Antigen-specific Tumor Immunotherapy- HPV-16 E7-expressing tumor cell line as a model

• Main Authors: Chien-Hung Chen, 陳健弘
• Other Authors: Ding-Shinn Chen
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/19248856911267393432

博士 === 國立臺灣大學 === 臨床醫學研究所 === 89 === Hepatocellular carcinoma (HCC) is one the most common malignancies in the world, especially in sub-Saharan Africa and Southeast Asia. Since 1984, it has been the leading cause of cancer death in Taiwan. About 6000-8000 people died of this cancer every year in Taiwan. Though regular sonographic examination can early detect small HCC and there are many therapeutic modalities for HCC, the therapeutic results remain unsatisfactory because of the high recurrent rate, especially intrahepatic recurrence. The survival rate can be improved if the recurrent foci can be early detected and even eradicated. Furthermore, metastasis is a common cause of death in many cancer patients. Conventional therapies, such as surgery, chemotherapy and radiotherapy, have limited success in controlling the cancer metastases. Antigen-specific immunotherapy is a potential alternative for the treatment of limited residual tumor burdens. To investigate the antigen-specific immunotherapy, a well-characterized tumor antigen is needed. However, the ideal tumor antigens of HCCs are still unknown. Thus, we choose a human papillomavirus-16 (HPV-16) E7 as a model antigen for the experimental vaccine development in this thesis. Immunotherapy as a potential alternative to cancer therapy The ideal cancer therapy should have the potency to eradicate systemic tumor in multiple sites in the body and the specificity to discriminate between neoplastic and non-neoplastic cells. In both aspects, immunotherapy is an attractive approach. The antitumor effects of the immune system is mainly mediated by cellular immunity. The cell mediated component of the immune system is equipped with multiple effector mechanisms capable of eradicating tumors, and most of these antitumor immune responses are regulated by T cells. T cells also possess the ability to recognize tumor-specific antigens which serve as targets that T cells can use to distinguish neoplastic from non-neoplastic tissues. Importance of cell mediated immune responses in controlling tumors Tumor-specific antigens, when efficiently presented by antigen presenting cells to both CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ helper T cells, are capable of inducing potent T cell mediated immunity. T cell-mediated immunity is considered to be the key player in the antitumor immunity. Activated T cells may function directly as effector cells, providing antitumor immunity through the lysis of tumor cells or through the release of cytokines capable of interfering with the propagation of tumors. While most of the focus in cancer immunology is on CD8+ CTL responses, recent evidence indicates that CD4+ T cells are an equally critical component of the antitumor immune response. Successful immunity to cancer will therefore require activation of tumor-specific CD4+ T cells. Thus, the ideal cancer vaccine would enhance both CD8+ and CD4+ T cell responses by delivering a tumor-specific antigen into both the major histocompatibility complex I (MHC-I) and MHC-II pathways of antigen presentation. Antigen processing and presentation pathways It is now clear that at least two distinct pathways exist for the processing of antigens recognized by T cells. CD8+ CTLs recognize antigens that are presented on major histocompatibility complex class I (MHC-I) molecules. MHC-I molecules are expressed on most cells of the body and carry peptide fragments of endogenously synthesized proteins. These proteins are digested by proteosomes to peptide fragments, which are then transported from the cytoplasm into the endoplasmic reticulum where they complex with newly assembled MHC-I molecules on their way to the cell surface. In this way, CD8+ CTL are capable of identifying novel foreign antigens derived inside the cell. In contrast, CD4+ T helper cells identify peptide antigens that are presented on MHC class II molecules (MHC-II) predominantly expressed on specialized APCs such as macrophages, dendritic cells (DCs), and activated B cells. For CD4+ T cells to recognize complex antigenic proteins, the exogenous antigens must first be engulfed by APCs and delivered to low pH endosomal and lysosomal compartments containing proteases, where they are degraded into peptide fragments. The peptide fragments are further sent to the compartments of peptide loading where they bind with MHC-II molecules and are presented to CD4+ T helper cell. Tumor specific antigens, when efficiently presented by APCs to both CD8+ CTLs and CD4+ helper T cells, are capable of inducing potent T cell mediated immunity. Dendritic cell as a central player for tumor vaccine development DCs are the most potent professional APCs that prime helper and killer T cells in vivo. DCs can stimulate T cells because of their high levels of MHC-I and MHC-II molecules, co-stimulatory molecules like B7, and adhesion molecules like intercellular cell adhesion molecule-1 (ICAM-1) and ICAM-3 and lymphocyte function-associated antigen-3. To effectively present antigens, DCs perform a series of coordinated tasks. Immature DCs develop from hematopoietic progenitors and are strategically located at body surfaces and in the interstitial spaces of most tissues. There, DCs are equipped to capture antigens and to produce large numbers of immunogenic MHC-peptide complexes. In the presence of maturation-inducing stimuli such as inflammatory cytokines or stimulation via CD40, DCs upregulate adhesion and costimulatory molecules to become more potent, terminally differentiated, stimulators of T cell immunity. At the same time, numerous intracellular MHC-II compartments seem to discharge MHC-II-peptide complexes to the cell surface where they can be unusually long lived. DCs also migrate to secondary lymphoid organs to select and stimulate rare antigen-specific T cells. Thus, vaccine strategies employing DCs to enhance T-cell mediated immunity against tumor have become extremely important and attractive. Identification of tumor specific antigen as a prelude for development of antigen-specific cancer immunotherapy The field of cancer immunotherapy is moving toward antigen-specific vaccines based on the recognition that there are tumor-specific antigens which can be identified by T cells. There are several advantages of using an antigen-specific cancer vaccine. It is less likely to generate non-specific autoimmunity. It is more flexible in deciding the amount of antigen administered and the methods of antigen presentation to the immune system. It is more reproducible from patient to patient. It has the potential to correlate clinical outcome to a specific immune response. Therefore, antigen-specific immunotherapy represent a desirable approach for controlling tumors. Several vaccines strategies aiming at controlling tumors have been developed. These strategies can arbitrarily be classified to five categories based on the form of vaccine administered: 1) vector-based vaccines; 2) peptide-based vaccines; 3) protein-based vaccines; 4) DNA vaccines; and 5) cell-based vaccines. HPV-16 as a model antigen In this thesis, we chose HPV-16 E7 as a model antigen for vaccine development because HPVs, particularly HPV-16, are associated with most cervical cancers. HPV oncogenic proteins E6 and E7 are coexpressed in most HPV-containing cervical cancers and are important in the induction and maintenance of cellular transformation. Therefore, vaccines or immunotherapies targeting E7 and/or E6 proteins may provide an opportunity to prevent and treat HPV-associated cervical malignancies. Furthermore, T.C. Wu has described a molecular approach that directly routed a nuclear/cytoplasmic antigen, HPV-16 E7, into the endosomal and lysosomal compartments by constructing a chimeric gene, Sig/E7/LAMP-1, in which E7 was linked to the endoplasmic reticulum translocation signal peptide (Sig) on its amino terminus and to the transmembrane and lysosomal targeting domains of the lysosome-associated membrane protein 1 (LAMP-1) on its carboxyl terminus. This specific targeting of HPV-16 E7 to the endosomal and lysosomal compartments allows antigenic peptides of E7 to complex with MHC class II molecules and enhances MHC-II-restricted CD4+ T cell response. In addition, a murine cell line of C57BL/6 background, designated TC-1, was also generated by co-transforming primary lung cells of C57BL/6 mice with HPV-16 E6 and E7 and activated ras oncogene. Specific aims Taking advantage of the established HPV-16 E7 system, we tried to ask the following questions: 1) Can Sig/E7/LAMP-1 vaccinia vaccine control TC-1 tumor grown in the liver ? 2) Can Sig/E7/LAMP-1 DNA vaccine control TC-1 tumor with lung and liver metastases ? 3) Can LAMP-1 strategy enhance the DNA vaccine potency and antitumor effect ? 4) What kind of combination for vaccine administration can lead to the most potent immune response ? 5) Is there any difference in the E7-specific immune response generated by DNA vaccine vs. vaccinia vaccine ? If the answer is yes, why ? 6) Can antigen-fused with heat shock protein increase the potency of DNA vaccine ? 7) Can we apply the new immunologic methods to analyze antigen-specific immune responses generated by the above vaccines ? Are the immunological results correlated with in vivo antitumor immunity ? In the past few years, we found the following results. Antigen-specific immunotherapy can control E7-expressing tumors grown in the liver. The liver is one of the most common sites for tumor metastasis and organ microenvironments may modulate the tumor cell responses to therapies. In addition, when developing clinical applications for the treatment of common solid malignancies, demonstrating efficacy of therapy within visceral sites such as the liver or lung is important. We used a recombinant vaccinia-based vaccine (vac-Sig/E7/LAMP-1) to control tumors grown in the liver. We found that vac-Sig/E7/LAMP-1 is an effective vaccine for controlling E7-expressing tumors grown in the liver. In the tumor prevention experiment (vaccination first, followed by tumor challenge), vac-Sig/E7/LAMP-1 generated a full protection against TC-1 grown in the liver, while vac-E7 generated only a 60% protection. In the tumor treatment experiment (tumor challenge first, followed by vaccination), vac-Sig/E7/LAMP-1 still provide 100% protection. In contrast, all vac-E7 vaccinated mice had TC-1 tumor grown in the mice. ELISPOT assay revealed that the highest number of E7-specific CD8+ T cell precursors was noted in the vac-Sig/E7/LAMP-1 vaccinated mice. The result of ELISPOT assay directly correlated with anti-tumor effect generated by Sig/E7/LAMP-1 vaccinia. These data implied that antigen-specific immunotherapy can be used to control tumors grown in the liver. Gene gun-mediated DNA vaccination induces antitumor immunity against E7-expressing murine tumor metastases in the liver and lungs. Though vaccinia vaccine can generate potent antitumor immunity, there are still some concerns about the safety of using vaccinia as vaccine vectors. DNA vaccination has emerged as an attractive approach for tumor immunotherapy because of the clear advantages of purity, simplicity of preparation, and stability. DNA-based vaccines can be prepared inexpensively and rapidly in large-scale. We used the gene gun method to vaccinate C57BL/6 mice intradermally with E7 DNA or Sig/E7/LAMP-1 DNA. We found that mice vaccinated with Sig/E7/LAMP-1 DNA generated the strongest E7-specific CTL activities, the highest numbers of E7-specific CD8+ T cell precursors (in both ELISPOT assay and intracellular cytokine staining), and the highest titers of E7-specific antibodies. While both E7 DNA and Sig/E7/LAMP-1 DNA generated potent antitumor immunity in the liver and lung metastases models, the Sig/E7/LAMP-1 DNA was more potent in a more stringent condition (vaccination once without booster). Therefore, DNA vaccination with E7-expressing plasmids were effective in controlling liver and lung metastases of an E7-expressing murine tumor. Based on the afore-mentioned results, antigen-specific immunotherapy (in either vaccinia form or naked DNA form) potentially can be applied to control liver and lung metastases of tumors with defined tumor- specific antigens. The LAMP-1 strategy can enhance the vaccine potency. Boosting with recombinant vaccinia increases HPV-16 E7-specific T cell precursor frequencies of HPV-16 E7-expressing DNA vaccines. We already demonstrated that both Vac-Sig/E7/LAMP-1 and Sig/E7/LAMP-1 DNA can generate strong antitumor immunity. To determine whether combination of Sig/E7/LAMP-1 DNA and Vac-Sig/E7/LAMP-1 can further enhance immune responses, sequential vaccination with Sig/E7/LAMP-1 DNA and Vac-Sig/E7/LAMP-1 was given. We compared seven groups of mice receiving different combination of vaccination (DNA priming/vaccinia boosting, DNA priming/DNA boosting, DNA priming/no boosting, vaccinia priming/DNA boosting, vaccinia priming/vaccinia boosting, vaccinia priming/no boosting, and control), and found that priming with Sig/E7/LAMP-1 DNA and boosting with Vac-Sig/E7/LAMP-1 generated the strongest E7-specific CD8+ T cell responses. The increase of E7-specific CD4+ T cell response by this combination was modest. However, the anti-E7 antibody titer did not increase by this strategy. Our results encourage the use of the DNA prime/ vaccinia booster regimen in future clinical trials. Recombinant DNA vaccines protect against tumors that are resistant to recombinant vaccinia vaccines containing the same gene. We hypothesized that different delivery systems may influence the pattern of antigen-specific immune response and the outcome of antitumor effect. We therefore evaluated recombinant vaccinia virus and naked DNA, two common gene delivery systems, for generation of antigen-specific immune responses and antitumor effects. In this experiment, we performed a head-to-head comparison of optimized delivery of vaccinia vaccines and DNA vaccines using dose-escalating tumor challenge. At a lower dose (5x104) of TC-1 dose, Sig/E7/LAMP-1 vaccinia and Sig/E7/LAMP-1 DNA generate a 100% tumor protection for up to 6 weeks after tumor challenge. However, at a higher dose of 1x106 TC-1 cells/mouse, Sig/E7/LAMP-1 DNA provided a 100% protection against subcutaneous growth of tumors while Vac-Sig/E7/LAMP-1 protected only 40% of the mice. Furthermore, only Sig/E7/LAMP-1 DNA vaccines, but not Vac-Sig/E7/LAMP-1, are capable of protecting against a challenge with a more stringent subclone of TC-1, designated TC-1 P2, a cell line established from TC-1 tumors that survived initial Vac-Sig/E7/LAMP-1 vaccination. In addition, immunological assays revealed that both vaccines induced comparable levels of E7-specific CD8+ T cell precursors (in both ELISPOT assay and intracellular cytokine staining) and anti-E7 antibody titers. Interestingly, Vac-Sig/E7/LAMP-1 induced E7-specific both IFN-g- and IL4-secreting CD4+ T cell precursors while Sig/E7/LAMP-1 DNA induced only E7-specific IFN-g-secreting CD4+ T cell precursors. In other words, Sig/E7/LAMP-1 DNA induced a Th1-predominant immune response, while Vac-Sig/E7/LAMP-1 induced a balanced one. Both Vac-Sig/E7/LAMP-1 and Sig/E7/LAMP-1 DNA can generate 100% protection against high-dose (1x106) TC-1 in IL-4 knock-out mice. These results implied that IL-4 generated by vaccinia vaccine may have a negative effect on the antitumor immunity. Our data implied that DNA vaccines may provide a better tumor protection than vaccinia vaccines employing the same gene, even against challenge using a more stringent tumor model. The results may have implications in the future design of antigen-specific cancer immunotherapy. Linking antigen gene to an HSP70 gene can enhance the potency of DNA vaccine. We have demonstrated that targeting E7 to MHC-II presentation can enhance the potency of DNA vaccine, enhancing the MHC-I presentation may be an alternative to increase the vaccine potency. Linkage of antigens to HSP represents a potential approach for increasing the potency of DNA vaccines. In the past few years, immunization with HSP complexes isolated from tumor or virus-infected cells has been shown to be able to induce potent antitumor immunity. These experiments demonstrate that (1) HSP-peptide complexes derived from tumor cells, but not from normal tissue, can stimulate tumor-specific immunity; (2) the specificity of this immune response is caused by tumor-derived peptides that are bound to the HSPs, not by the HSPs themselves; (3) the immune response can be induced in mice with MHC either identical or different to the MHC of donor HSPs. These investigations have made HSPs more attractive for use in immunotherapy. Using HPV-16 E7 as a model antigen, we evaluated the effect of linkage to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of antigen-specific immunity generated by naked DNA vaccines. We found that vaccines containing E7-HSP70 fusion genes increased the frequency of E7-specific CD8+ T cells by at least 30-fold relative to vaccines containing the wild type E7 gene. More importantly, this fusion converted a less effective vaccine into one with significant potency against established E7-expressing tumors. The E7-HSP70 DNA vaccine can provide a 100% protection against the growth of TC-1 tumor even in mice vaccinated once without further boosting. The enhanced potency needs E7 to be fused with HSP70 because vaccination with a mixture of E7 plasmid plus HSP70 plasmid did not enhance the immune response or the antitumor immunity. Using monoclonal antibodies to selectively deplete CD8+ T cells, CD4+ T cells, or NK cells, we found that E7-HSP70 fusion vaccines exclusively targeted CD8+ T cells; immunologic and antitumor effects were completely CD4-independent. These results indicate that fusion of HSP70 to an antigen gene may greatly enhance the potency of DNA vaccines via CD8-dependent pathways. ELISPOT assay and intracellular cytokine staining to analyze E7-specific T cell responses. The most common immunological assay used in most previous studies for T cell responses is chromium release assay and T cell proliferation assay. These techniques suffers from the drawback that they do not enable analysis of single cell responses in the context of an unselected cellular background. The chromium release assays may underestimate the real number of CTLs. In contrast, ELISPOT assay and intracellular cytokine staining are exquisitely sensitive and can detect low frequency T cells. They can detect the cytokine release from an activated T cell and can be used for enumeration of single cytokine-secreting cells. These assays are believed to be at least 30-100 times more sensitive than the chromium release assay, and is efficient and fast because it does not require any in vitro cellular proliferation. In this thesis, we successfully apply the ELISPOT assay and intracellular cytokine staining for the analysis of E7-specific CD4+ and CD8+ T cell responses. Compared with chromium release assay, both methods are more sensitive, less labor-intensive, and thus can be applied to analyze clinical materials. Conclusion and perspectives We demonstrated that antigen-specific tumor vaccine, in the form of vaccinia vaccine, DNA vaccine, could control the subcutaneous tumor growth or liver and lung metastases. Though we used HPV-16 E7 as a model antigen, the strategies described in this thesis can be applied to other tumors with know tumor-specific antigens. The immunological assays (ELISPOT assay and intracellular cytokine staining) described in this thesis can even have a broader application. They can be used to analyze any antigen for antigen-specific immune response. For example, we are currently applying these assays to investigate the hepatitis B virus (HBV)-specific T cell responses in patients infected with HBV. The initial encouraging results for experimental vaccination to treat tumors emphasize the need to accelerate the pace of exploration in these systems. Although it is still difficult to generate a dramatic therapeutic response by tumor vaccines in patients with bulky tumor burdens, cancer vaccines are likely to be effective in patients with limited residual tumor burdens. Another direction to improve the efficacy of tumor vaccine is to investigate how to break the immunosuppression induced by the tumors. With more understanding of basic immunological responses of the host on the occurrence of tumors, strategies using more potent tumor vaccine may be developed in the future.