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In previous studies we have shown that HSP70-peptide complexes (HSP70.PC) derived from the fusion of dendritic cells (DC) to tumor cells (HSP70.PC-F) possess superior properties compared to HSP70.PC from tumor cells. HSP70.PC-F are more effective in stimulation of DC maturation and induction of CTL that are able to provide protection of mice against challenge with tumor cells. To develop an improved formulation of HSP70.PC-based tumor vaccine for patient use, we extracted HSP70.PC-F from DC fused to patient derived ovarian cancer or established human breast cancer cells and examined their properties as tumor vaccines. HSP70.PC-F induced T cells that express higher levels of IFN-γ and exhibit increased levels of killing of tumor cells compared to those induced by HSP70.PC derived from tumor cells. Enhanced immunogenicity of HSP70.PC-F was associated with improved composition of the vaccine including increased content of tumor antigens and their processed intermediates, and the detection of other HSPs such as HSP90 and HSP110. The present study has therefore provided an alternative approach to preparation of HSP-based vaccines using DC/tumor fusion technology and gentle and rapid isolation of HSP.PC.
Heat shock proteins (HSPs) are members of a number of families of stress-induced proteins, whose main intracellular functions are as molecular chaperones (1–3). The HSPs possess the intrinsic property of recognizing unfolded/disordered sequences in target polypeptides, then aiding the folding/refolding of such sequences, targeting them to the proteasome for destruction (4). In addition, after exiting the cell and entering the extracellular environment, HSPs are capable of promoting antigen presentation of chaperoned peptides through interaction with the receptors on antigen presenting cells (APC) (5), a process called antigen cross-presentation. Thus, when HSPs form complexes with peptides (HSP.PC) that are derived from tumor cells, they possess the qualities of tumor vaccine. In previous animal studies, immunization with HSP.PC purified from tumor cells provided protection against tumors from which the HSP.PC were derived (6–9). In addition, treatment of mice with established tumor with the vaccines can slow the rate of tumor growth and stabilize disease (10, 11). In clinical trials, autologous tumor-derived HSP96.PC were used to treat a variety of malignancies including melanoma (12–14), colorectal cancer (15), renal cell carcinoma (16) with immunological and clinical responses in a subset of patients. Overall, the clinical response was muted in the randomized phase III trial (14, 16). These results suggest a need for enhancement in the potency of such vaccines.
We have attempted to produce an improved HSP70-based vaccine which might have application in the clinic. In our previous study, HSP70-peptide complexes extracted from fusions of dendritic cells (DC) and tumor cells (HSP70.PC-F) contained enriched antigenic peptides compared with HSP70.PC derived from tumor cells and possessed superior properties over its counterpart from tumor cells (17). To develop an HSP70.PC-based tumor vaccine with enhanced immunogenicity for patient use, we extracted HSP70.PC-F from DC fused to ovarian cancer cells from patients or established human breast cancer cells, respectively, and examined their properties as tumor vaccines. Our studies show that HSP70.PC-F carry increased levels of peptides of tumor antigens that stimulate enhanced T cell responses against tumor cells. In addition, the experiments provide a proof of principle indicating the use of alternative sources of DC and tumor cells to produce HSP70.PC vaccine.
Human breast carcinoma cells MCF-7 (HLA-A*0201+), BT-20 (HLA-A*0201−/A*1101−), ZR75 (HLA-A*1101+) and SKBR3 (HLA-A*1101+) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and maintained in medium according to the manufacture instruction.
Ovarian carcinoma cells (OVCA) were obtained from patients diagnosed with ovarian cancer (18). Briefly, the resected tumors were weighted and minced to small pieces (1–3 mm), and then mashed through a sterile 50-μm nylon mesher (Sigma, St. Louis, MO) in tissue culture hood. Single-tumor cell suspensions were obtained by processing a filter and serum column to remove dead tumor cells and other type cells. OVCA tumor cells (about 80–95% purity) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated human AB serum, and used as fusion partner, CTL targets or extraction of HSP70.PC from them.
Peripheral blood mononuclear cells (PBMC) were isolated from leucopacks obtained from healthy donors using Ficoll density gradient centrifugation (Ficoll-PaqueTM plus, GE healthcare Bio-Sciences AB, Sweden). PBMC were collected and plated in tissue culture dish with 5% human AB male serum (Sigma) in RPMI 1640 medium containing 2mM L-glutamine, 100U/ml penicillin, and 100μg/ml streptomycin for 1 hour in a humidified CO2 incubator. The adherent fraction was cultured in 1000U/ml GM-CSF (Genzyme, Framingham, MA) and 500U/ml IL-4 (R&D Systems, Minneapolis, MN) RPMI/AIM-V (1:1) medium with 1% of human AB male serum for 5 days. On day 4, the loosely adherent cells were collected and further enriched through repeated adherence method for fusion use. The non-adherent cells were frozen with 10% DMSO in human AB serum and used as source of T cells.
Tumor cells were mixed with DC preparations at 1:10 and washed in serum-free pre-warmed RPMI 1640 culture medium. The mixture cell pellet was resuspended in a Polyethylene glycol solution (PEG, MW1450, Sigma-Aldrich) for 5 min at room temperature, and then was progressively added pre-warmed serum-free RPMI medium to dilute the PEG in the next 5 min. After washing, fusion cells were resuspended in RPMI 1640 medium supplemented with 5% human AB serum and 500U/ml GM-CSF, and cultured in 5% CO2 at 37°C for 5 days.
Tumor and DC/tumor fusion cells were collected and incubated with lysis buffer (50mM Tris-HCl, pH 8.0, containing 50mM NaCl, 1% Nonidet P-40, 1mM PMSF) on ice for 30 min. The lysates were clarified by centrifugation, and the aqueous phase were collected and incubated with mAb against human HSP70 (5C1A12, Developed by ProMab Biotechnologies, Inc., Albany, CA) at concentration of 1/100 overnight at 4°C. Then, 50μl of protein A/G (1/1) agarose were added and incubated at 4°C for an additional 1.5 hours. After extensive wash with lysis buffer, the immunoprecipitates were eluted with PBS with high salt (500mM NaCl). The elute of HSP70.PC-F (derived from DC/tumor fusion cells) or HSP70.PC-Tu (derived from tumor cells) were used to stimulate the lymphocytes. The HSP.PC preparations were checked by limulus amebocyte lysate (LAL kit, Cambrex Bio Science Inc., Walkersville, MD) assay to ensure no contamination of endotoxin. For protein analysis, the immunoprecipitates were dissolved in Laemmli SDS sample buffer (0.1 Tris-Cl, 4% SDS, 20% glycerol, 0.05% bromphenol blue, 5% 2-ME) and analyzed by immunoblotting.
The proteins from cell lysates or immunoprecipitation with anti-HSP70 antibody (19) were subjected to SDS-PAGE and transferred on nitrocellulose membrane. The membranes were incubated with anti-HSP40 (SPA-400), anti-HSP90 (SPA-830), HSP110 (SPA-1101) antibodies (Stressgen, Ann Arbor, MI), anti-HSP70 (5G10, BD Pharmingen, San Diego, CA), anti-MUC1 (HMPV, BD Pharmingen), anti-MUC1 peptide antibody (BCP8, anti-DTR (20)), anti-c-ErbB2/c-Neu (Ab-3, Calbiochem, San Diego, CA) and β-actin (Sigma). The Ag/Ab complexes were visualized by ECL (ECL Detection System; GE). Densitometric analysis of the membranes was performed using GelDoc 2000 (Bio-Rad, Hercules, CA).
DCs, breast cancer cells and patient-derived ovarian carcinoma cells (OVCA) were incubated with anti-human mAbs against MUC1 (HMPV), HLA-DR (TU36), CD86 (IT2.2, anti-B70/B7-2), anti-HLA-ABC (W6/32) (BD Pharmingen); anti-HLA-A2 (0791HA) and HLA-A11 (0284HA) (One lambda, Canoga park, CA); anti-c-ErbB2/c-Neu (TA-1, Oncogene Research), and anti-CA125 (NCL-L-CA125, Novocastra Laboratories, UK). After 1 hour incubation with Abs, the cells were washed and incubated with FITC-conjugated anti-mouse IgG (Chemicon International Inc., Temecula, CA). To determine the efficiency of DC/tumor fusions, the fusion cells were dual stained with FITC-conjugated anti-MUC1 (HMPV) and PE-conjugated anti-HLA-DR (TU36) or anti-CD86 (IT2.2) (BD Pharmingen). Cells were fixed in 2% paraformaldehyde and analyzed by flow cytometry using CellQuest software (BD Biosciences).
Approximately 2×104 DC/tumor fusion cells were spin onto slides by Cytospin (Thermo Shandon, Waltham, MA). The cells were dual stained with FITC-conjugated anti-MUC1 (HMPV) and PE-conjugated anti-CD86 (IT2.2) for 1 hour at 4°C, respectively. The cells were washed, fixed and analyzed using a Laser Scanning Confocal microscope (TE2000-E, Nikon, Melville, NY).
T cells (nonadherent cell population, 1×107) were resuspended with 2μg/ml HSP70.PC extracted from tumor or fusion cells in RPMI-1640 medium containing 10% human AB serum, 10U/ml human IL-2, 15 mM HEPES, 2 mM L-glutamine, 100U/ml penicillin, 100μg/ml streptomycin, and 5×10−5 M β-mercaptoethanol. DC were added at 1:10 ratio into T cells and seeded in 96-well U bottom plates in a volume of 200μl/well for 5 day cultures. T-cell proliferation were assessed by [3H]thymidine incorporation after an additional 12-h incubation with 1μCi/well of [3H]thymidine. Radioactivity (mean ± SD of triplicates) was measured by liquid scintillation counting.
Lymphocytes (nonadherent cell population) were stimulated with 2μg/ml HSP70.PC extracted from tumor or fusion cells in the presence of 10U/ml human IL-2 in RPMI medium containing 10% human AB serum for 5 days. On day 5, the T cells were collected and purified through a nylon wool column to remove DC, and then stained with anti-human CD4/IFN-γ or anti-human CD8/IFN-γ (BD Pharmingen) according to the manufacture instruction. For tetramer staining, T cells obtained after passing through nylon wool column were incubated with PE-conjugated MUC1 tetramer 1 (HLA-A*0201, STAPPVHNV) or PE-conjugated HER2/Neu tetramer (HLA-A*0201, ILHNGAYSL) (PROIMMUNE, Oxford, UK) for 1 hour at 4°C. After wash, the T cells were further stained with FITC-conjugated anti-CD8 mAb for 40 min at 4°C. Cells were washed and fixed with 2% paraformaldehyde, and analyzed by flow cytometry using CellQuest software (BD Biosciences).
T cells (nonadherent cell population) from normal donor were mixed with autologus DC (DC/lymphocytes at 1:10 ratio) and place in 6 well plate with 2μg/ml HSP70.PC extracted from tumor or fusion cells in RPMI-1640 medium containing 10% human AB serum and 10U/ml human IL-2. After 5 day cultures, the cells were collected and T cells were purified through a nylon wool column using for effector cells. Breast tumor cells or OVCA isolated from ovarian cancer patient were labeled with 100μCi Na251CrO4 for 60 min at 37°C, and then washed to remove unincorporated isotope. In antibody blocking assay, the targets were incubated with indicated antibodies for 1 hour on ice. The effector T cells (E) or tumor target cells (T) were resuspended in CTL assay medium at indicated E: T ratios and placed in 96 well V-bottom plates for 5 hours at 37°C. After incubation, the supernatants were collected and radioactivity was quantitated in a gamma counter. Spontaneous release of 51Cr were determined by incubation of targets in the absence of effectors, and maximum or total release of 51Cr by incubation of targets in 0.1% Triton X-100. Percentage-specific 51Cr release was calculated using the following equation: percent specific release = [(experimental – spontaneous )/(maximum – spontaneous ) ] × 100.
Statistical significance was analyzed using χ2, One-way ANOVA or Student’s t test. P<0.05 indicates statistic significance.
Monocyte-derived DCs from donors were generated in the presence of GM-CSF and IL-4 and fused to human breast cancer cells including MCF-7 or BT-20, and analyzed for tumor or DC markers. The breast cancer cells were positive for BCP8 (anti-MUC1 peptide antibody) and expressed predictably high levels of HER2/neu and MUC1, but minimal levels of HLA-DR and CD86 molecules that were expressed at high levels by DC (Fig. 1A–C). In contrast, fusions of DC (HLA-A*0201) and breast cancer cells expressed both the tumor-derived antigens MUC1 and the DC-derived costimulatory molecules, CD86, and HLA-DR (Fig. 1B and C). In addition, DC/tumor fusion cells were also produced using ovarian cancer cells isolated from clinical samples. Ovarian cancer cells expressed high levels of tumor antigens HER2/neu, MUC1, CA-125 and HLA-ABC molecules (Fig. 1D and supplemental Table 1). As with the breast cancer cells, fusions of DC and OVCA cells expressed significant levels of MUC1, MUC1 peptides detected by BCP8, and/or CA-125 as well as HLA-DR or CD86 (Fig. 1E). When we examined the fusion efficiency between tumor and DC in these experiments, we observed 14.4–38% for DC fused to breast cancer cells (Fig. 1B and C), 14.9–44% for DC (DC9, HLA-A*0201+ and DC10, HLA-A*0201+/A*1101+) fused to patient-derived ovarian carcinoma cells (DC/OVCA) by two-color FACS analysis (Fig. 1E). We further characterized the fusion cells by confocal microscopy. Tumor-derived cell surfaces were identified by MUC1 expression (green color) (Fig. 1F, left panel), while DC were identified as CD86-postive DC stained orange cells (Fig. 1F, middle panel). Overlapping these images resulted in detection a subset of cells with yellow color (Fig. 1F, right panel), indicating DC/tumor fusion.
The fusion process introduces abundant tumor antigens into the efficient antigen processing milieu of the DC cytoplasm. It has been demonstrated that HSPs play a role in the processing of these antigens and chaperoning processed peptides (21). We thus hypothesized that we could potentially obtain enriched tumor antigens and their intermediates from human DC-tumor fusion cells. To test this hypothesis, we first determined the level of expression of HSPs in DC, ovarian cancer cells and their counterparts in fusion cells by immunoblotting with antibodies against HSPs. HSP70, HSP90 and MUC1 were detected in the lysates of ovarian cancer cells and fusion cells, whereas low levels of HSP90 and minimal MUC1 were detected in the lysates of DC. Notably, the expression of HSP90 was increased in the fusion cells (Fig. 2A). Indeed a three-fold increase of HSP90 was observed in the lysates of fusion cells compared with HSP90 from tumor cells (Fig. 2B). The relative levels of expression of HSP70 between OVCA2 (HLA-A*0201+/A*1101+) and DC/OVCA2 fusions was comparable (Fig. 2A and B).
To determine whether HSP70 is associated in the immunoprecipitates with tumor antigens, their processed intermediates and other HSPs proteins, lysates from OVCA and DC/OVCA fusion cells were precipitated with anti-HSP70 mAb (19) followed by immunoblotting with a panel of anti-HSP antibodies. HSP40, HSP90 and HSP110 were detected in the immunoprecipitates by anti-HSP70 antibody from lysates of DC/OVCA2 fusion cells and OVCA2 cells (Fig. 2C and D), suggesting intracellular association of HSP70 with these proteins. Interestingly, the expression of HSP90 was significantly increased in DC/OVCA2 fusions cells. In addition, the recovery of MUC1 and HER2/neu tumor antigens was significantly increased in the immunoprecipitates of HSP70 from DC/OVCA2 fusion cells compared with those from OVCA2 cells (Fig. 2E and F). A BCP8-positive band was observed in the immunoprecipitates, suggesting the existence of processed tumor antigen in HSP70.PC. Increased recovery of tumor antigens and their processed intermediates in the immunoprecipitates from DC/tumor fusion cells may be related to the increased expression of HSP90 in such cells.
The ability of HSP70.PC-F to stimulate T cell proliferation was assessed by the standard [3H]-thymidine incorporation assay. Incubation with HSP70.PC from DC/MCF7 fusion cells in the presence of IL-2 resulted in the proliferation of T cells (Fig. 3A). In contrast, a lower level of T cell proliferation was observed when T cells were incubated with IL-2 and either HSP70.PC-Tu or medium alone. Similar results were obtained in HSP70.PC from DC/BT-20 fusion cells (Fig. 3B). Increased T cell proliferation was observed in T cells stimulated by HSP70.PC from DC/BT-20 fusion cells compared with those stimulated by HSP70.PC from BT-20 tumor cells (Fig. 3B).
To characterize the T cells stimulated by these HSP70 preparations, they were double stained with antibodies against IFN-γ and either CD4 or CD8 prior to analysis by FACS. We found a subset of T cells stimulated by HSP70.PC extracted from MCF7 tumor cells or FC/MCF7 fusion cells expressed IFN-γ (Fig. 3C), suggesting activation of these T cell subpopulations. Furthermore, IFN-γ-positive CD4 or CD8 T cells were significantly increased when stimulated by HSP70.PC-F compared with HSP70.PC-Tu (4.3% ± 1.7 vs 2.03% ± 0.8 in CD4 T cells; 8.6% ± 2.6 vs 3.9% ± 1.9 in CD8 T cells) (Fig. 3D). To determine whether tumor antigen-specific T cells were induced, T cells were further analyzed with tetramers specific for either MUC1 peptide (HLA-A*0201, STAPPVHNV) or HER2/neu peptide (HLA-A*0201, ILHNGAYSL). MUC1 peptide-specific or HER2/neu peptide-specific T cells were induced by both HSP70.PC-Tu and HSP70.PC-F (Fig. 3E). Confluent with the findings on IFN-γ expression, MUC1- or HER2/neu-specific T cells were significantly increased in T cells stimulated by HSP70.PC-F, indicating that induction of tumor antigen-specific T cells is enhanced by HSP70.PC-F exposure (10.3% ± 3.9 vs 6.2% ± 4.1; 5.24% ± 0.59 vs 1.23% ± 0.3) (Fig. 3F). Thus stimulation with HSP70.PC-F enhances the quantity and quality of stimulated T cells.
To determine the influence of HSP70.PC on lymphocyte killing of tumor targets, the standard 51Cr-release assay was next used. T cells stimulated by HSP70.PC extracted from FC/BT20 breast carcinoma fusion cells showed higher CTL activity against BT20 tumor cells than those stimulated by HSP70.PC extracted from BT-20 tumor cells (Fig. 4A). Similar findings were observed when T cells stimulated by HSP70.PC extracted from FC/SKBR3 fusion cells were incubated with SKBR3 tumor targets (Fig. 4B). Furthermore, T cells stimulated by HSP70.PC extracted from patient-derived OVCA cells or from DC/OVCA1 or DC/OVCA2 fusion cells lysed OVCA tumor targets from which the HSP70.PC were derived. As with the experiments carried out on tumor cell lines, higher levels of CTL activity against OVCA cells were observed in T cells stimulated by HSP70.PC extracted from DC/OVCA fusion cells (HSP70.PC-F) than those stimulated by HSP70.PC extracted from OVCA cells (HSP70.PC-Tu). The difference in CTL activity induced by HSP70.PC-F or HSP70.PC-Tu was statistically significant (Fig. 4A and D).
To assess the antigen specificity of the CTL, multiple tumor targets were used. T cells (HLA-A*1101+) stimulated by HSP70.PC extracted from DC/SKBR3 (HLA-A*1101+) fusion cells were able to kill not only SKBR3 tumor cells but also relevant ZR75 (HLA-A*1101+) tumor cells (Fig. 4E). Interestingly, T cells (HLA-A*0201+) stimulated by HSP70.PC extracted from fusions of DC and OVCA1 (HLA-A*0201+) were effective in lysis of OVCA1 cells and, to a lesser extent, MCF7 breast cancer cells (HLA-A*0201+). Minimal CTL was observed in the killing of SKBR3 (HLA-A*1101+) or BT-20 (HLA-A*0201−/A*1101−) tumors with different HLA type (Fig. 4F). Similar results were observed in T cells (HLA-A*1101+) stimulated by HSP70.PC extracted from fusions of DC and OVCA2 (HLA-A*0201+/A*1101+) with highest killing to OVCA2 cells and, to a lesser extent, SKBR3 (HLA-A*1101+) tumor cells and minimal killing to K562 cells (HLA-A*0201−/A*1101−) (Fig. 4G). These experiments show that the induction of antigen-specific T cells is enhanced by the stimulation of HSP70.PC-F and that CTL capable of lysing the target from which HSP70.PC are extracted can also kill other tumor cells provided they share the expression of tumor antigens and restriction elements.
The data in Fig. 4 show that HSP70.PC extracted from DC/tumor fusion cells can lyse multiple targets with shared tumor antigens, suggesting broad applicability of this vaccine. We next determined whether the source of DC can be altered in the production of HSP70.PC. In these experiments, OVCA4 cells (HLA-A*0201+/A*1101+) were fused to DC derived from PBMC of two individual donors (DC9, HLA-A*0201+/A*1101− and DC10, HLA-A*0201+/A*1101+) to generate DC9/OVCA4 and DC10/OVCA4 fusions, respectively. Then HSP70.PC were extracted from these fusion cells and OVCA4 tumor, respectively. HSP70.PC from DC9/OVCA4, DC10/OVCA4 or OVCA4 tumor cells were incubated with T cells (HLA-A*0201+/A*1101−) isolated from the same donor from whom DC9 were generated. Comparable T cell proliferation was observed in T cells stimulated by HSP70.PC from DC9/OVCA4 or DC10/OVCA4 fusion cells (Fig. 5A). In contrast, HSP70.PC from OVCA4 stimulated minimal T cell proliferation. In addition, more CD4 and CD8 T cells stimulated by HSP70.PC from DC9/OVCA4 and, to a lesser extent, DC10/OVCA4 expressed IFN-γ than those stimulated by their counterparts from OVCA4 (Fig. 5B). Similar results were observed in the induction of CTL. HER2/neu and MUC1-specific CTL were induced by HSP70.PC from DC9/OVCA4 or DC10/OVCA4 fusion cells as demonstrated by tetramer staining (Fig. 5C), and importantly, they were effective in lysis of OVCA4 tumor cells (Fig. 5D). To determine the specificity and restriction elements of CTL, antibody blocking assay was performed. As shown in Fig. 5E, CTL activity against OVCA4 was almost completely blocked by mAb against HLA-ABC (upper panel) and partially blocked by mAbs against HLA-A2 or MUC1 (middle and lower panels). These results indicate that CTL induced by HSP70.PC from DC9/OVCA4 or DC10/OVCA4 fusion cells are antigen-specific. It is likely that polyclonal CTLs were induced against not only MUC1 but also other tumor antigens such as HER2/neu (Fig. 5C), and multiple restriction elements in addition to HLA-A2 were involved. To confirm these results, parallel experiments were performed using T cells (HLA-A*0201+/A*1101+) isolated from the same donor from whom DC10 were generated. Comparable results were observed in T cells from donor 9 or donor 10 stimulated by HSP70.PC. HSP70.PC from DC10/OVCA4 or DC9/OVCA4 fusion cells induced higher T cell proliferation (Supplemental Fig. 1A), higher level of IFN-γ expression (Supplemental Fig. 1B), frequency of CTL (Supplemental Fig. 1C) and CTL activity against OVCA4 T cells (Supplemental Fig. 1D) than their counterparts extracted from OVCA4 tumor cells. In addition, antibody blocking assay shows that CTL activity against OVCA4 was blocked or partially blocked by mAbs against HLA-ABC, HLA-A11 or MUC1 (Supplemental Fig. 1E). Together, these experiments indicate that HSP70.PC from fusion cells can be readily re-presented by DC with different HLA background, suggesting the potentially broad use of the HSP70.PC-F approach in the clinical setting.
Heat-shock protein-chaperoned antigenic peptides elicit antitumor immunity when used as a tumor vaccine (10, 22–25). Indeed, HSP-based vaccines (HSP70, HSP90, or GP96) derived from cancer cells have been widely studied in animal experiments (10, 26–29). In these elegant approaches, HSP.PC reproducibly induced CTL responses against the cancer cells from which the HSP.PC were purified (23, 24, 30, 31). Importantly, immunization of mice with HSP.PC provides protection against the challenge with the tumor cells from which HSP.PC are purified or slows the progression of that tumor (7, 10, 31). Based on these results, HSP-based vaccines have been used in human clinical trials (12–16, 32–34). In the early phase I and/or II trials with GP96.PC (vitespen) purified from patient-derived tumors, immunologic and clinical responses were obtained in a subset of patients with malignant tumors (12, 13, 15, 32). The randomized phase III trials, however, showed mixed results (14, 16). In a group of 728 patients with renal cell carcinoma that were enrolled into the trial with evaluable results, 361 patients received vitespen vaccination. No difference in recurrence-free survival was seen between patients given vitespen and the control cohort with no treatment (16). In the treatment of patients with melanoma, 133 patients received vitespen. Although the overall survival in the vitespen arm is statistically indistinguishable from that in the control cohort of 107 patients with physician’s choice of treatment, a subset of patients with early stage of disease (M1a and M1b) that received a large number of vitespen injection survived longer (14). In addition, these trials showed that clinical responses were limited by the amount of resected tumor available for HSP isolation, as responses to vaccines are related to the number of HSP injections (14).
We have attempted to produce an enhanced HSP70-based vaccine through rapid isolation of HSP70.PC from DC/tumor fusion cells. In the animal studies, HSP70.PC-F show superior properties such as enhanced induction of CTL against tumor cells and stimulation of DC maturation over its counterpart from tumor cells (17). In the present study, we aimed to extend the findings from animal to human samples and develop an HSP70-based vaccine for patient use with enhanced immunogenicity. We have shown that: i) HSP70.PC can rapidly be extracted from DC fused to either patient-derived ovarian carcinoma cells or established breast cancer cells and these extracts carry higher levels of tumor antigens and their processed intermediates; ii) Stimulation of T cells with HSP70.PC-F results in proliferation of T cells and induction of antigen-specific CTL that are able to lyse relevant tumor cells, thus improving the quality and quantity of T cells stimulated by HSP70.PC-F. Unlike allogenic cellular vaccine, HSP70.PC-F is a peptide vaccine and can be readily uptaken by APC and represented to antigen-specific T cells, thus minimizing activation of allo-reactive T cells; iii) HSP70.PC-F can be extracted from fusion cells with altered fusion partners, and DC or tumor cells can be interchanged while eliciting comparable CTL activity against tumor cells; iv) HSP70.PC-F carry multiple antigenic peptides with ability to induce polyclonal CTL to kill the tumor cells from which the HSP70 were extracted as well as tumor cells with shared antigen and restrict element.
Most HSP-based vaccines such as vitespen are purified from resected tumors of patient to whom the vaccine is applied. These customized vaccines possess certain advantages since they carry the repertoire of peptides of individual cancer without the need to identify them. This approach of vaccine preparation, though beneficial, also has certain limitations. For example, vitespen was prepared in 49% of patients enrolled in the trial after the 13% of patients with fewer than four injections were excluded (14). Studies show that four injections are minimal doses for administration of vitespen (14). To circumvent this problem, we have assessed the feasibility of extracting HSP.PC from both patient-derived and established cancer cells as fusion partners and using them to stimulate T cells. Although this approach has the drawback of introducing an extra step to the simple and elegant approach of using autologous vaccines based solely on affinity purified HSP, it does appear to have significant advantages. CTL induced by HSP70.PC-F kill not only the tumor cells from which they are derived but also other tumor cells with different genotypes but shared tumor antigens (Fig. 4). Thus, HSP70.PC-F may be effective in clinical settings where the resected tumor sample is insufficient for the preparation of multiple does of customized vaccine. In addition, HSP70.PC-F could be used as a prophylactic vaccine since immunotherapy is most effective in patients with minimal or no tumor. One further downside of this approach is that only relatively few shared tumor antigens have been identified at this time. With the advancement of molecular and biological technology, however, it is likely that more shared tumor antigens will be identified, thus increasing the applicability and potency of the vaccine.
The molecular basis for enhanced immunogenicity in HSP70.PC-F is not fully elucidated and our studies are ongoing. However, DC are the most potent APC in the body and their fusion to tumor cells introduces abundant tumor antigens into the efficient antigen processing and presentation of machinery of DC. It is thus likely that the fusion cells sort and produce a large repertoire of antigenic peptides than tumor cells for surface presentation. These antigenic peptides are likely to be chaperoned by HSPs during the antigen processing, thus maximizing the extraction of HSP chaperoned peptides and potentially increasing immunogenicity of HSP.PC vaccine. Indeed, the immunoprecipitates from fusion cells contain higher levels of MUC1 antigenic peptides than those from tumor cells (Fig. 2) (17). In addition, the gentle and rapid extraction of HSP70.PC used in the present study may also contribute to the retention of peptides and possible other HSPs or molecules that may facilitate the antigen cross-presentation through interaction with the receptors on APC.
In summary, we have presented an alternative method and source for the preparation of HSP-based vaccines. This approach relies on the efficient antigen processing and presentation machinery of DC to produce tumor antigenic peptides after fusion with tumor cells and gentle and rapid isolation of HSP70 complexes enriched in biologically active polypeptides. Enhanced enrichment and preservation of antigenic peptides and proteins associated with HSP70 thus increases the immunogenicity of this HSP-based vaccine.
A–E, T cells from were stimulated by HSP70.PC derived from DC9/OVCA4, DC10/OVCA4 fusion cells or OVCA4 cells. A–E, T cells (HLA-A*0201+/A*1101+) were stimulated by HSP70.PC derived from DC9/OVCA4, DC10/OVCA4 fusion cells or OVCA4 cells. T cells proliferation (A), intracellular expression of IFN-γ in CD4 and CD8 cells (B), HER2/neu peptide-specific T cells (C, left panel) or MUC1 peptide-specific T cells (C, right panel), CTL activity against OVCA4 cells (D) and antibody block assay (E) were assessed using the same methods as in Fig, 5 A–E.
Phenotype and selected HLA-type of ovarian cancer cells.
This work was supported by the Ellison Foundation (to J.G.) and NIH research grant R01CA119045 (to S.C.).
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