NOVEL MSLN TARGETING DNA VACCINE FOR CANCER IMMUNOTHERAPY
20180064794 ยท 2018-03-08
Inventors
Cpc classification
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to an attenuated mutant strain of Salmonella comprising a recombinant DNA molecule encoding Mesothelin. In particular, the present invention relates to the use of said attenuated mutant strain of Salmonella in cancer immunotherapy.
Claims
1. A method for treating or vaccinating a patient against a cancer comprising administering to the patient an attenuated mutant strain of Salmonella comprising at least one copy of a recombinant DNA molecule comprising an expression cassette encoding Mesothelin (MSLN).
2. The method of claim 1, wherein the attenuated mutant strain of Salmonella is of the species Salmonella enterica.
3. The method of claim 1, wherein the expression cassette is a eukaryotic expression cassette.
4. The method of claim 1, wherein MSLN is human MSLN.
5. The method of claim 3, wherein the recombinant DNA molecule further comprises the kanamycin antibiotic resistance gene, the pMB1 ori, and a eukaryotic expression cassette encoding human MSLN under the control of a CMV promoter, wherein the human MSLN has the nucleic acid sequence as found in SEQ ID NO 2.
6. (canceled)
7. (canceled)
8. (canceled)
9. The method of claim 1, wherein the method further comprises administration of one or more further attenuated mutant strain(s) of Salmonella comprising at least one copy of a recombinant DNA molecule comprising an expression cassette encoding a tumor antigen and/or a tumor stroma antigen.
10. The method of claim 9, wherein the attenuated mutant strain of Salmonella is co-administered with said one or more further attenuated mutant strain(s) of Salmonella.
11. The method of claim 1, wherein the method further comprises administering chemotherapy, radiotherapy or biological cancer therapy to the patient.
12. The method of claim 1, wherein the attenuated mutant strain of Salmonella is administered orally.
13. The method of claim 1, wherein the cancer is selected from mesotheliomas, ovarian and pancreatic cancers, squamous cell carcinomas of the cervix, head and neck, vulva, lung and esophagus, lung adenocarcinomas, endometrial carcinomas, biphasic synovial sarcomas, desmoplastic small round cell tumors and gastric adenocarcinomas.
14. The method of claim 1, wherein the single dose comprises from about 10.sup.5 to about 10.sup.11, particularly from about 10.sup.6 to about 10.sup.10, more particularly from about 10.sup.6 to about 10.sup.9 , more particularly from about 10.sup.6 to about 10.sup.8, most particularly from about 10.sup.6 to about 10.sup.7 colony forming units (CFU).
15. The method of claim 1 further comprising the step of assessing the MSLN expression pattern and/or the pre-immune response against MSLN of the patient, and wherein the treatment is individualized cancer immunotherapy treatment.
16. The method of claim 2, wherein the attenuated mutant strain of Salmonella is Salmonella typhi Ty21a
17. The method of claim 4, wherein MSLN has the amino acid sequence of SEQ ID NO 1 or is at least 80% identical to SEQ ID NO 1.
18. The method of claim 9, wherein said one or more further attenuated mutant strain(s) of Salmonella is Salmonella typhi Ty21a comprising a eukaryotic expression cassette.
19. The method of claim 18, wherein said one or more further attenuated mutant strain(s) of Salmonella comprise(s) an attenuated mutant strain of Salmonella encoding human VEGFR-2 and/or human Wilms' Tumor Protein (WT1).
20. The method of claim 11, wherein the attenuated mutant strain of Salmonella is administered before or during the chemotherapy or the radiotherapy treatment cycle, or before or during the biological cancer therapy, or before and during the chemotherapy or the radiotherapy treatment cycle or the biological cancer therapy.
Description
SHORT DESCRIPTION OF FIGURES AND TABLES
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[0097] Table 1: Pro5 Recombinant MHC Pentamers used in the Pentamer analysis.
EXAMPLES
Example 1
Preparation of Recombinant Plasmids pVAX10.mMSLN and pVAX10.hMSLN
[0098] Human MSLN (1893 bp, MSLN sequence according to NCBI reference sequence NM_013404.4) and murine MSLN (1878 bp, MSLN sequence according to NCBI reference sequence NM_018857.1) were cloned into the pVAX10 backbone derived of pVAX10.VR2-1. MSLN DNA fragments were generated by double-strand gene synthesis, where oligonucleotides were linked together using a thermostable ligase. The obtained ligation products were amplified by PCR. Upon amplification, the in vitro synthesized DNA fragments of human and murine MSLN were cloned into the pVAX10 backbone via NheI/XhoI (the VEGFR-2 coding region of recombinant plasmid pVAX10.VR2-1 was replaced by human or murine MSLN). For quality control, the entire plasmids were sequenced and aligned to the respective reference sequence after transformation into E. coli. Both sequences proved to be free of errors. The resulting plasm ids were designated pVAX10.mMSLN and pVAX10.hMSLN.
Example 2
Transformation of Attenuated Salmonella Strains with the Recombinant Plasmids
[0099] S. typhi Ty 21a was transformed with plasm id pVAX10.hMSLN. S. typhimurium SL7207 (aroA.sup.) was transformed with plasmid pVAX10.mMSLN. The transformation was performed by electroporation.
Preparation of Competent Salmonella Cells:
[0100] Glycerol cultures of S. typhi Ty21a and S. typhimurium SL7207 were inoculated on LB plates (animal component free [ACF] soy peptone). The plates were incubated at 37 C. overnight. One colony each was used for overnight-liquid-preculture. 3 ml LB medium (ACF soy peptone) inoculated with one colony each was incubated at 37 C. and 180 rpm overnight. To prepare competent cells, 2300 ml of LB medium (ACF soy peptone) were inoculated with 3 ml of the overnight culture and incubated at 37 C. and 180 rpm up to an OD.sub.600 of about 0.5. The cultures were then put on ice for 10 minutes. Subsequently, the bacteria were centrifuged for 10 minutes at 3000g at 4 C. and each pellet was resuspended in 500 mL of ice cold H.sub.2O.sub.dest. After a new centrifugation step, the bacterial pellets were washed twice in 10% ice cold glycerol. Both pallets were put together in 2 ml of 10% glycerol and finally frozen in aliquots of 50 L on dry ice. The used glycerol did not contain any animal ingredients (Sigma Aldrich, G5150).
Transformation of Competent Salmonella Cells:
[0101] For each transformation reaction, a 50 l aliquot of competent cells was thawed on ice for 10 minutes. After adding 3-5 L of plasmid DNA (pVAX10.hMSLN for competent S. typhi Ty21a cells and pVAX10.mMSLN for competent S. typhimurium SL7207 cells) the mixtures were incubated on ice for five minutes. Subsequently, the mixtures were transferred to pre-cooled cuvettes (1 mm thickness). The electric pulse was carried out at 12.5 kV/cm. Immediately afterwards, 1 ml of LB medium (ACF soy peptone) was added to the cells, the cells were transferred into a 2 ml Eppendorf tube and shaken for 1 hour at 37 C. After a short centrifugation step on a bench centrifuge (16600 rcf, 20 s), the bacterial pellet was resuspended in 200 l of LB (ACF soy peptone) antibiotic-free medium. The mixtures were applied with a Drigalski spatula on LB plates (ACF soy peptone) containing kanamycin (concentration=25 g/ml or 50 g/ml). The plates were incubated at 37 C. overnight.
Plasmid Preparation of Recombinant Salmonella Clones:
[0102] Three clones of each recombinant Salmonella strain were incubated overnight in 3 ml of LB medium (ACF soy peptone) containing kanamycin (50 g/ml) at 37 C. The bacterial culture was then pelleted by centrifugation (16600 rcf, 30 s). Plasmid isolation was performed using the NucleoSpin Plasmid Kit from Macherey-Nagel. The plasmid DNA was eluted from the silica gel columns with 50 l water. 5 l of the eluate was used in agarose gel electrophoresis for control.
[0103] For long-term storage, 1 ml glycerol cultures of the positive clones were produced. For this purpose, 172 l glycerol (no animal ingredients) was added to 828 l medium of a logarithmically growing 3 ml culture in a 1 low ml screw microtube. The samples were stored at 70 C. until further use.
Complete Sequencing of Recombinant Plasmid DNA Isolated from Salmonella:
[0104] 3 ml of liquid LB-Kan medium (ACF soy peptone) were inoculated with one colony of recombinant Salmonella (S. typhi Ty21a harboring pVAX10.hMSLN and S. typhimurium SL7207 harboring pVAX10.mMSLN) and incubated overnight at 37 C. and 180 rpm. The overnight culture was pelleted by centrifugation at 1300 rpm for 30 s on a bench centrifuge (Biofuge pico, Heraeus). The plasmid isolation was performed with the NucleoSpin Plasmid Kit from Macherey-Nagel. After alkaline lysis and precipitation of high molecular weight genomic DNA and cellular components, the plasmid DNA was loaded onto columns with a silica membrane. After a washing step, the plasmids were eluted from the column with 50 l of sterile water and sequenced. The sequences were then compared with the respective reference sequence by clone specific alignments, i.e. the plasmid sequences of each Salmonella clone was one by one aligned with the reference sequence. All sequences were in line with the respective reference sequences. The recombinant Salmonella strains were designated VXM04 (S. typhi Ty21a harboring plasmid pVAX10.hMSLN) and VXM04m (S. typhimurium SL7207 harboring plasmid pVAX10.mMSLN).
Example 3
Assessing Immunekinetics of VXM04m in Healthy C57Bl/6 Mice
[0105] The kinetics of specific immune activation against murine Mesothelin in healthy C57Bl/6 mice was evaluated by Pentamer analysis and ELISpot. As negative control, a vector control group (receiving VXM04m-empty=Salmonella typhimurium containing no expression plasmid) was included in the study setup to discriminate the desired immunologic effect from any unspecific background stimulation caused by Salmonella empty vector. Immune monitoring was carried out at D5, D7, D10, D14 and D21 post vaccination after 4-fold every second day vaccination with VXM04m and VXM04m-empty (each 10.sup.10 CFU/dose).
1. Animal Maintenance
[0106] 52 healthy female C57Bl/6 mice, 6 weeks old at reception, were obtained from JANVIER (Le Genest St Isle, France) and observed for 7 days in a specific-pathogen-free (SPF) animal care unit before starting the procedure. Animals were maintained in rooms under controlled conditions of temperature (232 C.), humidity (4510%), photoperiod (12 h light/12 h dark) and air exchange. Animals were maintained in SPF conditions. Room temperature and humidity were continuously monitored. The air handling system was programmed for 14 air changes/hour, with no recirculation. Fresh outside air was passed through a series of filters, before being diffused evenly into each room. A positive pressure (204 Pa) was maintained in the experimentation room to prevent contamination or the spread of pathogens within a rodent colony. Animals were housed in polycarbonate cages (Techniplast, Limonest, France) that were equipped to provide food and water. The standard area cages used were 800 cm.sup.2 with a maximum of 10 mice per cage (from the same group). Bedding for animals was sterile corn cob bedding (ref: LAB COB 12, SERLAB, Cergy-Pontoise, France), replaced twice a week. Animal wood was purchased from DIETEX (Saint-Gratien, France). Irradiated RM1 was used as sterile controlled granules. Food was provided ad libitum from water bottles equipped with rubber stoppers and snipper tubes. Water bottles were sterilized by sterile filtration and replaced twice a week. At D0, 50 mice out of 52 were distributed according to their individual body weight into 2 groups of 25 mice each using Vivo manager software (Biosystemes, Couternon, France). The mean body weight of both groups was not statistically different (analysis of variance).
2. Treatment Schedule
[0107] The mice from groups 1 to 5 received administrations of VXM04m-empty, the animals from groups 6 to 10 received administrations of VXM04m. Both VXM04m-empty and VXM04m were thawed and administered within 30 min, the working solutions were discarded after use. The treatment dose of VXM04m-empty and VXM04m was 10.sup.10 CFU in 100 l per administration. VXM04m-empty and VXM04m were administered by oral gavage (per os, PO) via a cannula with a volume of 0.1 ml. Regardless of animal groups, each animal received pre-dose application buffer to neutralize acid in the stomach prior dosing (100 l/animal/application). Thus buffer was composed by dissolution of 2.6 g sodium hydrogen carbonate, 1.7 g L-Ascorbic acid, 0.2 g lactose monohydrate in 100 ml of drinking water and was applied within 30 min prior application of VXM04m-empty and VXM04m. The treatment schedule was as follows:
[0108] The mice from groups 1 to 5 received daily PO administrations of VXM04m-empty at 10.sup.10 in CFU every two days for four consecutive times (Q2D4).
[0109] The mice from groups 6 to 10 received daily PO administrations of VXM04m at 10.sup.10 in CFU every two days for four consecutive times (Q2D4).
3. Animal Monitoring and Termination
[0110] The viability and behavior of the animals was recorded every day, body weights were measured twice a weak.
[0111] Irrespective of the administered Salmonella vaccine, mice were terminated after 5 (groups 1 and 6), 7 (groups 2 and 7), 10 (groups 3 and 8), 14 (groups 4 and 9) and 21 (groups 5 and 10) days post vaccination phase (5 mice per animal group and time point). Isoflurane (Baxter, France) was used to anaesthetize the animals before termination. Animals were terminated by cervical dislocation. An autopsy (macroscopic examination of heart, lungs, liver, spleen, kidneys and gastrointestinal tract) was performed on all terminated animals. At the time of mice termination, spleens were collected and placed individually into single ID labeled tubes containing chilled PBS (2-8 C.) each and stored over night at 2-8 C. Freshly isolated and purified splenocytes were used for Pentamer analysis. Freshly prepared CD8+ cells were used for ELISpot analysis.
4. Splenocyte Preparation
[0112] Splenocyte preparation was performed as follows: In a washing step a part of the PBS was discarded and replaced by fresh PBS. A 100 m nylon Cell Strainer (BD Falcon) was hung into the opening of a 50 ml Falcon containing 5 ml 1 PBS. The spleens were cut with a scalpel and then pushed through the cell strainer with the stamp of a 5 ml syringe. One strainer was used per spleen, the strainer was always rinsed in between with sterile 1 PBS. The cells were centrifuged at 1,500 rpm (approximately 450 g) for 10 min at 2-8 C. and the supernatant was discarded. 1 ml ACK-Ery-Lysis buffer (8.3 g/l NH.sub.4, 1 g/l KHCO.sub.3, 0.037 g/l EDTA; pH 7.2-7.4) was added per spleen to lyse the red blood cells. The solution was incubated for 30 sec at RT. 10 ml of PBS were added and the cells were again spun down at 1,500 rpm for 10 min at 2-8 C., the supernatant was discarded. The pellet was resuspended in 10 ml DMEM media. Live/dead cell staining was performed with trypan blue and the cell number was counted. The cell suspension was split for the subsequent analyses. About one third was used for Pentamer analysis, the rest was used for the ELISpot analysis.
5. Pentamer Analysis
[0113] Pentamer Analysis included a viability staining and the Pentamer staining. For the viability staining, one vial of the fluorescent reactive dye (Pacific Orangecomponent A) and the vial of anhydrous DMSO (component B) were brought to room temperature before the caps were removed. 50 l of DMSO (component B) was added to the vial of reactive dye (component A). Subsequently the well was mixed and it was confirmed visually that all of the dye had dissolved. The solution of reactive dye was used without delay, within a few hours of reconstitution. The suspension of cells containing at least 110.sup.6 cells was centrifuged and the supernatant was discarded. The cells were washed once with 1 ml of PBS and resuspended in 1 ml of PBS. The cells were counted and the density was adjusted with PBS to 110.sup.6 cells in a 1 ml volume. 1 l of the reconstituted fluorescent reactive dye was added to 1 ml of the cell suspension. The suspension was then mixed thoroughly and incubated at room temperature for 30 min, protected from light. The cells were washed once with 1 ml of PBS with 1% Fetal Calf Serum (FCS) and resuspended in 1 ml of PBS with 1% FCS.
[0114] For Pentamer staining, splenocytes prelabelled with Pacific Orange for viability gating were used. The Pro5 Recombinant MHC Pentamers used are listed in the following Table 1:
TABLE-US-00001 Pentamer description Pentamer mMesothelin including sequence nomenclature start position Unlabelled Pro5 MHC Pentamer H- MSLN-GAI-Penta 406 2Db GQKMNAQAI Mesothelin Unlabelled Pro5 MHC Pentamer H- MSLN-ASL-Penta 138 2Kb ACAHFFSL Mesothelin Unlabelled Pro5 MHC Pentamer H- MSLN-CLL-Penta 18 2Kb CSRSFLLL Mesothelin Unlabelled Pro5 MHC Pentamer H- MSLN-GSL-Penta 54 2Kb GAADFASL Mesothelin Unlabelled Pro5 MHC Pentamer H- MSLN-IQL-Penta 344 2Kb IPFTYEQL Mesothelin Pro5 Fluorotag R-PE
[0115] Pro5 Pentamers were centrifuged in chilled microcentrifuge at 14,000g for 5-10 minutes to collect any protein aggregates present in the solution at the bottom of the vial in order to avoid non-specific staining. The supernatant was used for Pentamer staining. All reagents were maintained on ice, shielded form light, until required. 110.sup.6 splenocytes were allocated per staining condition. The cells were washed with 2 ml wash buffer (PBS with 1% FCS) and spun down (500g for 5 minutes), the supernatant was discarded and the cells were resuspended in the residual volume (50 l). The tubes were kept chilled on ice for all subsequent steps, except where otherwise indicated. One test (2 l) of unlabeled Pentamer was added to the cells and the solution was mixed by pipetting and incubated at room temperature (22 C.) for 10 min, shielded from light. The cells were then washed with 2 ml wash buffer and resuspended in the residual liquid (50 l). Pro5 Fluorotag R-PE was spun in a chilled microcentrifuge at 14,000g for 3 minutes to remove protein aggregates that would otherwise contribute to non-specific binding. The reagents were maintained on ice, shielded from light, until required. The supernatant was used for Pentamer staining. 8 l Pro5 Fluorotag and 1 l of anti-CD8 FITC and 0.5 l anti-CD3 APC/Cy-7 antibodies were added and the solution was mixed by pipetting. The samples were incubated on ice for 20 minutes, shielded from light. The cells were washed twice with 2 ml wash buffer and each tube was mixed. 200 l of fix solution (1% FCS, 2.5% formaldehyde in PBS) was added and the tubes were vortexed. Thorough vortexing was important to avoid cell clumping. The tubes were stored in the dark in the refrigerator until ready for data acquisition. In any case the samples were left for 3 hours before proceeding with data acquisition due to morphology changes after fixing.
[0116] Specific binding to Pentamers each (manufactured by Proimmune) was investigated. mMesothelin specific CD8+ T cells were counted after selecting the appropriate gates. Ratios of mMesothelin specific CD8+ T cells were calculated based on binding affinity to the Pentamers. The ratios were compared between VXM04m and VXM04m-empty control group and within groups over time. All statistical analyses were performed using Vivo manager software (Biosystems, Dijon, France). A p value<0.05 was considered significant. The individual percentages of Meso-specific CD8+ cells are presented in