Patent Application
20090304637
The invention relates to a recombinant Mycobacterium strain expressing a mycobacterial FAP protein under the transcriptional control of a promoter active under hypoxia conditions and its use for the prevention and the treatment of epithelial tumors.
Bladder cancer is among the top five cancers in men and is three to four time less frequent in women. Over 80% of these cancers are superficial transitional cell carcinomas. Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) is, today, the reference for treatment and prophylaxis of superficial transitional cell carcinoma of the urinary bladder; it is the most effective therapy for preventing superficial tumor recurrences and treating residual tumors of the bladder after transurethral tumor resection (Lamm et al., N. Engl. J. Med., 1991, 325, 1205-).
While the anti-tumor effect of BCG is well-established, the mechanism through which it occurs is less clear. Some studies suggest that the cell mediated immune response to BCG plays a critical role in the BCG anti-tumor effect. At the same time, other studies identify a direct anti-tumor effect of BCG. The relative contribution of these two possible mechanisms to BCG's clinical anti-tumor activity remains unknown.
The instillation of BCG into the bladder provokes a complex inflammatory response during which lymphocytes and various cytokines are released into the urine. Local granulomas formed in the suburothelial stroma are proposed to play a key role in the control of carcinoma cells (Alexandroff et al., The Lancet, 1999, 353, 1689-1694). NK cells have been hypothesized to mediate a part of this control. The production of different cytokines (IL-2, IL-12, IL-18 and interferons α) is over-expressed after BCG therapy and may activate NK cells (Suttmann et al., The Journal of Urology, 2004, 172, 1490-1495). However, the intense immune responses observed in some patients did not correlate with a better disease control and the immunological mechanisms allowing the proliferation control of carcinoma cells are still unclear.
A number of studies demonstrate that BCG exerts a direct anti-proliferative effect on human urothelial carcinoma cells. The alanine and proline rich secreted protein APA (also named FAP-B, antigen MPT-32, 45-kDa glycoprotein, or 45/47-kDa antigen) of the mycobacterial fibronectin attachment protein (FAP) family is a necessary protein for in vivo BCG attachment to the bladder epithelial cells and mediation of BCG-induced anti-tumor activity (Zhao et al., Int. J. Cancer, 2000, 86, 83-88). Mycobacterial FAP proteins constitute a family of highly homologous proteins that bind fibronectin in a unique manner. FAP proteins from at least five mycobacterial species including M. leprae (FAP-L), M. avium (FAP-A), M. bovis BCG (FAP-B; GenBank accession number AF013569), M. smegmatis (FAP-S) and M. tuberculosis (APA, antigen MPT-32, 45-kDa glycoprotein, or 45/47-kDa antigen; EMBL accession number X80268) have been cloned and characterized. The fibronectin binding region (positions 269-292 of FAP-A) contains a conserved RWFV tetrapeptide (positions 273 to 276 of FAP-A) that is necessary for fibronectin binding function. The minimal binding sequence is the 12 amino acid peptide, 269-280 (positions 269 to 280 of FAP-A; Zhao et al., The Journal of Biological Chemistry, 1999, 274, 4521-4526). The APA protein functions as an opsonin, linking BCG to cell surface integrins, via a fibronectin bridge. FAP-mediated BCG adherence to the urothelial carcinoma surface α5β1 integrin, which is the predominant FAP receptor on urothelial cells, induces signaling and gene transactivation pathways involving NF-κB and AP-1. Cell-cycle arrest at the G1/S interface, rather than apoptosis, was reported as being the mechanism contributing to BCG's anti-proliferative effect (Chen et al., BMC Urology, 2005, 5:8).
However, the use of BCG for bladder cancer does not come without drawbacks, both in terms of efficacy and toxicity. First, the response to BCG is unpredictable and not linear. As a result, thirty percent of patients are BCG refractory and there are currently no reliable prognostic factors that accurately predict treatment success or failure. In addition, the long-term durability of response to BCG is limited and the use of maintenance (three weekly treatments every three to six months) or booster therapy (once a month) for up to 36 months is necessary to reduce recurrence of bladder cancer.
Second, BCG has side effects. Most patients experience local symptoms of cystitis including frequency, urgency, dysuria and occasional haematuria. Mild systemic symptoms of high temperature, malaise, and a transient influenza-like illness are also common. Severe side-effects occur in 5% of patients, roughly 10% of which involve frank BCG sepsis (Alexandroff et al., precited).
Recent advances in recombinant mycobacteria technology, have open new horizons for bladder cancer immunotherapy. Several approaches have been suggested to produce a recombinant alternative to BCG which is less toxic and/or more effective than the existing BCG (Alexandroff et al., precited):
In addition, it has been suggested to use a non-pathogenic strain of mycobacteria, such as M. smeginatis.
However, to date, none of these approaches has provided an alternative to BCG immunotherapy. It is in this context that the invention was made.
Knowing that the PO2 in urine is low (Leonhardt et al., New Engl. J. Med., 1963, 269, 115-121), i.e. the local PO2 of the surface of the bladder epithelium where carcinoma cells are growing, the inventors have engineered a recombinant BCG (BCG Apa++) overexpressing the APA protein under hypoxic conditions. In this construct, expression of the APA open reading frame is driven by the mycobacterial α-crystallin promoter. The α-crystallin is a protein which is specifically synthesized during M. tuberculosis late exponential and stationary phase growth in vitro, following a shift to oxygen-limiting conditions, and may play a role in long-term survival of M. tuberculosis, in vivo (Yuan et al., J. Bacteriol., 1996, 178, 4484-4492). In vivo administration of BCG Apa++, leads to overexpression of the APA protein in tissues were the partial pressure of oxygen is low, such as the bladder (Leonhardt et al., N. England J. Medecine, 1963, 269, 115-121). As a result, BCG Apa++ is more active than wild-type BCG to control human bladder carcinoma cells proliferation, both in vitro and in vivo. In addition, BCG Apa++ is a potent activator of apoptosis pathways. This recombinant BCG should lower the doses of BCG used and minimize the side-effects associated with BCG immunotherapy.
Therefore, the invention relates to a recombinant vector comprising a mycobacterial FAP protein coding sequence under the transcriptional control of a promoter sequence that is active under hypoxia conditions.
by “functional FAP protein” is intended a protein able to bind fibronectin, to inhibit cell proliferation and to induce apoptosis. A functional FAP protein may be a FAP protein fragment or a FAP protein variant having one or more mutation (insertion, deletion, substitution of one or more amino acids) in the FAP protein sequence.
In a first embodiment, the invention features a recombinant vector, wherein said mycobacterial FAP protein is Mycobacterium tuberculosis FAP protein (M. tuberculosis APA protein).
In a second embodiment, the invention features a recombinant vector, wherein the promoter is a mycobacterial promoter which is active in hypoxic conditions. Examples of such promoters are described in Florczyk et al., Infect. Immun., 2001, 69, 5777-5785 and Florczyk et al., Infect. Immun., 2003, 71, 5332-5343. Among said promoters, the one specified here above may advantageously be used (see chapter definitions, hspX gene); another promoter may also be advantageously used: it corresponds to the promoter of the TB31.7 gene. The promoter sequence may be amplified with the pair of primers SEQ ID NO: 5 and SEQ ID NO: 6, derived from those described in Table 1 of Florczyk et al., (Infect. Immun., 2003, precited), by addition of respectively a BamHI and a NcoI restriction site, at the 5′ end.
The TB31.7 gene of M. tuberculosis corresponds to the locus_tag:
Rv2623; Gene ID: 887442; gi2104288. By reference to accession number BX842580 (Mycobacterium tuberculosis H37Rv complete genome; segment 9/13), the TB31.7/Rv2623 promoter region (SEQ ID NO: 14) is included between positions 176190 and 176560.
By reference to M. bovis BCG Strain Pasteur 1173P2 (accession number NC—008769), the TB31.7 gene (locus tag BCG-2650) corresponds to positions 1459592 to 1460485 on the DNA minus strand. Promoter region of the TB31.7 gene includes promoter region of every TB31.7 mycobacterial homolog gene that can be amplified with the pair of primers SEQ ID NO:5 and SEQ ID NO:6.
Preferably, the mycobacterial promoter is the promoter of the acr gene (hspX promoter); expression of the alpha-crystallin heat shock protein homolog HSPX (14 kDa antigen; HSP16.3) encoded by the mycobacterial acr gene, is specifically induced to very high level under hypoxic conditions.
More preferably, the vector comprises a M. tuberculosis APA protein coding sequence under the transcriptional control of the hspX promoter.
A vector according to the present invention comprises, but is not limited to, a YAC (yeast artificial chromosome), a BAC (bacterial artificial), a phage, a phagemid, a cosmid, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non chromosomal, semi-synthetic or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids” which refer generally to circular double-stranded DNA loops which, in their vector form are not bound to the chromosome. Large numbers of suitable vectors are known to those of skill in the art.
The vector according to the present invention comprises an expression cassette, wherein the sequence encoding the FAP protein is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said protein. More particularly, the vector may comprise additional features which are well-known in the art, including:
In a fourth embodiment, the invention features a vector which is a plasmid (double-stranded circular DNA). Preferably, said plasmid contains a bacterial origin of replication and an antibiotic resistance gene. In particular, the invention features a plasmid, identified as pYAPA2031, wherein the M. tuberculosis APA protein coding sequence under the transcriptional control of the hspX promoter is inserted in the EcoRV site of the pYUB295 plasmid (Dussurget et al., Infect. Immun., 2001, 69, 529-533; Gomez et al., Mol. Microbiol., 1998, 29, 617-628).
The invention concerns also an expression cassette derived from the recombinant vector according to the present invention, consisting of a polynucleotide fragment comprising the promoter sequence which is active under hypoxia conditions operatively linked to the mycobacterial FAP protein coding sequence, as defined above. Examples of expression cassettes are SEQ ID NO: 18 and SEQ ID NO: 19 wherein the M. tuberculosis APA coding sequence (SEQ ID NO: 15) is under the control of respectively the M. bovis AF2122/97 hspX/acr promoter (SEQ ID NO: 16) and the M. tuberculosis TB31.7/Rv2623 promoter (SEQ ID NO: 17). Said expression cassette may further comprise additional features as defined above (cloning site, tag, downstream termination signal).
The invention relates also to a host cell which is genetically modified by a recombinant vector as defined above (recombinant cell).
Particularly, the invention relates to bacteria, preferably mycobacteria transformed by a recombinant vector as defined above.
Preferred mycobacteria include those of M. tuberculosis complex, such as M. bovis BCG strains, for example the Pasteur BCG strain. Other preferred mycobacteria are those which are fast growing and non pathogenic for humans such are for example, Mycobacterium smegmatis, Mycobacterium fortuitum and Mycobacterium microti.
In particular, the invention features a recombinant Mycobacterium bovis BCG strain, stably transformed with the plasmid pYAPA2031, deposited at the Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, 75724 Paris Cedex 15, on Jul. 21, 2006, under accession number 1-3659, identified hereafter as BCG Apa++.
The invention relates also to an antitumoral composition comprising a suitable amount of a recombinant vector or host cell as defined above, in an acceptable carrier, such as a stabilizer, a buffer and the like.
The composition according to the present invention may comprise living or killed vector/cell. The killed vector/cell compositions are prepared by any means, known to those of ordinary skill in the art.
A pharmaceutical composition or formulation refers to a form suitable for administration (oral, topical, by injection or inhalation) into a subject, for example a mammal, and in particular a human. Suitable forms, in part, depend upon the use or the route of entry. A preferred formulation is for topical administration, more preferably for intravesical, intravaginal, rectal administration, or for inhalation.
A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence or treat (alleviate a symptom to some extent, preferably all the symptoms) of a disease or state. The pharmaceutically effective dose of the vector/host cell depends upon the composition used, the route of administration, the type of mammal 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 effective dose of recombinant mycobacteria for human, is between 106 and 1010 CFU (Colony Forming Unit) or CFU equivalent.
Preferably, the tumor is an epithelial tumor. More preferably, the tumor is a superficial tumor. In particular, the tumor is a transitional cell carcinoma of the bladder or a lung, colon or cervix carcinoma.
In a first embodiment, the antitumoral composition according to the present invention, comprises a recombinant mycobacteria strain as defined above, in particular the BCG Apa++ strain.
Preferably, the recombinant mycobacteria strain is killed, more preferably by extended freeze-drying. The method of preparation of extended-freeze-drying mycobacteria strains is described in the PCT International Application WO 03049752.
In a second embodiment, the antitumoral composition according to the present invention, further comprises an immunostimulatory agent.
The invention relates also to the use of a recombinant vector or host cell as defined above for the manufacture of a medicament intended for the prevention or the treatment of a tumor in a subject.
The invention relates also to a product containing a recombinant vector or a host cell as defined above, and an immunostimulatory agent, as combined preparation for simultaneous, separate or sequential use in antitumoral therapy.
The invention relates also to a method for preventing or treating a tumor in a subject, comprising: administering to the subject, an effective amount of a composition, as defined above, by any appropriate means. Particularly, the composi-tion is administered intravesically, intravaginally, rectally, or by inhalation and comprises between 106 and 1010 CFU (Colony Forming Unit) or CFU equivalent of recombinant mycobacteria according to the invention.
The invention relates also to the use of the expression cassette or recombinant vector, as defined above for the preparation of a modified host cell, preferably a recombinant mycobacteria strain wherein the expression cassette is integrated in the genome of the recombinant mycobacteria.
The invention also relates to a host cell which is genetically modified and has the expression cassette as defined above, integrated in its genome.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of microbiology, molecular biology and immuno-biology within the skill of the art. Such techniques are explained fully in the literature.
The recombinant vector according to the invention is constructed and introduced in a host cell by the well-known recombinant DNA and genetic engineering techniques using classical methods, according to standard procedures as those described in: Current Protocols in Molecular Biology (Frederick M. A USUBEL, 2000, Wiley and son Inc, Library of Congress, USA) and Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).
In addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, which refers to examples illustrating the BCG recombinant strain and its use according to the invention, as well as to the appended drawings in which:
To construct a recombinant BCG able to produce APA molecules when the bacteria are under hypoxia, a plasmid containing the appropriate sequences was tailored. This plasmid contains the apa gene of M. tuberculosis (EMBL accession number X80268 and Laqueyrie et al., Infection and Immunity, 1995, 63, 4003-4010) coding for the APA protein, cloned under the transcriptional control of the hspX (GenBank accession number S79751; Yuan et al., J. Bacteriol., 1996, 178, 4484-4492; Florczyk et al, Infect. Immun., 2001, 69, 5777-5785 and Florczyk et al., Infect. Immun., 2003, 71, 5332-5343), and the aph gene (kanamycin resistance). Bacteria of the wild-type BCG 1173P2 Pasteur strain, were then electroporated with the plasmid, and recombinant BCG overexpressing APA (BCG Apa++) was isolated in Sauton medium containing kanamycin (
The M. tuberculosis APA coding sequence was amplified by PCR from pLA34-2 (Laqueyrerie et al., Infect. Immun. 1995, 63, 4003-4010). The apa sequence was modified to create a NcoI site via the forward primer (5′-catgccatggtacaggtggaccccaacttgaca-3′: SEQ ID NO: 1) and a BamHI site in the reverse sequence (5′-ttaggtcggccggtaaggtccgctgcggtgt-3′: SEQ ID NO: 2) in order to subclone the NcoI-BamHI PCR product into the pQE60 expression vector (QIAGEN; (Horn et al., J. Biol. Chem., 1999, 274, 32023-32030).
Two inducible BCG promoters with BamHI and NcoI cohesive ends were generated by PCR performed on Pasteur M. bovis BCG DNA, using primers BamhspX R (5′-ttaggatccgtccggcatgatcaacctcc-3′: SEQ ID NO: 3) and NcohspX F (5′-catgccatggggtggccatttgatgcctcc-3′: SEQ ID NO: 4) for alpha-crystalline (acr) promoter and the primers BamRv2623F (5′-ttaggatccgggccatggactggtcgtcg-3′: SEQ ID NO: 5) and NCoRV2623 R (5′-catgccatggacatcgcggtcctcctgtcg-3′: SEQ ID NO: 6) for Rv2623 promoter (Florczyk et al., 2001 and 2003, precited).
These fragments were T4 ligated and after two Klenow treatments, acr-apa or Rv2623-apa cassettes were inserted into EcoRV restricted plasmid pYUB295 (Dussurget et al., Infect Immun., 2001, 69, 529-533; Gomez et al., Mol. Microbiol. 1998, 29, 617-628;
In particular, the invention features a recombinant Mycobacterium bovis BCG strain, stably transformed with the plasmid pYAPA2031, deposited at the Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, 75724 Paris Cedex 15, on Jul. 21, 2006, under accession number I-3659, identified hereafter as BCG Apa++.
Low-oxygen liquid cultures were grown in vented-cap tissue culture flasks (25 cm2 Corning) in an incubator which allowed to control the oxygen tension. Vented-cap tissue culture flasks allowed for the exchange of gases between the flasks and the controlled environment of the incubator. Oxygen levels were maintained by injecting prepared gas mixture (Carboxique) into the incubators. Low-oxygen cultures were grown under atmospheres of 1.3% total O2. High-oxygen cultures were grown in ambient air in 25 cm2 tissue culture flasks with the caps tightly sealed. When necessary, kanamycin was used at 25 μg/ml.
Overexpression was confirmed by Western blotting using a rabbit polyclonal antibody raised against the M. bovis BCG APA (Laqueyrerie et al., Horn et al., precited).
The BCG apa gene was mutated by allelic exchange (Pelicic et al., Proc. Natl. Sci. USA, 1997, 94, 10955-10960). Two DNA fragments containing 1000 bp of apa flanking sequences were generated by PCR using primers Sma15up (5-tcccccgggggtgttgacccgacac-3; SEQ ID NO: 7)) and Pst23up (5-aactgcagggcgaagaacctacc-3; SEQ ID NO: 8) for upstream fragment (U) and Pst35down (5-aactgcagccaagtgatacccct-3; SEQ ID NO: 9) and Hin43down (5-cccaagcttggagatcggtgcggc-3; SEQ ID NO: 10) for downstream fragment (D). These fragments were cloned into pBluescript II KS (INVITROGEN), constructing pUD1. pApa::Kan was created by cloning the HincII-flanked aph gene (kanamycin resistance) of pUC4K into the Apa KpnI site of pLA34-2 (Horn et al., J. Biol. Chem., 1999, 274, 32023-32030). A 3.3 kb blunt ended fragment of pApa::Kan containing Apa::aph was cloned into the PstI site of pUD1 creating pUDApa::Kan whose SmaI-HindIII fragment was subcloned in SmaI site of pXYL4, a plasmid bearing the xylE gene (Pelicic et al., precited). The 6-kb BamHI fragment containing U, D, Apa::aph and xylE was isolated and ligated at the BamHI site of pPR27, a vector which contains the counterselectable sacB gene and the thermosensitive origin of replication of pAL5000 (Pelicic et al., precited). The resulting plasmid pPR27::Xyl::UDApaKan was electroporated into BCG and transformants were selected at 32° C. on 7H11 medium containing kanamycin (25 μg/ml) and then grown in 7H9 broth containing kanamycin. Gene replacement accompanied by plasmid loss was selected on 7H11-kanamycin-2% sucrose at 39° C. (Pelicic et al., precited). Loss of the plasmid was confirmed in 100% of the resultant colonies by spraying with catechol, a chromogenic substrate of XylE (Pelicic et al., precited). Gene replacement of apa was verified by PCR of genomic DNA from three colonies, using the primers of apa gene (Horn et al., precited) and the primers for aph gene (Norman, E., Gene replacement in Mycobacterium bovis BCG. Methods in Molecular Biology Vol: 101, Mycobacteria Protocols Edited by T. Parish and N. G. Stoker. Humana Press 1998). One mutant clone was designated BCG Apa−. The absence of APA from the mutants was confirmed by Western blotting using a rabbit polyclonal antibody raised against the M. bovis BCG Apa (Laqueyrerie, Infect Immun., 1995, 10, 4003-4010; Horn et al., precited).
In vitro studies were performed in two cell lines established from human bladder carcinomas: T24 cells, derived from a human transitional cell carcinoma (ATCC # HTB-4) and RT112 or RT112/84 cells (ECACC 85061106; Marshall C. et al., J. Natl. Cancer Inst., 1977, 58, 6, 1743-1751), derived from a human papillary non-metastatic bladder carcinoma using standard cell culture protocols, which are well-known to those skilled in the art.
T24 and RT112 cells were cultivated at 37° C., in a humid atmosphere with 5% CO2, in DMEM (GIBCO) cell culture medium supplemented with 10% decomplemented fetal bovine serum (FBS, EUROBIO) and 1% non-essential amino acids.
In the following studies, Mycobacterium bovis BCG 1173P2 (The Pasteur strain) will be named BCG or classic BCG, recombinant BCG Apa++ will be named BCG Apa++ and the mutant BCG Apa−, BCG Apa−.
T24 cells (106) were seeded in 25 cm2 culture flasks and cultivated in 10 ml culture medium. After 48 hours of culture, 5.106 BCG bacilli (BCG, BCG Apa− or BCG Apa++) were added to cell culture medium. After 24 or 48 hours of culture in the presence of BCG, the cells were harvested and living cells, excluding trypan blue, were counted in a Malassez cell.
2) Inhibition of Cell Viability
The effect of BCG and BCG Apa++ on cell viability was evaluated by MTT assay, using Cell Proliferation Kit I (MTT) from ROCHE DIAGNOSTICS. The MTT assay is based on the cleavage of the yellow tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) to purple formazan crystals by metabolic active cells. This cellular reduction involves the pyridine nucleotide cofactors NADH and NADPH. The formazan crystals formed are solubilized and the resulting colored solution is quantified using a scanning multiwell spectrophotometer.
T24 cells were cultivated for 48 hours in the presence of 5.106 or 107 BCG, BCG Apa− or BCG Apa++ bacilli (equivalent to 5 and 10 bacilli per cell, respectively). At the end of the incubation period, the cells were transferred into microplates (tissue culture grade, 96 wells, flat bottom) in a final volume of 100 μl culture medium per well. Twenty four hours after cell transfer, 10 μl of the MTT labeling reagent (0.5 mg/ml) was added to each well. After an incubation period of 4 h, at 37° C. in 5% CO2, 100 μl of the solubilization solution was added into the wells. The plates were incubated overnight at 37° C. and the reaction products were quantified by measuring the absorbance at 570 nm using a scanning multiwell spectrophotometer.
The effect of BCG and BCG Apa++ on cell proliferation was evaluated by BrdU incorporation assay, using Cell Proliferation ELISA BrdU (calorimetric) kit (ROCHE DIAGNOSTICS).
T24 cells (2.5×104/well) were grown in presence of 2.5×105 BCG or BCG Apa++ bacilli (10 bacilli/cell) for 12 hours, 24 hours, 36 hours or 48 hours at 37° C. At the end of each measuring time, 10 μM BrdU was added to the cells for 3 hours. During this labelling period, the pyrimidine analogue BrdU was incorporated in place of thymidine into the DNA of proliferating cells. After removing of the culture medium, the cells were fixed with a hair drier and their DNA was denatured by 200 μl FixDenat for 30 minutes at room temperature. The antibody anti-BrdU-peroxydase was bound (2 h at room temperature) to the BrdU incorporated in newly synthesized cellular DNA. The immune complexes were detected with substrate reaction (TMB—TetraMethyl Benzidine). After an acidic stop, the reaction products were quantified by measuring the absorbance at 450 nm by a scanning multiwell spectrophotometer. The developed colour and thereby the absorbance values directly correlate to the amount of DNA synthesis and hereby to the number of proliferating cells in the respective cultures.
T24 and RT112 cells (105/ml) were incubated for 48 h with 106 CFU/ml of BCG or BCG Apa++ or without bacteria. The cells were cooled at 4° C. during 15 min and incubated for 2 h at 4° C. with or without fibroblast growth factor (FGF-1, PROMEGA; 50 ng/ml). After covalent cross-linking with 1 mM bis-(sulfo-succinimidyl)-suberate and 15 mM disuccinimidyl-suberate (PIERCE) for 30 min at room temperature, the cells were lysed, immunoprecipitated with anti-fibroblast growth factor receptor (FGFR-3) C-terminal antibody (C-15 sc-123; SANTA CRUZ BIOTECHNOLOGY INC), and probed on an immunoblot with a polyclonal antibody to the cytoplasmic domain of FGFR-3 (P-18 sc-31162; SANTA CRUZ BIOTECHNOLOGY INC.). The density obtained for the dimers (˜250 kDa) relative to the sum of the density of monomers (˜120 kDa) and dimers were determined.
The dimerisation of FGF receptor (FGFR-3) observed in T24 and RT112 carcinoma cells incubated with FGF is partly inhibited in presence of BCG and totally inhibited in presence of BCG Apa++ (
Analysis of subsequent stimulation pathways was performed. Therefore, T24 cells cultivated in six-well plates were incubated 48 hours with 106 CFU of BCG or BCG Apa++ in serum-free DMEM, then 15 minutes with or without EGF (100 ng/ml), and lysed. The presence of phosphorylated mitogen-activated protein kinase p42/p44 (pMAPK) and phosphorylated Akt 1-2 were determined by total protein ELISA by PhosphoDetect ERK1/2 (pThr185/pTyr187) (CALBIOCHEM) and PhosphoDetect Akt (pSer473) (CALBIOCHEM) ELISA Kits, respectively.
The results reporting phosphorylation of MAPK (
Morphological observation of T24 and RT112 human bladder carcinoma cells treated with BCG or BCG Apa++ revealed the presence of abundant cytoplasmic vesicles and nuclear abnormalities indicative of apoptosis.
The switch to apoptotic pathways is under the control of the Bcl-2 family of proteins and involves cysteine-aspartic-acid-proteases (caspases), (For a review, see J. M. Adams and S. Cory Oncogene 2007, 26, 1324-1337). Among the Bcl-2 family members, Bcl-2 promotes cell survival and at the opposite, Bid, Bak and Bax promote cell death. Caspases are a group of cysteine proteases, enzymes with a crucial cysteine residue that can cleave other proteins or peptides after an aspartic acid residue. Caspase-8, a protease that recognizes the amino acid sequence IETD (Ile-Glu-Thr-Asp) is a key element in the external apoptosis pathway. Caspase-9, a protease that recognizes the amino acid sequence LEHD (Leu-Glu-His-Asp) is a key element in the internal apoptosis pathway.
Therefore, the activation level of Bcl-2, Bid, Bak, Bax was evaluated in T24 and RT112 cells treated with BCG or BCG Apa++ or untreated, using RNase protection assay (RPA). In addition, caspase-8 and caspase-9 levels were evaluated in T24 cells treated with BCG or BCG Apa++ or untreated, using a colorimetric assay.
1) RNase Protection Assay
The cells (106 per flask) were cultivated with 5.106 CFU of BCG or BCG Apa++ or without bacteria. Twenty-four hours after treatment, the cells were harvested after cold-PBS washes. Cell suspensions were homogenized in TRIzol and RNA was extracted from the lysates using the chloroform-isopropanol method, according to the manufacturer's instruction (INVITROGEN). Dried RNA precipitates were resuspended in DEPC-H2O (0.1% DiEthylPyroCarbonatein water, INVITROGEN).
mRNA expression was measured by using the RiboQuant multiprobeRNA protection assay (BD BIOSCIENCES PHARMINGEN), following the manufacturer's instructions. Briefly, antisense RNA probes were transcribed using the cDNA template set of Human Apoptosis hAPO-2c (BD BIOSCIENCES PHARMINGEN). For transcription, 1 μl (50 ng) of the template was incubated for 2 h at 37° C. in a 19 μl aqueous mixture containing 2 μl of the enzymes (80 U of RNasin and 40 U T7 RNA polymerase), 4 μl of the nucleotide pool (5 mM) containing 3.25 mM Biotin-16-UTP (ROCHE DIAGNOSTICS), 2 μl of DTT (100 mM) and 4 μl of 5× transcription buffer. Except for Biotin-16-UTP, all reagents were supplied by the manufacturer in RiboQuant Non-Rad In Vitro Transcription Kit (BD BIOSCIENCES PHARMINGEN). The reaction was terminated by adding 2 μl (2 U) DNase, followed by a 30 min incubation at 37° C. DNase was inactivated by 20 mM EDTA and labeled RNA probes were extracted using Lithium Chloride (LiCl 4 M), followed by ethanol precipitation on dry ice.
For hybridization, 20 μg RNA samples precipitated by ethanol and dried using a vacuum evaporator centrifuge, were resuspended in an 8 μl hybridization buffer (80% formamide, 1 mM EDTA, 400 mM NaCl, and 40 mM Prpes (pre-eazine-N,N′-bis-ethanesulfonic acid; BD BIOSCIENCES PHARMINGEN) at 56° C., mixed with 2 μl (30 ng) of probe prepared as previously described, heated to 90° C., and then incubated at 56° C. for 18 h.
For RNase digestion, 10 volumes of a mixture containing RNase A and RNase T1 (100 U) was added and reacted for 45 min at room temperature. After digestion, the samples were mixed with proteinase K and an appropriate buffer supplied in RiboQuant Non-Rad RPA Kit (BD BIOSCIENCES PHARMINGEN) for 15 min at 37° C., after which they were extracted by LiCl and precipitated with ethanol. The samples were then air-dried, resuspended in loading buffer, and size-separated using Polyacrylamide Gel Electrophoresis (PAGE). The bands were electrotransferred to a positively charged nylon membrane (BD BIOSCIENCES PHARMINGEN) by using a Semi-dry Electroblotter (100 mA for 20 min) and crosslinked by UV.
Chemiluminescent probe detection, was performed by using BD RiboQuant Non-Rad Detection kit (BD BIOSCIENCES PHARMINGEN). Nylon membranes were blocked with blocking buffer and conjugated with Streptavidin-Horseradish peroxidase for 15 min at room temperature. Conjugated membranes were washed (supplied Wash Buffer and Substrate Equilibration Buffer) and incubated for 10 min in a freshly prepared equal volume mixture of Stable peroxide solution and Luminol/Enhancer at room temperature. Revealed membranes were exposed to CL-XPosure films (PIERCE) and nucleotide lengths versus migration distances were compared with the standards (30 ng transcribed biotin-labeled probes) on a logarithmic grid. Ribosomal protein L-32 mRNA was used as control.
T24 cells were incubated 6 hours, 12 hours, 24 hours, 36 hours or 48 hours with BCG or BCG Apa++ or without bacteria and lysed. Caspase-8 and caspase-9 were measured on cell lysates using colorimetric assay kits (R&D SYSTEMS). Synthetic peptides (IETD) or (LEHD) conjugated to the chromophore pNA (β-nitroanilide) were added to cell lysates. Upon cleavage of the substrate IEDA-pNA by Caspase-8 or LEHD-pNA by Caspase-9, free pNA is released, resulting in an increase of absorbance at 405 nm, that is measured with a spectrophotometer. Comparison of the absorbance of reaction mixtures made from treated cells with reaction mixtures made from untreated controls allows determination of the increase in caspase activity.
Both caspase-8 (
Thirty female Wistar rats (JANVIER), 7 weeks of age at the beginning of the experiment, were used in this study. 24 rats were given 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN; KASEI INDUSTRY COMPANY) in the drinking water for 19 weeks. 6 rats were given (normal) drinking water as controls and maintained without any treatment up to 30 weeks. One week after the end of BBN treatment (20th week), the rats received 6 weekly intravesical instillations (0.5 ml) of PBS alone, or PBS containing 108 CFU/ml of BCG or BCG Apa++ (
The urines were harvested at week 30 and their content in haemoglobin measured using the Drabkin's method. This colorimetric assay is based on a measure of cyanmethemoglobin; the total hemoglobin present in urine is rapidly converted to the cyanoderivative at alkaline pH. Drabkin's solution containing alkaline ferricyanide and cyanide reacts with all forms of hemoglobin. 25 μl of the ready to use Hemoglobin reagent from BIOLABO (#3502200, 82250, 82200) was added to 100 μl of rat urines. The mixture was incubated at least 15 minutes at room temperature. The absorbance of sample against blank is read at 540 nm. This non-invasive assay allows to control tumour development and to adjust BCG treatment (dose, frequency of administration) in order to optimize the antitumoral treatment.
The decrease in haemoglobin present in urine observed after BCG therapy was more marked with BCG Apa++.
Rats were euthanized at the 30th week (one month after the last BCG treatment), bladders were collected and subjected to a macroscopic examination.
Clinical observation of the bladders indicated the presence of tumors in all the animals of the PBS-treated group and half of the animals of the BCG treated group. By contrast, the bladder was normal in all the animals of the BCG Apa++-treated group (
Rats were euthanized at the 30th week (one month after the last BCG treatment). Bladders were collected, homogenized and nuclear protein extracts were prepared by Activemotif nuclear extract kit (Active motif, CARLSBAD). Activated wild type p53 were measured by an Enzyme Immunometric Assay Kit (TiterZyme®, EIA900-117, Ann Arbor, Mich.).
Analysis of wild-type p53 tumor suppressor factor concentration in the tumor cells nucleus, demonstrates no reduction in the BCG Apa++ treated group, compared to a 50% reduction in the PBS-treated group and a 25% to 30% reduction in the BCG treated group (
RNA extraction was performed as described in example 4. Human cancer pathway finder microarray kits (GEArray™ Oligo OHS-033) were obtained from SUPERARRAY BIOSCIENCE CORP. They included nylon membranes printed by specific cDNA fragments of 113 genes associated with different cancer cascades and classified in six groups (Table II). Probe synthesis and probe biotin labelling were performed by using GEArray AmpoLabeling-LPR Kit (SUPERARRAY BIOSCIENCE CORP.) and Biotin-16-dUTP (ROCHE DIAGNOSTICS), following the manufacturer's instructions. Biotinylated and amplified cDNA probes were hybridized overnight at 60° C. with different array membranes and AP-streptavidin chemiluminescent detection was performed by SuperArray Detection Kit (SUPERARRAY BIOSCIENCE CORP.). Membranes were exposed on CL-XPosure films (PIERCE) and image acquisition was accomplished by a desk scanner using 200 dpi. Data acquisition was performed by using the ScanAlyze software version 2.50 (http://rana.lbl.gov/EisenSoftware.htm). Data analysis was completed by GEArray Analyzer software (www.superarray.com). Raw data were subtracted from the mean signals of three negative controls as areas without spotted gene sequences (blanks) or areas spotted by the genes not expressed in human cells (pUC18). For adjusting of loading (1 μg total RNA of stimulated or control T24 cells) these subtracted data were normalized by their ratio to averaged signals of three positive controls (housekeeping genes: β-actin, GAPDH and Ribosomal protein S27a). Additionally, the data were also normalized according to another criterion of lowest signal. Experiment was performed three times to ensure reproducibility of results.
Transcriptome analysis of T24 cells treated with different BCG strains confirms the results from functional studies (examples 3, 4 and 5) and indicate that genes coding for integrins, apoptosis and cell cycle were modulated by BCG and BCG Apa++ but not by BCG Apa− (Table I, Table II and
CDKN1A (p21Waf1), CDKN1B (p27Kip1),
E2F1, MDM2, PRKDC (DNA-PK), PTEN,
APAF1, BAD, BAX, BCL2, BCL2L1 (bcl-X),
(telomerase), TNFRSF1A (TNF-a receptor),
AKT1, CTNNB1 (β-catenin), ERBB2, ETS2,
(MEK), MYC, NFKB1 (NFκB), NFKBIA
(IκBa
), PIK3CB (PI3K p110b), PIK3R1
(PI3K p85a
), RAF1, RASA1, SNCG, SRC.
(integrin a1), ITGA2 (integrin a2), ITGA3
(integrin a3), ITGA4 (integrin a4), ITGA5
(integrin a5), ITGA6 (integrin a6), ITGAV
(integrin β3), ITGB5 (integrin β5), MCAM,
(angiopoietin-2), BAI1, COL18A1 (endostatin),
TGFB1, TGFBR1 (ALK-5), THBS1
MMP2 (gelatinase A), MMP9 (gelatinase B),
MTA1, MTA2, NME1, NME4 (Nm23), PLAU,
| Number | Date | Country | Kind |
|---|---|---|---|
| 06291205.0 | Jul 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB07/03300 | 7/25/2007 | WO | 00 | 4/26/2009 |
