The invention relates to an improved cancer treatment by immunotherapy with BCG, antigenically related non-pathogenic mycobacteria, or immunogenic component(s) thereof, as well as to a method for monitoring cancer treatment by immunotherapy with BCG, antigenically related non-pathogenic mycobacteria, or immunogenic component(s) thereof.
The use of infectious agents for achieving an anti-tumor effect dates back to the 1700s, but it was William Coley who made the first attempt at extracting an active agent for the purpose of achieving immune-mediated regression of inoperable sarcomas. His extract, called Coley's toxin, was a streptococcal extract that was later supplemented with mycobacterial cell wall preparations. Remarkably, he reported ˜10% response rates in patients with advanced stage disease (Wiemann, B. and Starnes, C. O., Pharmacol. Ther., 1994, 64, 529-564).
Bacillus Calmette Guérin (BCG) is a live attenuated strain of Mycobacterium bovis generated by the repetitive passage of a virulent strain of M. bovis. BCG was initially developed as a vaccine for tuberculosis (Mycobacterium tuberculosis (M. tuberculosis or Mtb) infection; Zwerling et al., PLoS Med., 2011, 8, e1001012) and following the work of William Coley, BCG was evaluated for use as an anti-cancer therapeutic vaccine. In fact, it has been injected into many solid tumors and while there were reports of some success, controlled clinical trials did not provide statistical significance (Brandau, H. Suttmann, Biomed. Pharmacother., 2007, 61, 299; Mathe et al., Lancet, 1969, 1, 697; Morton et al., Ann. Surg., 1970, 172, 740). Nonetheless, animal studies with BCG continued and it was recognized that long lasting direct contact with the live bacteria resulted in optimal tumor immunity (Zbar et al., J. Natl. Cancer Inst., 1971, 46, 831). These results prompted Morales et al. to evaluate BCG as an adjuvant intravesical treatment for carcinoma of the bladder, (Morales et al., J. Urol., 1976, 116, 180; Herr et al., J. Urol., 2008, 179, 53).
Carcinoma of the bladder is the most common cancer of the urinary tract and the fourth most common malignant disease in the developed world (Jemal, A. et al, CA Cancer J Clin., 2011, 61, 69-90). Most tumors are diagnosed at a superficial stage and are surgically removed by transurethral resection (Babjuk, M. et al., Eur. Urol., 2011, 59, 997). Depending on the stage and grade of the non-muscle invasive tumors, adjuvant therapy is recommended as a strategy for both reducing recurrence and diminishing risk of progression.
The initial treatment schedule was established empirically by Morales and colleagues in 1976 (Morales et al., J Urol., 1976, 116, 180): 120 mg lyophilized BCG Pasteur was reconstituted in 50 mL saline and instilled via a catheter into the bladder. Patients were asked to retain the solution for at least 2 hours, and they additionally received 5 mg BCG intradermally. Treatments were given weekly over 6 weeks, and altered favorably the pattern of recurrence in 9 patients.
Since then, BCG therapy has been the standard of care for high-risk urothelial carcinoma, namely carcinoma in situ, and high-grade Ta/T1 bladder lesions (Babjuk et al., Eur. Urol., 2011, 59, 997). It is also the most successful immunotherapy applied in the clinics, with response rates ranging 50-70% in patients with non-muscle invasive bladder cancer.
Modifications of the initial regimen have mainly focused on the elimination of the concomitant intradermal dose, introduction of maintenance BCG dosage schedule and introduction of other substrains of BCG. Importantly, two clinical studies performed in the 1990s have investigated the combined use of intravesical and intradermal (or scarification) routes for treating patients with BCG (Herr H. W., J Urol, 1986, 135, 265-267; Lamm et al., J. Urol., 1991, 145, 738). These trials showed no evidence of enhanced clinical response so that nowadays the regimen recommended by the European and American guidelines is as follows (Babjuk et al., Eur. Urol., 2011, 59, 997; Gontero et al., Eur. Urol., 2010, 57, 410):
While now in use for over 35 years, many questions remain about the mechanism of action by which BCG mediates the observed clinical response (Brandau et al., Biomed. Pharmacother., 2007, 61, 299), additionally, there is a need to identify strategies for optimizing therapy.
While success of therapy is known to rely on repeated instillations of live BCG administered as adjuvant therapy shortly after tumor resection, its precise mechanisms of action remain unclear. In the context of bladder cancer, repeated instillation with clinical-grade BCG is known to trigger a strong innate immune response, followed by the influx of type 1 polarized lymphocyte subsets (Alexandroff et al., Immunotherapy, 2010, 2, 551; Brandau et al., Biomed. Pharmacother. 2007, 61, 299). Using orthotopically-transplanted urothelial tumors in mice, several groups have reported that BCG-mediated anti-tumor activity relies on a functional immune system of the tumor-bearing host (Ratliff et al., J. Urol., 1987, 137, 155; Ratliff et al., J Urol., 1993, 150, 1018; Brandau et al., Int. J. Cancer, 2001, 92, 697; Suttmann et al., Cancer Res. 2006, 66, 8250). In particular, CD4+ and CD8+ T lymphocytes seem to be essential effector cells for eliminating the tumor in a mouse model (Ratliff et al., J Urol., 1993, 150, 1018), and correlates have been established between T cell infiltration and clinical response in patients (Prescott et al., J. Urol. 1992, 147, 1636). The inventors have recently reported that repeated intravesical instillations with BCG were required in order to trigger a robust inflammatory response (Bisiaux et al., J. Urol., 2009, 181, 1571).
The purified protein derivative (PPD) skin test is a standard assay that is used to detect an active immune response to BCG in subjects previously vaccinated with BCG. Tuberculin skin testing (TST) has been used for years as an aid in diagnosing latent tuberculosis infection (LTBI) and includes measurement of the delayed type hypersensitivity (DTH) response 48-72 hours after intradermal injection of PPD. While not typically attributed to inflammation in the bladder mucosa, DTH reactions are known to be mediated by antigen-specific effector T cells (e.g., induration induced by PPD challenge in the skin of a primed individual). This reaction is defined by antigen-specific T cells mediating the rapid recruitment of inflammatory cells to the site of injection (Marchal et al., J. Immunol., 1982, 129, 954; Milon et al., J. Immunol., 1983, 130, 1103). The PPD skin test has been assessed as potential predictor of BCG response. However, current evidence does no support its use as predictor of BCG response in clinical practice (Gontero et al., Eur. Urol., 2010, 57, 410).
Using an experimental model, the inventors have demonstrated that BCG dissemination to bladder draining lymph nodes and priming of interferon-γ-producing T cells could occur following a single instillation. However, repeated instillations with live BCG were necessary for a robust T cell infiltration into the bladder. Subcutaneous immunization with BCG prior to instillation overcame this requirement, triggering a more robust acute inflammatory process following the first intravesical instillation and accelerating T cell entry into the bladder, as compared to the standard protocol. Moreover, subcutaneous immunization with BCG prior to intravesical treatment of an orthotopic tumor dramatically improved response to therapy (
These data thus provide critical new insight into a long-standing clinically effective immunotherapeutic regimen and suggest strategies that may improve patient management.
The inventors have demonstrated that inducing BCG-specific immunity prior to local therapy improves anti-tumor response. However, BCG-specific immunity can be induced, not only by immunization with BCG, but also by immunization with antigenically-related non-pathogenic mycobacteria or immunogenic component(s) thereof. For these reasons, like BCG, other antigenically-related non-pathogenic mycobacteria could be used for cancer immunotherapy.
In addition, the drastic anti-tumor response improvement observed by using BCG pre-immunization in bladder cancer treatment, suggest that an anti-tumor response could also be observed by using the same strategy for treating other cancers for which standard BCG immunotherapy may have failed thus far. Therefore, BCG pre-immunization should increase the number of cancer types that can be treated using BCG immunotherapy.
Therefore, the present invention relates to a first and a second identical or different mycobacterial immunogenic compositions, each comprising at least a Mycobacterium bovis bacillus Calmette-Guérin (BCG), an antigenically related non-pathogenic mycobacteria, or one or more immunogenic component(s) thereof, as therapeutic active ingredient(s) for use in the treatment of cancer by parenteral or oral administration of the first composition to a cancer patient before local administration of the second composition at tumor site.
According to the present invention, a non-pathogenic mycobacteria antigenically related to BCG refers to an avirulent or attenuated mycobacteria which induces a BCG-specific immune response. The tumor site refers to the site of tumors, before and after tumor resection.
The present invention encompasses the use of whole cell, live or killed, non-pathogenic mycobacteria. Non pathogenic mycobacteria include naturally avirulent Mycobacterium species, attenuated strains (genetically modified or not) of Mycobacterium sp., and recombinant strains derived from the preceding strains.
A mycobacteria for use in the present invention may be a recombinant BCG (rBCG) improved through addition of relevant genes such as Th1 cytokines (IL2, GM-CSF, IFN-γ, IFN-α2, IL-18, MCP-3, IL-15, TNF-α), BCG or Mycobacterium tuberculosis immunodominant antigens such as M. tuberculosis Ag85B (rBCG30), or listeriolysin (rBCGΔureC:Hly or VPM1002; Kaufmann et al., Lancet, 2010, 375, 2110-2119). rBCG30 and VPM1002 are candidate vaccines for tuberculosis that have entered clinical trials. It may also be a recombinant mycobacteria expressing a mycobacterial FAP protein under the control of a promoter active under hypoxia conditions (International PCT Application WO 2008/012693).
Another mycobacteria for use in the present invention may be a genetically modified M. tuberculosis that has been attenuated through deletion of virulence genes such as phoD and fadD26 (MTBVAC01; Kaufmann et al., Lancet, 2010, 375, 2110-2119).
Yet another mycobacteria for use in the present invention may be a naturally avirulent or attenuated mycobacteria such as M. microti, M. smegmatis, M. fortuitum, M. vaccae, M. hiberniae, M. terrae, M. triviale, M. triplex, genavense, M. kubicae, M. heidelbergense, M. cookii, M. haemophylum, M. botniense, M. conspicuum, M. doricum, M. farcinogenes, M. heckeshornense, M. monacense, M. montefiorense, M. murale, M. nebraskense, M. saskatchewanense, M. scrofulaceum, M. shimnodei, M. tusciae, M. xenopi, M. chelonae, M. boletii, M. peregrinum, M. porcinum, M. senegalense, M. houstonense, M. mucogenicum, M. mageritense, M. austroafricanum, M. diernhoferi, M. hodleri, M. frederiksbergense, M. aurum, M. chitae, M. fallax, M. confluentis, M. flavescens, M. madasgkariense, M. phlei, M gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. duvalii, M. elephantis, M gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M. pulveris, M arosiense, M. aubagnense, M. chlorophenolicum, M. fluoranthenivorans, M. kumamotonense, M. novocastrense, M. parmense, M. phocaicum, M. poriferae, M rhodesiae, M. seoulense, and M. tokaiense, and subspecies.
BCG for use in the present invention is preferably a commercial available BCG strain which has been approved for use in humans such as Pasteur, Frappier, Connaught (Toronto), Tice (Chicago), RIVM, Danish 1331, Glaxo-1077, Tokyo-172 (Japan), Evans, Prague, Russia, China, Sweden, Birkhaugh, Moreau, and Phipps.
Killed mycobacteria, either killed but metabolically active or killed and metabolically inactive, are prepared according to methods well-known in the art which include treating mycobacteria with physical agents such as for example heat, UVA or gamma radiations and/or chemical agents such as formalin and psoralen. Killed but metabolically active mycobacteria refers to mycobacteria that are viable and able to express their genes, synthesize and secrete proteins but are not culturable (i.e., not capable of colony formation) because they are not replicative. Killed but metabolically active mycobacteria include for example nucleotide excision repair mutants which have been inactivated by photochemical treatment with psoralen and/or UV light. Killed and metabolically inactive include for example gamma-irradiated mycobacteria, heat-killed mycobacteria and extended freeze-dried killed mycobacteria (International PCT Application WO 03/049752).
The present invention encompasses also the use of immunogenic components such as subcellular fractions and recombinant antigens (proteins and vectors encoding said proteins) from BCG or antigenically related non-pathogenic mycobacteria. Said immunogenic components are well-known in the art and include for example: (i) mycobacterial cell wall fraction, eventually complexed with DNA (MCC; Morales et al., J. Urol., 2009, 181, 1040-1045; Morales et al., J. Urol., 2001, 166, 1633; Chin et al., J. Urol., 1996, 156, 1189; Uenishi et al., Chemical and Pharmaceutical Bulletin, 2007, 55, 843-852; Azuma et al., J. Natl. Cancer Inst., 1974, 52, 95; Takeya, K and Hisastsuna, K., J. Bacteriol., 1963, 85, 16: Fox et al., J. Bacteriol., 1966, 92, 1) or R8 liposome (Joraku et al., BJU Int., 2009, 103, 686-693) and (ii) M. tuberculosis or BCG recombinant immunodominant antigens such as Ag85A, Ag85B, TB10.4, Mtb32, Mtb39, ESAT-6, used as fusion proteins consisting of one or more antigens, or expressed by a recombinant vector such as a replication-deficient vaccinia virus or E1-deleted adenovirus (Kaufmann et al., Lancet, 2010, 375, 2110-2119). The recombinant proteins are formulated in a vaccine adjuvant such as a mixture of oligodeoxynucleotides and polycationic amino acids or monophosphoryl lipid A and QS21.
In a particular embodiment of the present invention, the first composition comprises a live or killed but metabolically active non-pathogenic mycobacteria. Preferably, said composition is for parenteral or oral administration to a patient having no active immune response to BCG, as assessed by example by a weak positive or a negative PPD skin test. More preferably, the composition comprises a live non-pathogenic mycobacteria selected from the group consisting of: BCG, a rBCG expressing Th1 cytokines, BCG or M. tuberculosis immunodominant antigens, or listeriolysin, and a genetically modified M. tuberculosis that has been attenuated through deletion of virulence genes.
In another particular embodiment of the present invention, the first composition comprises one or more immunogenic component(s) of the non-pathogenic mycobacteria. Preferably, said composition is for parenteral or oral administration to a patient having an active immune response to BCG, as assessed by example by a positive PPD skin test. More preferably, the composition comprises one or more M. tuberculosis or BCG recombinant immunodominant antigens or recombinant vector(s) expressing said antigens, or a mycobacterial cell wall fraction.
The parenteral or oral administration is at any time after cancer diagnosis. It is usually before tumor resection but can be concomitant with tumor resection. It is preferably performed, just after the diagnosis. It may be subcutaneous (s.c.), percutaneous, intradermal, intramuscular or oral, more preferably subcutaneous (s.c.), percutaneous or intradermal. It usually comprises one single administration.
In another particular embodiment of the present invention, the second composition comprises live or killed (killed but metabolically active or killed and metabolically inactive) non-pathogenic mycobacteria or one or more immunogenic components thereof as defined above.
According to the present invention, the first and the second composition may be the same composition or different compositions. When different compositions comprising different mycobacteria or different immunogenic components are used for the parenteral or oral and the local administration, the mycobacteria or immunogenic components are chosen so that they have B, T CD4+ and/or T CD8+ epitopes in common. Using this type of compositions will ensure that the local administration will boost the specific immune response induced by the parenteral or oral administration.
The local administration is usually after tumor resection and at least seven days after the parenteral or oral administration. Preferably, it is at least three weeks after the parenteral or oral administration. The local administration at tumor site will depend on the type of cancer. For example, for bladder cancer it is intravesical. It usually comprises at least one series of at least three separate administrations, usually between three to six administrations, at an interval of one to three weeks. For the maintenance therapy, additional series of repeated administrations are generally performed using a similar administration regimen. The intravesical administration regimen recommended for BCG by the European and American guidelines comprises an induction course of 6 weekly intravesical instillations, followed by maintenance therapy. The recommended maintenance regimen consists in 3 weekly instillations at 3 months, 6 months, and then every 6 months up to 3 year. This recommended administration regimen can be used for the composition of the present invention in the treatment of bladder cancer.
The composition(s) for use in the present invention comprise a pharmaceutically effective dose of a non-pathogenic mycobacteria or one or more immunogenic component thereof. A pharmaceutically active dose is that dose required to prime or boost a BCG specific immune response in a patient and improve the anti-tumor response leading to a better recurrence-free survival compared to untreated patients or patients treated by local administration only. The pharmaceutically effective dose depends upon the composition used, the route of administration, the type of mammal (human or animal) being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors, that those skilled in the medical arts will recognize. Generally, the dose of live non-pathogenic mycobacteria in the composition depends on the age of the patients. For human adults, it is in the range of 107 to 1010 CFUs (Colony Forming Units) for the local and parenteral or oral administrations, preferably about 108 to 109 CFUs. The dose of killed non-pathogenic mycobacteria in the composition is in the range of 50 to 150 mg, which corresponds to the amount of killed mycobacteria obtained starting with 109 to 1011 CFUs before the killing. The dose of mycobacterial cell wall fraction is in the range of 1 to 10 mg, preferably formulated in an emulsion.
The composition(s) for use in the present invention may further comprise one or more additional agents like: (i) pro-inflammatory agents such as inflammatory cytokines (IL-2, IFN-α, TNF-α, GM-CSF), (ii) T-cell stimulatory molecules such as agonist antibodies directed against T-cell activating co-stimulatory molecules (CD28, CD40, OX40, GITR, CD137, CD27, HVEM) and blocking antibodies directed against T-cell negative co-stimulatory molecules (CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3), (iii) antibiotics, and (iv) chemotherapy drugs.
Alternatively, the composition (s) comprising a non-pathogenic mycobacteria or immunogenic component(s) thereof may be used in combination (separate or sequential use) with such additional agents.
For example, antibiotic(s) such as ofloxacin may be used in combination with the composition comprising live BCG or antigenically related non-pathogenic mycobacteria strain, to reduce side-effects in patients.
The composition(s) for use in the present invention usually comprises a pharmaceutically acceptable carrier. The composition is further formulated in a form suitable for parenteral, oral, and/or local (intravesical, intravaginal or epicutaneous) administration into a subject, for example a mammal, and in particular a human.
Examples of cancer that can be treated using the treatment of the invention include with no limitation: bladder, melanoma, cervical, colon, prostate, ovarian and breast cancer.
In another particular embodiment of the invention, the cancer is a mucosal cancer including with no-limitation, non-muscle invasive (Ta, carcinoma in situ (Tis), T1) and muscle invasive (T2, T3, T4) transitional cell carcinoma of the bladder, cervical and colon cancers.
Preferably said mucosal cancer is superficial or non-invasive, i.e., low-stage tumor. In a more preferred embodiment, said mucosal cancer is a non-muscle invasive bladder cancer selected from the group consisting of: carcinoma in situ (Tis) and high-grade Ta or T1 transitional cell carcinoma of the bladder.
According to a first preferred embodiment of the invention, a first and a second composition comprising live BCG are used for the treatment of bladder cancer. Preferably, the first composition comprising 107 to 109 CFUs of BCG Pasteur or Danish strains is injected intradermally (ID) to a PPD negative patient, shortly after bladder cancer diagnosis. The second composition comprising 107 to 109 CFUs of BCG Connaught strain is then instilled intravesically after tumor resection, three weeks after the ID injection, using the intravesical administration regimen recommended for BCG by the European and American guidelines.
Another aspect of the present invention relates to a method in vitro for monitoring cancer treatment by immunotherapy with BCG or antigenically related non-pathogenic mycobacteria, comprising:
BCG-specific immune response may be assayed by standard assays well-known in the art. For example, BCG-specific antibodies may be detected by ELISA, BCG-specific CD4+ T-cells and CD8+ T-cells may be detected by proliferation assays (CSFE assay), cytokine assays (ELISPOT, Intracellular cytokine staining) or immunolabeling assays (FACS assay). Antigens that can be used for assaying a BCG specific immune response are well-known in the art and include the purified protein derivative (PPD) from M. tuberculosis and isolated immunodominant antigens of BCG or antigenic fragments thereof comprising B, CD4+ or CD8+ T-cells epitopes. For example, BCG Antigen 85 may be used to detect a BCG specific immune response and the HLA-2 restricted peptides Ag85A(6-14) and Ag85A(200-208) may be used to detect CD8 specific responses.
The assay is performed on a biological material containing antibodies and T-cells. For example, it may be performed on a whole body fluid such as blood or urine, or on a fraction thereof.
For example, the QuantiFERON®-TB test which is based on the quantification of interferon-gamma (IFN-γ) released from sensitized lymphocytes incubated overnight with purified protein derivative (PPD) from M. tuberculosis and control antigens can be used to assay BCG-specific T-cell response in patients.
The method may comprise the detection of antibodies, CD4+ T-cells, or CD8+ T-cells specific to BCG.
The assay is performed before to initiate the immunotherapy, as well as during the immunotherapy, to optimize the administration regimen and in turn improve the anti-tumor response in the patient.
According to another preferred embodiment, the BCG-specific immune response is assayed before to initiate the immunotherapy, in order to determine which composition should be administered by the parenteral route (live mycobacteria or subunit vaccine), and eventually, just before or after tumor resection, before the first local administration, and/or at the end of the local administrations of the BCG, antigenically related non-pathogenic mycobacteria, or immunogenic component(s) thereof at tumor site.
For a better understanding of the invention and to show how the same may be carried into effect, there will now be shown by way of example only, specific embodiments, methods and processes according to the present invention with reference to the accompanying drawings in which:
There will now be described by way of example a specific mode contemplated by the Inventors. In the following description numerous specific details are set forth in order to provide a thorough understanding. It will be apparent however, to one skilled in the art, that the present invention may be practiced without limitation to these specific details. In other instances, well known methods and structures have not been described so as not to unnecessarily obscure the description.
For intravesical instillations, 7-12 week-old C57BL/6 female mice (Charles Rivers) were water starved for 7-8 hours, reflecting the clinical practice of patients being asked not to drink prior to treatment. Mice were anesthetized (125 mg/kg ketamine and 12.5 mg/kg xylazine intraperitoneally) and drained of any urine present by application of slight digital pressure to the lower abdomen. The urethral orifice was disinfected with povidone-iodine and a 24 Ga-catheter (BD Insyte Autoguard, Becton Dickinson) adapted to a 1 mL tuberculin syringe (Braun) containing 50 μL of either phosphate-buffered saline (PBS, Invitrogen) or BCG (about 3×106 CFUs) was carefully inserted through the urethra. The injection was made at a low rate to avoid trauma and vesico-ureteral reflux, and there was no dead volume in the catheter. Mice were kept under anesthesia for 2 hours, with catheter and syringe maintained in place to retain the intravesical solution. For tumor challenge, mouse bladders were pre-treated with 0.1 mg/mL poly-L-lysin (Sigma-Aldrich) for 20 minutes, prior to instillation of 80,000 MB49 cells in 50 μL PBS, which were retained for 1 hour into the bladder. For subcutaneous (s.c.) immunization, mice received a single injection of 2-5×106 CFUs BCG. Mice were housed under specific-pathogen free conditions and used under approved protocols.
For instillations, Immucyst (Sanofi Pasteur) was reconstituted in 3 mL PBS following the manufacturer's instructions. Heat-killed BCG was obtained by autoclaving Immucyst preparation 20 min at 121° C. For s.c. administration, either Immucyst (once) or frozen aliquots of BCG Pasteur (1137P2) were used with similar results. BCG-Pasteur was grown at 37° C. in Middlebrook 7H9 medium supplemented with bovine albumin, dextrose and catalase (ADC, Difco), harvested in exponential growth phase, washed, dispersed with 3 mm glass-beads, resuspended in PBS, aliquoted and then frozen at −80° C. A defrosted aliquot was used to determine the lot titer on 7H11 medium supplemented with oleic acid, albumin dextrose and catalase (OADC, Difco). In addition, all preparations used for intravesical or subcutaneous injections were titrated. For organ bacterial load, bladders were resected in sterile PBS, homogenized 2 min at 25 Hz in a Tissue Lyzer II (Qiagen) while draining lymph nodes (LN) were mashed with the back of a syringe in sterile PBS. Five-fold serial dilutions of the homogenates were plated on 7H11 supplemented with OADC and colony forming units (CFUs) were assessed after 17-28 days of growth at 37° C.
For FACS, CD16/CD32 (clone 2.4G2, Fc block), CD45.2 (clone 104), CD3ε (clone 145-2C11), NK1.1 (clone PK136), CD8α (clone 53-6.7), CD44 (clone IM7), CD45RA (clone 14.8), CD45R/B220 (clone RA3-6B2), CD11c (clone HL3), CD86 (clone GL1), Ly-6C (clone AL-21), Ly-6G (clone 1A8) antibodies (Abs) were purchased from BD Pharmingen; CD4 (clone GK1.5), CD11b (clone MAC-1), pan-γδ TCR (clone GL3), IAb-IEb (clone M5), F4/80 (clone BM8) Abs were from eBioscience and CD45.2 (clone 104-2) from Southern Biotech. Dead cells were stained either with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) or with live/dead fixable Aqua dead cell staining kit (Invitrogen). Cells were enumerated using Accucheck counting beads (Invitrogen). For histology, CD3ε (clone 500A2) and CD45.2 (clone 104) Abs were obtained from BD Pharmingen; α-smooth muscle actin (α-SMA, clone 1A4) from Sigma-Aldrich and syrian-Hamster secondary Ab from Jackson ImmunoResearch Laboratories. Abs used in the IFN-γ ELISPOT assays were purchased from Mabtech. H2-Db-restricted Mtb32309-318 peptide (GAPINSATAM) an I-Ab Ag85A241-260 peptide (QDAYNAGGGHNGVFDFPDSG) were obtained from PolyPeptide. Depleting anti-CD4 (clone GK1.5) and anti-CD8 (clone YTS169.4) as well as rat IgG2b isotype control mAbs were purchased from Bio X Cell. MB49 cells were received from the Brandau group and cultured in D-MEM (Invitrogen), complemented with 10% fetal calf serum (FCS, Eurobio) and 1% Penicilin/Streptomycin (Invitrogen). Poly-L-lysin was purchased from Sigma-Aldrich.
Bladders were resected and incubated in DMEM (Invitrogen) containing 1 mg/mL collagenase D (Roche), 0.17 U/mL liberase TM (Roche) and 1 U/mL Deoxyribonuclease 1 (Invitrogen) at 37° C. for two successive cycles of 30 min. Tissue suspensions were washed in DMEM+10% FCS, pressed through a 70-μm mesh, washed in PBS+2% FCS, pressed through a 40-μm mesh and pelleted for FACS staining. Spleens were mashed, incubated at 37° C. in 1.66% ammonium chloride (VWR International) in water for 5 min for red blood cell lysis and filtered through a 70-μm mesh. All cells were preincubated with Fe block (and Aqua, if used), washed and incubated with appropriate Abs for 20 min in PBS+0.5% FCS. Samples were run on a BD FACSCantoII cytometer (BD Biosciences) and analyzed using FlowJo (Treestar) software.
BCG-specific T cell responses were tested by IFN-γ ELISPOT assays. For IFN-γ ELISPOT assays with CD4+ or CD8+ T cells, at indicated time points, spleens and bladder draining LN were harvested and combined, CD4+ and CD8+ T were purified using microbeads and MS columns (Miltenyi Biotec) and ELISPOT assays for IFN-γ-producing cells were performed as previously described (Blachere et al., PLoS. Biol., 2005, 3, e185).
In particular, for IFN-γ ELISPOT assays with CD4+ T cells, purified CD4+ T cells from spleen and draining lymph nodes were restimulated ex vivo for 20 h using splenocytes pulsed with Ag85A241-260 peptide. Unpulsed splenocytes served as a negative control. The number of spot forming cells (SFC) per 106 CD4+ T cells was then determined for individual mice.
For IFN-γ ELISPOT assays with CD8+ T cells, purified CD8+ T cells from spleen and draining LN were restimulated ex vivo for 20 h using splenocytes pulsed with Mtb32309-318 peptide. Unpulsed splenocytes served as a negative control. The number of spot forming cells (SFC) per 106 CD8+ T cells was then determined for individual mice.
The ELISPOT plate evaluation was performed in a blinded fashion by an independent evaluation service (Zellnet Consulting).
For tetramer staining, soluble Db-Mtb32309-318 monomers were produced using a modified version of that described (Bousso et al., Immunity, 1998, 9, 169) and conjugated using premium grade streptavidin-PE (Invitrogen), added for 1 hour at room temperature.
Tissues were processed as previously described (Peduto et al., J. Immunol., 2009, 182, 5789). Briefly, samples were fixed overnight at 4° C. in a fresh solution of 4% paraformaldehyde (Sigma-Aldrich) in PBS, embedded in OCT compound (Sakura Finetek) and frozen at −80° C. Frozen blocs were cut at 8-μm thickness and sections collected onto Superfrost Plus slides (VWR International). Slides were dried one hour and processed for staining or stored at −80° C. For staining, slides were first hydrated in PBS-XG (PBS containing 0.1% Triton X-100 (Sigma-Aldrich) and 1% FCS) for 5 min and blocked with 10% FCS in PBS-XG for 1 hour at room temperature. Slides were then incubated with primary antibodies in PBS-XG overnight at 4° C., washed, incubated with secondary antibodies for 1 hour at room temperature, incubated with DAPI for 5 min at room temperature, washed and mounted with Fluoromount-G (Southern Biotech). Slides were examined under an Axiolmager M1 fluorescence microscope (Zeiss) equipped with a CCD camera and images were processed with AxioVision software (Zeiss).
Mice were injected intraperitoneally with a mixture of 100 ug anti-CD4 and 100 ug anti-CD8 antibody, or with 200 ug isotype control, 48 hrs prior to instillation. Depletion efficiency was controlled on blood and splenocytes.
Fifty-five patients with non-muscle invasive bladder cancer and previously treated with BCG were retrospectively evaluated. Patients had been tested for their purified protein derivative (PPD) status prior to therapy as a means of assessing potential adverse effects (e.g., allergic response to BCG). All patients had been treated by transurethral resection and were eligible for BCG therapy (tumor stage and grade is reported in Table 1, and subsequently received weekly instillations of BCG. Patients were followed according to clinical guidelines and tumor recurrence was defined based on biopsy or urine cytology. Kaplan-Meier curves indicate recurrence-free survival. Collection and use of clinical data was in accordance with the host institutional ethical review board and all patients provided informed consent.
Unless otherwise indicated, two-tailed Mann-Whitney non-parametric tests were employed for statistical analyses using Prism software (Graphpad). Differences with a p value of 0.05 or less were considered significant. For the mouse tumor challenge (
Based on the clinical practice of resecting the tumor shortly prior to adjuvant BCG therapy, the inventors began their studies in tumor-free mice. Therefore, to determine the dynamics of T cell infiltration into the bladder, age-matched female C57BL/6 mice were intravesically instilled with either phosphate-buffered saline (PBS; control) or clinical-grade BCG (Immucyst, Sanofi-Pasteur) once a week for a total of three instillations (
Early attempts to use BCG as an anti-cancer agent have reported anti-tumor activity after a single intratumoral injection with BCG (Zbar et al., J. Natl. Cancer Inst., 1971, 46, 831). However, the inventors observed that T cell frequency in the bladder never increased much above the basal level following a single instillation of BCG (
Interestingly, early clinical investigation in humans suggested that live BCG was required in order to achieve tumor immunity (Kelley et al., J. Urol., 1985, 134, 48; Zbar et al., Natl. Cancer Inst. Monogr., 1972, 35, 341), despite the fact that the clinical-grade lyophilized preparation contains only 5-10% live organisms (Behr M. A., Lancet Infect. Dis., 2002, 2, 86). However, mechanistic reasons for the failure of heat-killed organisms to provoke a response remain unclear. To evaluate the possibility that T cell recruitment was dependant on live bacilli, repeated intravesical instillations of clinical-grade BCG (containing live BCG) versus heat-killed (HK) BCG was compared. As expected, the latter did not result in T cell recruitment to the bladder (
To assess the requirement for live BCG to activate adaptive immunity components, the inventors have evaluated BCG dissemination with the hypothesis that its entry into the bladder draining LN is a pre-requisite for priming and subsequent T cell infiltration of the bladder. Such a requirement has indeed been well documented in the context of low-dose Mtb lung infection (Wolf et al., J. Exp. Med., 2008, 205, 105; Reiley et al., Proc. Natl. Acad. Sci. USA, 2008, 105, 10961; Chackerian et al., Infect. Immun., 2002, 70, 4501). They first assessed decay of BCG after intravesical injection, assaying colony-forming units (CFUs) that remain in the bladder after first voiding (at removal of catheter) at 2 hours, and on day 1 post-instillation. Consistent with what has been suggested for human treatments (Durek et al., J. Urol., 165, 2001, 1765; Siatelis et al., J. Clin. Microbiol., 2011, 49, 1206), the inventors demonstrated that the BCG load in the bladder rapidly decreased to 1% of the instilled dose and was barely detectable by 24 h post-instillation (
The inventors next evaluated the priming of BCG peptide-specific T cells, assessed using H2-Db-Mtb32309-318 tetramers (also known as PepA or GAP; Irwin et al., Infect. Immun., 2005, 73, 5809) (
To investigate if dissemination of BCG also correlated with local adaptive immunity, the inventors examined lymphocyte populations in the bladder. While they observed low levels of T cell infiltration in CFU+ animals, the level of infiltration was significantly lower in mice that had received single versus repeated instillations (
To further test the dissociation of priming from T cell trafficking, the inventors evaluated whether the activation of BCG-specific T cells prior to bladder instillations would impact T cell recruitment during the first intravesical instillation. Mice were injected subcutaneously (s.c.) with BCG, and after 21 days, intravesical instillations were initiated—comparing single vs. repeated BCG challenge. In mice primed by s.c. BCG, they observed a robust T cell infiltration as early as 12 days following a single instillation (
To further characterize the differential bladder T cell trafficking following different BCG regimens, the inventors evaluated the local inflammation of the bladder mucosa. Shortly after the first and the third instillation, they observed a rapid but short-lived (less than 42 hours post instillation) influx of neutrophils (characterized as Ly-6G+ leukocytes,
Given the robust inflammatory process observed in mice immunized s.c. with BCG, the inventors hypothesized that the existence of BCG-specific T cells at the time of instillation was impacting upon the acute inflammatory process. To test this possibility, mice previously immunized s.c. with BCG were subjected to anti-CD4 and anti-CD8 depleting antibodies 48 h prior to intravesical instillation. Following T cell depletion, they demonstrated a decrease in the number of neutrophils and inflammatory monocytes infiltrating the bladder (
Repeated intravesical instillations with HK-BCG do not result in T cell recruitment to the bladder. In a preliminary experiment, the inventors wondered if this could be due to a different inflammatory response, but the influx of inflammatory monocytes was found to be similar after the first and the third instillation with either live (clinical-grade) or HK-BCG (
Based on the ability to achieve stronger inflammation and earlier T cell recruitment to the bladder microenvironment, the inventors reasoned that s.c. exposure to BCG prior to intravesical BCG therapy might improve the anti-tumor response. To test that hypothesis they employed an orthotopic tumor model—implantation of syngeneic MB49 tumor cells into the bladders of C57/BL6 mice. Although the derived epithelial MB49 tumors grow in an aggressive manner (Chan et al., B.J.U. Int., 2009, 104, 1286), this model remains, to their knowledge, the only mouse model in which intravesical BCG treatment has been shown to induce anti-tumor responses, when initiated 1-2 days after tumor implantation (Gunther et al., Cancer Res., 1999, 59, 2834). To evaluate the impact of pre-existing BCG-specific T cells, mice were immunized s.c. with BCG and, after 3 weeks, 80,000 MB49 cells were implanted into the bladder mucosa, as described in the materials and methods. Two days later, intravesical BCG therapy was initiated, and mice were monitored twice daily for survival. Strikingly, 100% of mice that received BCG s.c prior to intravesical therapy survived as late as 70 days post tumor challenge; in comparison, 80% of mice with no prior BCG immunization succumbed within 50 days, despite intravesical BCG therapy (
These results prompted them to investigate the relevance of pre-existing BCG-specific immunity in patients with high-risk non-muscle invasive bladder cancer undergoing BCG therapy. Retrospective analysis of clinical data was performed, in which patients underwent a purified protein derivative (PPD) skin test prior to intravesical therapy (
A positive skin test is the signature of previous exposure and active immune response to BCG, M. tuberculosis or other mycobacteria. The inventors therefore stratified patient outcome data according to their PPD status prior to treatment, and observed that patients with a positive PPD had a significantly better recurrence-free survival than patients with a negative PPD skin test (
Together these data suggest that boosting BCG-specific immunity prior to intravesical therapy might improve clinical response and tumor immunity.
The BCG-specific CD4+ T cell response in mice that had received single or repeated intravesical instillation with BCG was assessed using IFN-γ ELISPOT assay (
While prior efforts have evaluated immunologic response during therapy in human observational studies or in experimental mouse models, the inventors study provides the first systematic evaluation of BCG-induced T cell infiltration of the bladder mucosa. Using histological and cytometric analyses, and paying careful attention to mycobacterial persistence and antigen-specific T cell priming, the inventors have defined the parameters required for achieving effective adaptive immune responses in the bladder. They have identified a requirement for live bacteria that disseminate to local draining lymph nodes in order to achieve T cell priming (
Based on experimental work in humans, the inventors have focused their attention on the BCG induced inflammation and activation and recruitment of T lymphocytes after intravesical instillations in mice. Based on their observations of a delayed influx of T cells, they hypothesized that parenteral exposure to BCG prior to standard-of-care might accelerate the kinetics of bladder inflammation. They demonstrate that such an approach provides an optimized strategy for T cell recruitment and that this treatment protocol improves the host anti-tumor response.
From the perspective of the host response to infection, one striking observation is that bladder T cell infiltration following repeated BCG instillations occurs only after day 29 (
The inventors also discovered that BCG dissemination to the regional lymph node is critical for achieving efficient T cell priming, again showing similarity to what has been shown in the lung Mtb infection model. T cell priming, however, was not sufficient to achieve T cell recruitment to the bladder, as shown by the relatively low level of T cell infiltration following a single instillation, even in the presence of measurable BCG-specific T cell responses (
To apply their insights into dynamics of bladder inflammation, they tested their modified treatment regimen using an orthotopic bladder tumor model. Most strikingly, they demonstrate the ability to achieve up to 100% survival as compared to 80% lethality at day 70 (median survival time being ˜45 days) (
In summary, the inventors have demonstrated that while BCG dissemination to regional LNs and priming of IFNγ-producing T cells can occur following a single instillation, repeated instillations of live BCG are necessary to achieve robust bladder T cell infiltration. Strikingly, parenteral exposure to BCG prior to instillation overcomes the requirement for repeated instillations, triggering a more robust acute inflammatory process at the first instillation and accelerating the recruitment of T cells to the bladder. Moreover, parenteral exposure to BCG prior to orthotopic tumor challenge dramatically improves response to BCG therapy. Importantly, patients with pre-existing immunity to BCG responded significantly better to therapy. Together these data suggest that checking patients' immunity to BCG prior to intravesical therapy, and boosting it if necessary, might improve BCG-induced clinical responses.
Number | Date | Country | Kind |
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12305086.6 | Jan 2012 | EP | regional |
Filing Document | Filing Date | Country | Kind |
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PCT/IB2013/050611 | 1/24/2013 | WO | 00 |