) gene family, is recognized as an ideal candidate for tumor vaccine because it is expressed in a significant proportion of tumors of various histological types and can induce antigen-specific immune response in vivo. There is now substantial evidence that heat shock proteins (HSPs) isolated from cancer cells and virus-infected cells can be used as vaccines to produce cancer-specific or virus-specific immunity. In this research, we investigated whether M. tuberculosis HSP70 can be used as vehicle to elicit immune response to its accompanying MAGE-3 protein. A recombinant protein expression vector was constructed that permitted the production of fusion protein linking amino acids 195–314 of MAGE-3 to the C terminus of HSP70. We found that HSP70-MAGE-3 fusion protein can elicit stronger cellular and humoral immune responses against MAGE-3 expressing murine tumor than those elicited by MAGE-3 protein in vivo, which resulted in potent antitumor immunity against MAGE-3-expressing tumors. Covalent linkage of HSP70 to MAGE-3 was necessary to elicit immune response to MAGE-3. These results indicate that linkage of HSP70 to MAGE-3 enhanced immune responses to MAGE-3 in vivo and HSP70 can be exploited to enhance the cellular and humoral immune responses against any attached tumor-specific antigens.

Heat shock protein 70 / MAGE-1 tumor vaccine can enhance the potency of MAGE-1–specific cellular immune responses in vivo

The cancer-testis antigen encoded by the MAGE-1 gene is an attractive antigen in tumor immunotherapy because it can be processed as a foreign antigen by the immune system and generate tumor-specific cellular immune response in vivo. However, increase of the potency of MAGE-1 DNA vaccines is still needed. The high degree of sequence homology and intrinsic immunogenicity of heat shock protein 70 (HSP70) have prompted the suggestion that HSP70 might have immunotherapeutic potential, as HSP70 purified from malignant and virally infected cells can transfer and deliver antigenic peptides to antigen-presenting cells to elicit peptide-specific immunity. In this research, we evaluated the enhancement of linkage of Mycobacterium tuberculosis HSP70 to MAGE-1 gene of the potency of antigen-specific immunity elicited by naked DNA vaccines. We found that vaccines containing MAGE-1-HSP70 fusion genes enhanced the frequency of MAGE-1–specific cytotoxic T cells in contract to vaccines containing the MAGE-1 gene alone. More importantly, the fusion converted a less effective DNA vaccine into one with significant potency against established MAGE-1–expressing tumors. These results indicate that linkage of HSP70 to MAGE-1 gene may greatly enhance the potency of DNA vaccines, and generate specific antitumor immunity against MAGE-1–expressing tumors.

MAGE-1/Heat shock protein 70/MAGE-3 fusion protein vaccine in nanoemulsion enhances cellular and humoral immune responses to MAGE-1 or MAGE-3 in vivo

Tumor vaccines, which can elicit antigen-specific antitumor immunity and play an important role in prevention and therapy of tumor, are regarded as the most attractive method. Its underlying mechanism is that tumor antigen, especially the tumor specific antigen (TSA), can induce tumor-specific cytotoxic T lymphocyte (CTL) accordingly to damage tumor cells. The melanoma antigen (MAGE) was the first reported example of TSAs. MAGE-1 and MAGE-3 are two important members of MAGE. Whereas the expressions of MAGE family proteins deviate in different tumors, MAGE-1 and MAGE-3 are two most common tumor antigens. They are expressed in most tumors, respectively or jointly but not in normal tissues except the testes and placenta. Moreover, MAGE-1 and MAGE-3 antigens are termed as tumorrejection antigens because tumors expressing these antigens on appropriate human leukocyte antigen (HLA) class I molecules are rejected by host CTLs [16]. Heat shock proteins (HSPs), named as ‘‘molecular chaperone’’, participate in the folding, transporting and assembling of proteins. Besides, HSPs are important in the processing and presenting of antigens, which could conjugate proteins or peptides and improve their antigenicity. Moreover, the HSP receptors expressed on the surface of antigen-presenting cells (APCs) can facilitate APCs uptaking tumor antigens and improve the presentation. Related research had shown that HSP70 could be exploited to enhance the cellular and humoral immune responses against any attached tumor-specific antigens [7]. To find more effective and more comprehensive vaccine, our colleagues have constructed and purified a recombinant MAGE-1/HSP70/MAGE-3 (MHM) fusion protein successfully. In recent years, significant effort has been devoted to develop nanotechnology for drug delivery, since it offers a suitable means of delivering drugs with small molecular weights, as well as macromolecules such as proteins, peptides or genes by either localized or targeted delivery to the tissue of interest [10]. A nanoemulsion is a system of water, oil and amphiphile, which is a single optically isotropic and thermodynamically stable liquid solution. Therefore, a nanoemulsion is capable of encapsulating significant amounts of drugs. When compared to available traditional emulsions, a nanoemulsion is characterized by its smaller size (limited to 1–100 nm), higher efficiency, and elegant compatibility with tissues as well as with proteins. In addition, it can be prepared with oil and surfactant components that are generally regarded as safe, having less side effect, and toxicity. Moreover, W/O (water-in-oil) nanoemulsions can improve absorption of water-soluble drugs or peptides following intraduodenal administration. In this study, we used this novel artificial lipoprotein delivery system – nanoemulsion as delivery vehicle of the MHM fusion protein vaccine and determined its humoral and cellular immune responses and the antitumor effects. The goal was to investigate the potential benefits of using W/O nanoemulsion as an alternative carrier of the vaccine. W. Ge Æ Y.-F. Sui (&) Æ Y.-J. Sun Æ G.-S. Chen Æ Z.-S. Li S.-Y. Si Æ P.-Z. Hu Æ Y. Huang Æ X.-M. Zhang State Key Laboratory of Cancer Biology, Department of Pathology, Xi Jing Hospital, Fourth Military Medical University, Xi’an, 710032 Shaanxi Province, China E-mail: suiyanf@fmmu.edu.cn Tel.: +86-29-83374597 Fax: +86-29-83255697

In vivo tumor co-transfection with superantigen and CD80 induces systemic immunity without tolerance and prolongs survival in mice with hepatocellular carcinoma

Background: Since transfection of established tumors with immunostimulatory genes can elicit antitumor immunity, we treat mouse HCC with in vivo transfection of superantigen SEA and/or costimulatory molecule CD80 and evaluated the safety and efficacy. Methods Mice with HCC were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin A and/or CD80. Then the mice were evaluated for tumor regression, systemic immunologic responses, survival times and treatment-associated toxicity. Results Of all treated mice, the overall response rates (complete or partial remission) for SEA, CD80 and SEA/CD80 treated mice in this study were 65%, 60% and 75% separately, and were significantly higher than that of untreated mice. Most of the treat mice completed the therapy without any significant reaction. CTL activity increased with time of treatment and correlated temporally with an objective tumor response. Also our results indicated that local intratumoral expression of SEA did not lead to detectable deletion or anergy of SEA-reactive spleen T cells. Survival times for hepatoma mice in this study treated by intratumoral injection of SEA, CD80 and SEA/CD80 were prolonged significantly (P < 0.01) compared with the control mice. Conclusions Thus, local tumor transfection with superantigen and CD80 genes was capable of inducing strong systemic antitumor immunity.

Identification of HLA-A2-restricted CTL epitope encoded by the MAGE-n gene of human hepatocellular carcinoma.

Background: Identification of the cytotoxic T lymphocytes (CTL) restricted epitopes of tumor antigens opens up possibilities of developing a new cancer vaccine. For the MAGE-n has been demonstrated closely associated with hepatocellular carcinoma (HCC) and HLA-A2.1 is found in over 50% of HCC patients in China, we aim at identifying MAGE-n-encoded peptide presented by HLA-A2.1. Methods: A HLA-A2.1-restricted CTL epitope was identified by using an improved “reverse immunology” strategy: (a) computer-based epitope prediction from the amino acid sequence of MAGE-n antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward HCC cells expressing MAGE-n antigen and HLA-A2.1. Results: Of the five tested peptides, effectors induced by a peptide of MAGE-n at residue position 159-167(QLVFGIEVV) lysed HCC cells expressing both MAGE-n and HLA-A2.1. Our results indicated that peptide QLVFGIEVV was a new HLA-A2.1-restricted CTL epitope capable of inducing MAGE-n specific CTLs in vitro. Conclusions: Identification of the MAGE-n /HLA-A2.1 peptide QLVFGIEVV may facilitate peptide-based specific immunotherapy for HCC. The combination of epitope prediction, epitope reconstruction method and immunological methods can improve the efficiency and accuracy of CTL epitope studies.

Immunization with truncated sequence of Telomerase Reverse Transcriptase induces a specific antitumor response in vivo

To select the MHC-I-binding epitope-rich sequence of mice telomerase reverse transcriptase (mTERT) and study the antitumor immune response induced by truncated TERT through mRNA-transfected dendritic cells (DCs) immunization in mice. The MHC-I-binding epitopes of TERT were predicted using bioinformatics software. The selected sequence of TERT (Truncated mTERT, TERTt, mTERT cDNA 1776 bp-2942 bp encoding 584 aa-969 aa) was cloned from B16 mouse melanoma cells and inserted into pBluescriptIIKS(+) plasmid downstream of the T7 promoter. TERTt RNA was prepared through in vitro transcription. Bone marrow-derived DCs were electroporated with TERTt RNA and used to immunize syngeneic naïve mice. The quantity and cytotoxic activity of TERT-specific cytotoxic T lymphocytes (CTLs) in mice spleen were evaluated using IFN-γ enzyme-linked immunospot (ELISPOT) and Lactate dehydrogenase release assay. The immunoprophylactic effects against TERT positive tumor induced by TERTt RNA transfected DC in vivo were evaluated through an immunized-challenged mouse model. TERTt was cloned and in vitro transcribed into TERTt mRNA. As shown in FCM analysis, the efficiency of DC electroporation is 35.1% (29.7–41.2%). After electroporation, a subtle increase of costimulator and MHC-II molecules were expressed on the cell surface. Immunization of TERTt mRNA transfected DCs induced IFN-γ-secreting CTLs which manifested specific cytotoxic activity against TERT-positive target cells. In a cancer mouse model, vaccination of TERTt mRNA-transfected DCs suppressed the growth of TERT positive tumors (p=0.001) and prolong the survival time of tumor-bearing animals (p=0.029). TERTt evokes an antitumor immune response in vivo which is targeted to TERT. TERTt can be used as an antigeneic sequence to produce anti-TERT tumor vaccine.