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Recently, the emerging studies have revealed that the 78 kDa glucose-regulated protein (GRP78) plays a crucial role in cancer progression. GRP78 was first discovered as a cellular protein induced by glucose starvation (1). It is also recognized as the immunoglobulin heavy chain binding protein (Bip) or heat shock 70 kDa protein 5 (Hsp70-5) (1, 2). GRP78 belongs to HSP70 protein family, which comprises homologous conserved chaperon proteins, facilitating protein folding and mediating protection against damage under stress. The gene HSPA5 is located on chromosome 9 and encodes a constitutively expressed compartment-specific protein (3). Distinct from other proteins in the family, GRP78 contains an ER-location signal sequence at N-terminal and a "KDEL" ER-retention signal at C-terminal, thus normally, GRP78 resides primarily in the ER of all cells, functioning as a ER chaperon as well as an ER stress signalling regulator with anti-apoptotic properties (4). However, there is a growing body of research have found that GRP78 is overexpressed in the cytoplasm and also on the cell surface in malignant cells, such as breast cancer (5), gastric cancer (6), hepatocellular cancer (7), and prostate cancer (8). This increased expression of GRP78 is associated with cancer proliferation, malignancy, metastasis, and resistance to a variety of therapy (9).
GRP78 in Prostate Cancer
Prostate cancer, a typical epithelial adenocarinoma, progresses slowly through a series of clinical stages. It can be initially detected by monitoring the level of prostate-specific antigen (PSA) in the blood, and sequentially diagnosed based on microscopic evaluation of the biopsy. Most patients are diagnosed with localized disease, which is potentially curable, can be treated with surgery or radiotherapy. If prostate cancer returns with rising PSA level, the optional treat for this biochemical recurrence is androgen ablation includes chemical castration or surgical castration. However, once prostate cancer becomes resistant to this treatment, it spreads to other tissues and develops to the stage as known as castration-resistant metastatic disease, which is generally incurable. At present, the primary therapy in this state of prostate cancer is chemotherapy (10).
GRP78 was initially identified through eptiope fingerprinting of circulating immunoglobins from the serum of prostate cancer patients. The reactivity against GRP78 of serum showed an apparently increased percentage as prostate cancer progressed relative to other malignant tumors (11). Furthermore, a large population of prostate cancer patients biopsy staining have shown that GRP78 expression was significantly higher in the primary tumour sites compared with benign prostatic tissue (8), and its expression was correlated with castration-resistance, great risk of clinical recurrence, and poor survival (12). Therefore, considering the intensity of GRP78 in castration-resistant and metastatic prostate cancer, GRP78 may sever as an ideal cancer antigen to induce a therapeutic anti-tumour immune response and prevent cancer recurrence after conventional treatments. Thus, combing GRP78-based cancer vaccine with androgen ablation could lead to better response in advanced cancer patients.
Induction of Tumour Specific Cytotoxic T Cells
Cellular immune response is considered to be more effective to kill tumour cells. In principle, the goal of this vaccine is to stimulate cytotoxic T cells (TC) of sufficient magnitude to eliminate pre-existing tumour, and also to maintain a memory population preventing its recurrence. This requires helper T cells (TH) support, which are crucial for the induction and generation of immune memory. For activation of naÃ¯ve T cells, DNA vaccine encoded antigen must be processed and presented on MHC molecules by dendritc cells (DCs), which can be recognized by T cell receptors (TCRs) on T cells. Costimulation is also required for optimal activation via the ligation of CD28 on T cells and B7 molecule on DCs. Moreover, license of DCs, which is essential for generative high avidity TC response, can be stimulated either by TH through the interaction of CD40-CD40-ligand (CD40L), or by pathogen materials through Toll-like receptors (TLRs) (13). Once DCs are activated, they are able to promote CD8+ T cells and generate anti-tumour effector TC, a proportion of which become memory TC. When encountering with recurred cancer cells expressing the same target peptide, memory T cells can reproduce stronger immune response rapidly. However, the second expansion of TC requires IL-2 which are secreted by TH, and once induced, memory TC rely on IL-15 for maintaining their numbers and functions (14).
DNA Vaccine Design
In order to stimulate both CD4+ TH cells and CD8+ TC cells with high-avidity as possible. This vaccine contains genes which encoding a 9 amino acid long peptides that binds to HLA-A2 (HLA-A*0201) and a 15 amino acid long one that binds to HLA-DR4 (HLA-DRB1*04). The HSPA5 gene is highly conserved in eukaryotes, hence the epitope sequence of GRP78 binding MHC molecules, which are expressed both in mice and human, are predicted by the SYFPEITHI Database. Those with intermediate affinity scores are applicable owing to that self-tolerance may lead to deletion of T cells specific for the high affinity self-peptides during thymic selection. In addition, immunity response tends to focus on immunodominant peptides, low affinity peptides might not be presented by MHC molecules (14).
DNA vaccine, compared with other types of vaccine, is highly flexible, allowing to encode several immunological components of interest, is precisely synthesized in large-scale production, are low cytotoxic and stable in vivo. Additionally, DNA vaccine have been proved to elicit strong CD4+ TH cells and CD8+ TC response (15). However, the main disadvantage of DNA vaccine is its low immunogenicity. In order to improve this, the epitope sequence is incorporated into a bacterial plasmid and under control of a mammalian promoter such as CMV immediate/early promoter and its adjacent intron A sequence (CMV-inA) (14). This bacterial derived plasmid includes the unmethylated cytosine-phosphate-guanine (CpG) motif, which can stimulate innate immunity by binding to TLR9 on DCs, can trigger adaptive immunity and help TC priming (16).
Other molecules which can help stimulating immune response are also incorporated into this DNA vector, including chemokine GM-CSF to enhance DCs proliferation and maturation (17), costimulatory molecules TRICOM (LFA3-CD8-ICAM1) to facilitate T cell activation (10), and cytokines IL-2 and IL-15 to activate T cells proliferation and maintain its memory functionality (14). Furthermore, in order to block the immunosuppresion of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) which inhibit CD8+ T cell function through the secretion of suppressive cytokines in the tumour microenvironment, anti-CTLA4 antibodies and anti-PD1 antibodies are co-injected (18).
Manipulation of vaccine
DNA vaccine is injected intradermally by gene gun, which has been shown to activate higher TC response and anti-tumour effects with the least dose (19). DNA plasmid contains CpG which binds to TLR9 on DCs stimulating innate immunity. Additionally, direct transfection of DNA into epidermal keratinocytes or Langrhans cells contributes to antigen presentation. In the former route, encoded proteins are then expressed in keratinocytes, a proportion of which will undergo cell death. DCs then capture cell debris, including the vaccine-encoded GRP78 peptides, and present it. In the other route, transfected Langerhans cells then migrate to lymph node and become mature, allowing direct presentation. While DCs present HLA-A2 binding 9-mer peptide to CD8+ T cells, HLA-DR4 binding 15-mer peptide activate anti-GRP78 CD4+ T cells, which in turn license of the DCs through CD40 ligation and secreted cytokines. Then the licensed DCs are capable of priming and boosting anti-GRP78 CD8+ T cells. Furthermore, anti-CTLA4 antibodies and anti-PD1 antibodies are co-injected intraperitoneally to inhibit MDSCs and Tregs and to increase the infiltration of anti-GRP78 T cells into tumour site and hence causing tumour lysis. Other encoded protein includes chemokine, costimulatory moleculues, and cytokines also facilitate this immune response and activate high-avidity anti-tumour TC.
In vitro. Several epitope sequences with intermediate affinity to MHC molecules are chosen. To evaluate whether these 9-mer peptides bind to HLA-A2, T2 stabilisation assay are used to examine peptide binding. Then, to identify which 9-mer peptide bound HLA-A2 can be recognized by a large population of T cells from HLA-2A transgenic, HLA-DR4 transgenic or C57Bl/6 mice (4-6-week-old male) through MHC tetramer analysis. Also, CD4+ T cells or CD8+ T cells are isolated to test their proliferation respectively in the presence of each MHC-restricted GRP78 peptide-pulsed DCs by in vitro proliferation assay. The haplotype of MHC molecules is confirmed by flow cytometry.
Moreover, to determine the combination of peptides with higher efficacy to stimulate optimal TC response, mice are immunized repeatedly with GRP78-based DNA plasmid via gene gun. The frequency and timing of immunization need to be comfirmed. To test effector TC function, splenocytes are isolated from mice and are measured the secretion of IFN-ï§ and granzyme B by ELISPOT assay ex vivo against each specific 15-mer peptide. The effector TH function is also checked for IL-2 and IFN-ï§ with each specific 9-mer peptide. TC are analyzed the ability of lysing the GRP78-overexpressed TRAMP-C1 cells by Cr release assay. TRAMP-C1 cell is a cell line derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) which is tumourigenic in syngeneic C57Bl/6 mice (20). Owing to lack of GRP78 overexpression in TRAMP-C1, cells could treated with thapsigargin, which is a stress-induced agent to up-regulate GRP78 expression (21). Alternatively, cells could transfected with grp78 using lentiviral vector, and utilizing drug selection to select a stable GRP78-overpressed clone. The best combination of peptides, including 9-mer and 15-mer, with most ability of activating TC is selected for further study.
In a preclinical model. To verify whether this vaccine enhances priming and prevent the tolerance of anti-tumour TC response, mice are injected orthotopically with GRP78-overexpressed TRAMP-C1 cells and then are immunized with DNA vaccine via gen gun and the indicated antibody intraperitoneally. The timing and dose of antibody injection need to be further confirmed. The effector CD4+ T cells and CD8+ T cells of the vaccine and prostate draining lymph nodes as well as prostate are isolated, and are analyzed their percentage and ration to Tregs and MDSCs, which may serve as predictor of therapeutic efficacy. Their effector functions are also evaluated by measure the secretion of cytokine with each GRP78 peptide-pulsed DCs. In addition, to minor tumour growth and lymph node metastasis, genomic DNA from prostate or draining lymph nodes of mice are screened for the SV40 transgene by PCR (22). Survival of mice is also compared with untreated group.
In patients. The aim of clinical study is to evaluate safety and efficacy of GRP78-based vaccine combined anti-CTLA4 antibodies and anti-PD1 antibodies in cancer patients. In phase I study, toxicity in advanced prostate cancer patients who have failed both first-line (radical prostatectomy or radiation) and second-line (radiation, androgen ablation, and/or chemotherapy) treatments is determined, in which physical examination, complete blood count, liver and kidney function, diagnostic imaging, and electrocardiogram are monitored. Owing to the vaccine -induce response is specialized against patients with HLA-A2 and HLA-DR4 MHC molecules, patients characterization need to be confirmed prior to vaccination. Also, binding of peptide-bound HLA-A2 against T cells of patients can be verified by MHC tetramer analysis. Then, anti-tumors immune response of patients is examined, which includes antibodies measurement and T cells analysis. Serial dilution of patients serum are incubated with a GRP78-overexpressed prostate cancer cells and are analyzed the titer of anti-GRP78 antibodies by ELISA or flow cytometry. To verify the anti-GRP78 TC response, lymphocytes of patients are isolated and detected the production of IFN-ï§ by ELISPOT against purified GRP78 protein, and lysis of GRP78-overexpressed prostate cancer cells by Cr release assay. Moreover, the clinical assessment is determined by the level of serum PSA and patient survival. In phase II study, the maximum tolerated dose and efficacy of vaccine are determined. Patients are separated into groups with different dose of vaccination. If no significant toxicities are detected with the initial dose, the dose escalation may continue until reach a maximum tolerated dose. In phase III study, patients with minimal residual prostate cancer (castration-resistant) are conducted in a double blind randomized study. The same examinations are administered as mentioned before. Furthermore, efficacy of this vaccine will compare to conventional treatments such as chemotherapeutic drugs.