Immunodominant PstS1 antigen of mycobacterium tuberculosis is a potent biological response modifier for the treatment of bladder cancer
© Sänger et al; licensee BioMed Central Ltd. 2004
Received: 18 February 2004
Accepted: 26 November 2004
Published: 26 November 2004
Bacillus Calmette Guérin (BCG)-immunotherapy has a well-documented and successful clinical history in the treatment of bladder cancer. However, regularly observed side effects, a certain degree of nonresponders and restriction to superficial cancers remain a major obstacle. Therefore, alternative treatment strategies are intensively being explored.
We report a novel approach of using a well defined immunostimulatory component of Mycobacterium tuberculosis for the treatment of bladder cancer. The phosphate transport protein PstS1 which represents the phosphate binding component of a mycobacterial phosphate uptake system is known to be a potent immunostimulatory antigen of M. tuberculosis. This preclinical study was designed to test the potential of recombinant PstS1 to serve as a non-viable and defined immunotherapeutic agent for intravesical bladder cancer therapy.
Mononuclear cells (PBMCs) were isolated from human peripheral blood and stimulated with PstS1 for seven days. The activation of PBMCs was determined by chromium release assay, IFN-γ ELISA and measurement of lymphocyte proliferation. The potential of PstS1 to activate monocyte-derived human dendritic cells (DC) was determined by flow cytometric analysis of the marker molecules CD83 and CD86 as well as the release of the cytokines TNF-α and IL-12. Survival of presensitized and intravesically treated, tumor-bearing mice was analyzed by Kaplan-Meier curve and log rank test. Local and systemic immune response in PstS1-immunotherapy was investigated by anti-PstS1-specific ELISA, splenocyte proliferation assay and immunohistochemistry.
Our in vitro experiments showed that PstS1 is able to stimulate cytotoxicity, IFN-γ release and proliferation of PBMCs. Further investigations showed the potential of PstS1 to activate monocyte-derived human dendritic cells (DC). In vivo studies in an orthotopic murine bladder cancer model demonstrated the therapeutic potential of intravesically applied PstS1. Immunohistochemical analysis and splenocyte restimulation assay revealed that local and systemic immune responses were triggered by intravesical PstS1-immunotherapy.
Our results demonstrate profound in vitro activation of human immune cells by recombinant PstS1. In addition, intravesical PstS1 immunotherapy induced strong local and systemic immune responses together with substantial anti-tumor activity in a preclinical mouse model. Thus, we have identified recombinant PstS1 antigen as a potent immunotherapeutic drug for cancer therapy.
Urothelial carcinoma of the bladder accounts for about 4% of all cancer related death in man. The large majority of tumors (70–80%) is superficial at diagnosis and has a high rate of local recurrence (70%) and progression (30%) after local surgical therapy. Therefore, patients require lifelong medical follow-up examinations and effective prophylactic treatment to prevent recurrences and progression of the tumor.
In this type of cancer, the immunotherapeutic use of Mycobacteria – specifically Bacillus Calmette-Guerin (BCG), a non-pathogenic strain of Mycobacterium bovis – has a well-documented and successful clinical history. Immunotherapy with BCG is performed by six weekly instillations of viable Mycobacteria (for induction course) into the bladder of patients after initial transurethral resection of the tumor. Until now various clinical trials have shown that this type of therapy is superior to topical chemotherapy and transurethral resection of the tumor alone to prevent recurrences and local progression especially in patients with high risk tumors [1–3]. Despite several clear advantages of BCG immunotherapy for the treatment of bladder cancer, several problems and limitations compromise its use. Although BCG is the most effective agent against superficial transitional cell carcinoma (TCC), currently there are still 30 to 40% of patients not responding to the therapy . Furthermore, in the case of muscle invasive bladder cancer, BCG has not been shown to be effective . BCG's activity appears to be strictly localized and as a living organism, BCG poses unique toxicity problems associated with its use. Although only 5% of these problems are severe, most if not all patients experience some irritable bladder symptoms (cystitis) during BCG therapy . Roughly 40% develop hematuria and 30% experience flu-like symptoms including fever, malaise and nausea or vomiting. Actual BCG sepsis is a rare event and has been reported in only 0.4% of all cases. In addition, some of these cases have been fatal [7, 8]. Thus, the reported side effects limit the clinical applicability and acceptance of this effective immunotherapy and underscore the need for alternative forms of treatment.
In order to limit toxicity recent endeavors are therefore focused on the development of alternative non-viable products and some of those have already been tested in preclinical and clinical studies [9–12]. The mycobacterial antigen PstS1 is known as a highly immunogenic and immunostimulatory component of the mycobacterial cell membrane . PstS1 is the phosphate binding subunit of the inorganic phosphate uptake system from M. tuberculosis belonging to the family of ABC (ATP-binding cassette) transporters [14, 15]. It is a glycosylated lipoprotein which can be found both, intracellularly and secreted into the extracellular culture supernatant [16, 17]. Moreover PstS1 represents one of the most immunogenic antigens in active multibacillary tuberculosis .
We hypothesized that this highly immunogenic protein antigen could function as an effective biological response modifier in immunotherapy of bladder cancer. To test the tumortherapeutic potential of recombinantly expressed PstS1  we performed a detailed analysis of the immunostimulatory capacity of this antigen in a well-defined human in vitro system and in a previously described murine model of experimental bladder cancer therapy . Because the role of prior exposure of bladder cancer patients to mycobacterial antigens for the effectiveness of BCG therapy is controversely discussed [21–23], we performed intravesical PstS1-immunotherapy with and without prior sensitization of mice.
The data reported herein demonstrate that PstS1 is a potent activator of human tumor-cytotoxic MNCs, induces maturation and activation of human dendritic cells and most importantly is very effective in the treatment of experimental orthotopic bladder cancer. While local and systemic immune responses were observed in sensitized and non-sensitized mice, immunotherapy of cancer was only successful in non-sensitized animals.
The human bladder tumor cell line T-24 was cultured at 37°C and 5% CO2 in RPMI 1640 (PAA Laboratories, Linz/Austria) containing 10% FCS (Linaris, Bettingen/Germany), 1% glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The murine bladder tumor cell line MB-49 was cultured at 37°C and 5% CO2 in DMEM (PAA Laboratories, Linz/Austria) containing 10% FCS (Linaris, Bettingen/Germany), 1% L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.
Stimulation of human PBMCs
MNCs from heparinized blood of healthy human donors were obtained by discontinuous gradient centrifugation using Biocoll Separating Solution (Biochrom, Berlin/Germany) and adjusted to a concentration of 1 × 106/ml in RPMI-1640 medium containing 5% human serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Recombinant PstS1 in concentrations from 0.1 μg/ml – 100 μg/ml, BCG (Connaught substrain, Immucyst, 4 × 104 cfu/ml), or PBS (100 μl) was added and the cells were cultured for 7 days in 6-well microtiter plates at 37°C and 5% CO2.
Chromium release assay
Cytotoxicity was determined in a standard 4-hour chromium release assay. Target cells were labeled with Na2 51CrO4 (ICN, Irvine/USA) for 1 h at 37°C, washed and resuspended at 5 × 104 cells/ml. Effector cells were added to a total of 100 μl of target cells at an effector:target ratio of 40:1. The radioactive content of the supernatant was measured in a gamma-counter (Berthold, Wildbad/Germany). The specific lysis was determined according to the formula: spec. lysis (%) = 100 × (Exp – Spo)/ (Max – Spo) where Exp is the experimental release, Spo the spontaneous release and Max the maximum release.
Human IFN-γ ELISA
Supernatants of PstS1, BCG and PBS stimulated PBMCs were recovered at days 2, 5 or 7 and examined for the presence of IFN-γ. Each experiment was carried out several times with different donors. Detection was performed with an anti human IFN-γ ELISA-Kit according to the manufacturers' instructions (eBioscience, San Diego/USA).
Proliferation of human PBMC
PBMCs were stimulated for 2 to 7 days with PstS1, BCG, PBS or PHA (Sigma). 2 × 105 cells / well of the stimulated PBMCs were cultured in a microwell plate and 1 μCi 3H-Thymidine (five wells per sample) (Amersham, Freiburg/Germany) was added during the last 18 h of stimulation. DNA was harvested on filter membranes and thymidine incorporation was measured by liquid scintillation counting (counter: LKB Wallace 1205 beta-plate, Turku/Finland).
Generation and stimulation of human monocyte derived dendritic cells
After separating peripheral blood mononuclear cells (PBMCs) from heparinized blood of healthy donors by Ficoll-Paque centrifugation, monocytes were elutriated by counterflow centrifugation. For generation of immature DCs, 2 × 106 monocytes were cultured for seven days with 2 ml RPMI 1640, 10% FCS (Linaris, Bettingen/Germany) 1% penicillin/streptomycin and IL-4/GM-CSF (500 U/ml each) (TEBU/PeproTech, Offenbach/Germany) in 24-well cell culture plates (Nunc, Wiesbaden/Germany). Exchange of medium was carried out at day three and day five. This procedure resulted in full differentiation of monocytes with no undifferentiated monocytes present in the cultures after seven days. Immature DCs were stimulated for three days with 10 μg/ml PstS1 or BCG (MOI = 0.01) and subjected to flow cytometry. Debris was excluded from the analysis according to FSC/SSC gating.
Expression of cell surface molecules was analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes/USA) using phycoerythrin (PE) conjugated monoclonal antibodies (mAbs): anti-CD1a (Biosource, Camarillo/USA), anti-CD83 (Pharmingen, San Diego/USA), anti-CD14 and anti-CD86 (Dianova, Hamburg/Germany).
1 × 105 DCs suspended in PBS containing 5% human serum and 0.1 % sodium acide were incubated with mAbs for 30 minutes on ice. Then cells were washed with PBS and resuspended in 400 μl 1.5 % paraformaldehyde in PBS.
human TNF-α and IL-12 ELISA
Supernatants of PstS1, BCG and PBS stimulated DCs were recovered at day 3 after stimulation and examined for the presence of TNF-α and IL-12. Each experiment was carried out several times with different donors. TNF-α detection was performed with a quantitative ELISA, provided by Dr. H. Gallati (Intex, Muttenz/Switzerland).
IL-12 detection was carried out with an ELISA-Kit from eBioscience (San Diego/USA).
PLG-Particle preparation and protein loading
PLG-particles (10 mg/ml) (Lionex, Braunschweig/Germany) were diluted 4:1 with PBS and the pH was adjusted to 7.0 with NaOH. PstS1 was recombinantly expressed in E. coli and purified by standard chromatography.
250 μg of PLG-particles were loaded with PstS1 or BSA by coincubation with 60 μg of the respective protein for 15 h at RT on a shaker (overall volume 100 μl). Afterwards the particles were spun down and washed for 5 min with 100 μl PBS. The supernatant was removed and the particles were diluted in 100 μl of PBS. 10 μl of loaded particle solution were centrifuged and the pellet was treated for 30 min at RT with 0.1 M NaOH/ 10% SDS. After centrifugation in a minifuge, supernatant was removed and analyzed on a 10% SDS page. Using recombinant PstS1 as a calibration standard it was determined that 250 μg PLG-particles bound approximately 50 μg of PstS1 protein.
PLG-particle vaccination and immunotherapy of experimental bladder cancer
Female C57BL/6 mice were purchased from Charles-River Laboratories (Sülzfeld/ Germany) at the age of 6–8 weeks. To test efficacy of PstS1 therapy in C57/BL6 mice a published syngeneic, orthotopic bladder cancer model was used . Trial I: 15 animals per group were subcutaneously injected with 250 μg PLG-particles coated with 50 μg PstS1 or 250μg PLG-particles alone. Ten days later the mice were anesthetized by intraperitoneal treatment with Pentobarbital (0.067 mg/g body weight). After insertion of a 24-gauge teflon intravenous catheter (Insyte-W, Becton Dickinson, Franklin Lakes/USA) transurethrally into the bladder, electrocoagulation was performed with a guide wire. Thereafter 6 × 104 MB-49 cells were instilled into the bladder. Intravesical immunotherapy with 100 μg PstS1 in 100 μl PBS was performed on days 1, 8, 15 and 22 after tumor implantation. Control groups were instilled with PBS (100 μl) alone. The viability status of the mice was checked daily. Surviving mice were sacrificed on day 70. Survival of mice was compared using Kaplan-Meier analysis and log-rank test. Trial II: Performance similar to trial I with the following differences. 11 animals per group were subcutaneously injected with a) 250 μg empty PLG-particles b) 250 μg PLG-particles coated with 50 μg BSA c) 100μl of PBS. Intravesical treatments were performed with 100 μg PstS1 or PBS only.
Immunohistochemistry of murine bladders
Serum antibody response after PstS1 immunotherapy
S.C. / Intravesical Treatment
Responders in %
anti-PstS1-IgG ELISA from murine blood
Murine blood was obtained immediately after sacrificing mice by heart punctation and centrifuged in a microcentrifuge to obtain the serum for antibody analysis which was performed with the PstS1-ELISA-kit (Lionex GmbH, Braunschweig/Germany) according to the manufacturer's instructions.
Splenocyte restimulation assay
Splenocytes were isolated, washed with PBS and erythrocytes were lysed with H2O. Afterwards 2 × 105 cells / well were cultured in a microwell plate and stimulated with 10 μg/ml PstS1 for a period of five days (five wells per sample). Then 1μCi 3H-Thymidine (Amersham, Freiburg/Germany) was added and cells were incubated for 15 h at 37°C and 5% CO2. Finally the DNA was harvested on filter membranes and 3H-Thymidine incorporation was measured by liquid scintillation counting (counter: LKB Wallace 1205 beta-plate, Turku/Finland).
PstS1 activates human PBMCs
To assess the immunostimulatory properties of our PstS1-preparation we analyzed several human PBMC in vitro systems. As in these systems the immunostimulatory mechanisms of whole BCG bacteria have been thoroughly studied in the past [9, 25–27] we used BCG as a positive control and reference stimulus.
Dendritic cell activation by PstS1
Dendritic cells are central cellular mediators for the induction of anti-tumor immune responses. We tested the potential of PstS1 to induce activation and maturation of human monocyte-derived DCs.
Intravesical immunotherapy with PstS1 in sensitized and non-sensitized mice
After we have shown profound immunostimulatory properties of PstS1 in vitro we conducted a series of studies to test its immunotherapeutic potential in vivo. Prompted by the controversy about the role of prior exposure to mycobacterial antigens in BCG immunotherapy we wanted to assess the in vivo activity of PstS1 in sensitized and non-sensitized mice. For this purpose two independent in vivo experiments were carried out. PLG-particles have previously been described as useful agents for sensitization of mice to mycobacterial antigens and were used as such in our study .
Local and systemic immune response in PstS1-immunotherapy
After we had demonstrated successful immunotherapy of bladder cancer by local instillation of PstS1 we next analyzed the local and systemic immune response of the various treatment groups to obtain initial insight into the immunological basis of this novel immunotherapy. To achieve this we analyzed the systemic antibody response by a semiquantitative anti-PstS1 IgG ELISA, the systemic lymphocyte response by splenocyte restimulation assays and the local immune response in the bladder by immunohistology.
Local immune response in the bladder wall after PstS1 immunotherapy
S.C. / Intravesical Treatment
3 ± 2
28 ± 24
8 ± 9
2,2 ± 1,7
6 ± 7
3 ± 2
17 ± 6
4 ± 3
1,6 ± 1,4
24 ± 20
4 ± 7
69 ± 60
16 ± 15
22,2 ± 24,4
86 ± 46
44 ± 30
45 ± 18
10 ± 4
1,9 ± 2,1
114 ± 25
56 ± 20
56 ± 16
13 ± 14
3 ± 2,4
143 ± 84
49 ± 15
75 ± 29
36 ± 29
4,5 ± 4,2
BCG therapy is a clinically successful therapy in the treatment of bladder cancer but its acceptance is hampered by hazards and side effects related to the use of viable mycobacteria. The aim of this study was to evaluate the anti-tumor potential of the well-defined, non-viable mycobacterial antigen PstS1.
To this end we first tested the potential of recombinant PstS1 to activate human PBMC cultures. In this series of experiments PstS1 induced strong cytotoxicity, proliferation and IFN-γ production in human PBMC. Ten μg/ml PstS1 were found to be optimal for activation of human PBMC. We used BCG lyophilisate as a reference stimulus in these experiments as we have previously described BCG as a potent activator of human PBMC functions . PstS1 followed a similar time kinetic as BCG in the stimulation of human PBMC, albeit activation of PBMC functions was somewhat lower with PstS1 compared with BCG. PstS1 was previously shown to induce profound cellular immunity in different vaccination studies [28–30]. Therefore, our results further confirm the potential of PstS1 to function as a potent inducer of T-helper 1 and cell-mediated immune responses.
While IFN-γ is a key cytokine in TH1 immune responses produced by activated lymphocytes, IL-12 is mainly produced by monocytes/macrophages as well as dendritic cells and important in the early phase of cellular immunity . IL-12 is a potent inducer of anti-tumor immunity  and acts together with IFN-γ and TNF-α in a positive feedback loop [25, 33, 34]. Although BCG mycobacteria are relatively weak inducers of bioactive IL-12p70 in human dendritic cells in the absence of additional costimulation like CD154 or IFN-γ (figure 3b), IL-12 has been shown to be essential for therapeutic efficacy in experimental BCG immunotherapy of bladder cancer . Given the importance of dendritic cells for the initiation of anti-tumor immune responses  and as a source of IL-12 and TNF-α we tested the capacity of recombinant PstS1 to activate human monocyte-derived dendritic cells. Our results clearly identified PstS1 as a potent stimulator of human DCs as it provoked upregulation of CD83 and CD86 surface expression as well as induction of cytokines. Of interest is the expression of high amounts of bioactive IL-12 after stimulation of DCs with PstS1 as only small amounts of this cytokine are produced after challenge with BCG or other mycobacterial antigens (figure 3 and unpublished observations).
The encouraging in vitro experiments then prompted us to evaluate the immunotherapeutic potential of PstS1 in a well-established murine model of experimental bladder cancer . The effect of prior sensitization or responsiveness to mycobacterial antigens for the effectiveness and outcome of subsequent immunotherapy with BCG is still a matter of intensive debate [21–23]. Taking this into account we took advantage of the fact that we had in hand a well-defined, recombinant antigen already evaluated in vaccination studies against mycobacterial infections and included a prime-boost treatment regimen into our experimental immunotherapy protocol. The main objective of our series of in vivo experiments was to determine the anti-tumor potential of local instillations of recombinant PstS1 and to compare the effect of intravesical PstS1 in sensitized and non-sensitized mice. Because adsorption of PstS1 to L-particles has previously been described as very efficient in inducing specific cellular immunity to this antigen , we adopted this method of sensitization for our treatment protocol. Using a protocol of four weekly instillations of PstS1 into the bladder (adopted from the treatment schedule in use for immunotherapy with BCG) we observed a strong therapeutic effect of PstS1 instillations. Intravesical PstS1 significantly prolonged survival of mice and induced systemic immune responses and cellular infiltration of the bladder with different subpopulations of leukocytes. The anti-tumor effect of PstS1 was already evident after intravesical instillation of PstS1 only but was further enhanced after s.c. injection of empty L-particles (Fig. 5). Unexpectedly, sensitization of mice with PstS1-loaded particles almost completely abrogated the therapeutic effect of intravesical PstS1 (Fig. 4). In order to test whether the negative effect of prior sensitization was specific for PstS1 or could also be induced by s.c. injection of L-particles loaded with an irrelevant antigen, we compared the therapeutic effect of intravesical PstS1 combined with a) s.c. sensitization with BSA-loaded L-particles, b) s.c. injection of empty L-particles and c) s.c. injection of PBS only (Fig. 5). Clearly, the injection of BSA-loaded particles did not influence the therapeutic effect of intravesical PstS1 indicating that only PstS1-specific previous sensitization is detrimental (Fig. 5a and 5c). We analyzed the systemic serum antibody response, activation of splenocytes and the local cellular infiltration of the bladder wall in sensitized and non-sensitized mice (Fig. 6) in order to understand and explain the remarkable therapeutic potential of intravesical PstS1 as well as the negative effect of prior specific sensitization. Interestingly, even without sensitization, instillation of PstS1 into the bladder provoked a systemic anti-PstS1 response visualized by murine anti-PstS1 serum antibodies and splenocyte proliferation after rechallenge in vitro. In addition to this systemic immune activation, intravesical PstS1 also induced the local influx of lymphocytes, macrophages and granulocytes into the bladder. While only a minimal influx of CD8-cells could be observed after instillation of PstS1, a considerable infiltration of the bladder with granulocytes, macrophages and CD4-cells was noted. Although prior specific sensitization with PstS1 completely abrogated the anti-tumor effect of intravesical PstS1, the cellular infiltration of the bladder wall remained essentially unchanged when comparing sensitized and non-sensitized animals. In the splenocyte restimulation assay an enhanced response of two out of four mice was noted in the group of mice which received s.c. PstS1 followed by intravesical PstS1 compared with the two groups which received either treatment alone (Fig. 6). This indicates that the combination of s.c. sensitization and intravesical treatment indeed augmented the systemic immune response at least in some animals. Surprisingly, this enhanced systemic immune response coincided with an abrogation of tumor-therapeutic efficacy. A possible explanation for this phenomenon could be that the anti-PstS1 antibodies, which were induced after s.c. priming would bind to the recombinant PstS1 shortly after instillation into the bladder and thereby neutralize its function and anti-tumor effect. However, as mentioned earlier, even in the PstS1-specific prime-boost treatment regimen an unchanged cellular infiltration of the bladder wall was noted. Alternatively, the sensitization might render the subsequent local immunotherapeutic immune response insufficient, because the host immune system is still actively responding to the priming at the onset of immunotherapy. This situation might be called "immune exhaustion" making the host unable to mount a sufficient local anti-tumor immune response while still responding to the specific priming. Further experiments are needed to clarify whether a modification of the time schedule of such a "prime-immunotherapy" protocol could prevent the negative effects observed in this study and might even enhance immunotherapy with PstS1.
Nonetheless, our combined in vitro and in vivo analyses clearly identified PstS1 from M. tuberculosis as a potent immunostimulant and a potential immunotherapeutic anti-cancer agent for topical treatment strategies. Our data do not show and do not imply that PstS1 is the major or only immunostimulatory component of whole BCG mycobacteria in BCG immunotherapy of bladder cancer.
We have identified the immunodominant mycobacterial PstS1 antigen as a potent biological response modifier for tumor immunotherapy. Using a human in vitro system of PBMC activation and a murine model of experimental bladder cancer immunotherapy we could show strong immunostimulatory capacity of PstS1 as well as significant anti-tumor activity. In a model of prime-boost immunotherapy we observed that antigen-specific sensitization might jeopardize the positive effects of topical immunotherapy and therefore has to be considered and evaluated with caution. GMP-production of PstS1 and a clinical trial in humans is currently being established and might open the door for an efficient and safe alternative in the field of bladder cancer immunotherapy.
This work was supported by the ProInno program of the BMWA of Germany.
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