Salmonella-based vectors for cancer immunotherapy targeting Wilms' tumor gene WT1

Abstract

The present invention relates to an attenuated mutant strain of Salmonella comprising a recombinant DNA molecule encoding Wilms' tumor Protein I. In particular, the present invention relates to the use of said attenuated mutant strain of Salmonella in cancer immunotherapy.

Claims

1. A method of treating a Wilms' Tumor Protein (WT1) expressing cancer, comprising administering orally to a subject a DNA vaccine comprising an attenuated mutant strain of Salmonella enterica typhi Ty21a comprising at least one copy of a recombinant DNA molecule comprising a eukaryotic expression cassette encoding WT1 in cancer immunotherapy, wherein the vaccine is not HLA-restricted.

2. The method of claim 1, wherein WT1 is human WT1.

3. The method of claim 1, wherein the recombinant DNA molecule comprises a kanamycin antibiotic resistance gene, a pMB1 ori, and a eukaryotic expression cassette encoding human WT1 under the control of a CMV promoter.

4. The method of claim 1, wherein the cancer immunotherapy 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.

5. The method of claim 4, wherein the attenuated mutant strain of Salmonella is co-administered with said one or more further attenuated mutant strain(s) of Salmonella.

6. The method of claim 1, wherein cancer immunotherapy is accompanied by chemotherapy, radiotherapy or biological cancer therapy.

7. The method of claim 1, wherein the cancer is selected from leukemia and solid tumors.

8. The method of claim 1, wherein the attenuated mutant strain of Salmonella is administered as the single dose comprising from about 10.sup.5 to about 10.sup.11 colony forming units (CFU).

9. The method of claim 1, for individualized cancer immunotherapy comprising a step of assessing a tumor antigen expression pattern and/or stroma antigen expression pattern of the subject.

10. The method of claim 1, wherein WT1 is truncated and the truncated WT1 has the amino acid sequence as found in SEQ ID NO 1.

11. The method of claim 4, wherein said one or more further attenuated mutant strain(s) of Salmonella is/are Salmonella typhi Ty21a comprising a eukaryotic expression cassette.

12. The method of claim 11, wherein said one or more further attenuated mutant strain(s) of Salmonella comprise(s) an attenuated mutant strain of Salmonella encoding human VEGFR-2.

13. The method of claim 7, wherein the leukemia is selected from acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL).

14. The method of claim 7, wherein the solid tumor is selected from lung cancer, breast cancer, esophageal, colon, colorectal, gastric, cholangioductal, pancreatic cancer, glioblastoma, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, cervical, endometrial, ovarian cancer, neuroblastoma, rhabdomyosarcoma and prostate cancer.

Description

SHORT DESCRIPTION OF FIGURES AND TABLES

(1) FIG. 1: Amino acid sequence of truncated human WT1 encoded by WT1 cDNA contained in plasmid pVAX10.hWT1

(2) FIGS. 2A and 2B: Nucleic acid sequence of pVAX10.hWT1

(3) FIG. 3: Plasmid map of pVAX10.hWT1

(4) FIG. 4: Kaplan-Meier survival curves of mice bearing FBL-3 leukemia treated with VXM0m_empty, VXM06m and VXM06

(5) FIG. 5: Mean survival of mice bearing FBL-3 leukemia treated with VXM0m_empty, VXM06m and VXM06

(6) Table 1: In vitro gene synthesis: phosphorylation reaction setup

(7) Table 2: In vitro gene synthesis: amplification of ligation productPCR profile

(8) Table 3: Vaccine compositions

(9) Table 4: Assessment of antitumor activityexperimental design

(10) Table 5: Survival of mice bearing FBL-3 leukemia treated with VXM0m_empty, VXM06m and VXM06

(11) Table 6: Number of dead animals after tumor challenge over time

(12) Table 7: Mean and median survival of mice bearing FBL-3 leukemia treated with VXM0m_empty, VXM06m and VXM06

EXAMPLES

Example 1: Preparation of Recombinant Plasmids pVAX10.mWT1 and pVAX10.hWT1

(13) Human truncated WT1 (1116 bp, WT1 sequence according to UniProt reference sequence P19544-7, truncated by the zinc finger domain) and murine truncated WT1 (1101 bp, WT1 sequence according to UniProt reference sequence P22561-5, truncated by the zinc finger domain) were cloned into the pVAX10 backbone derived of pVAX10.VR2-1. WT1 DNA fragments were generated by double-strand gene synthesis, where oligonucleotides were linked together using a thermostable ligase.

(14) Oligo Design and Synthesis:

(15) In a first step, the gene sequence of truncated human and truncated murine WT1 (truncated by the zinc finger domain) were subdivided into individual oligonucleotides of 40 to 50 bases using the software SeqEditor (Entelechon). The defined oligonucleotides overlapped and corresponded to both DNA strands. After synthesis of the oligonucleotides of both DNA strands, the oligonucleotides were diluted with 10 mM Tris (pH 8.5) to a final concentration of 50 pmol/l.

(16) Kinase Reaction:

(17) The in vitro synthesized oligonucleotides were then phosphorylated by incubation with T4 polynucleotide kinase in order to allow for subsequent ligation of the oligonucleotides. Forward and reverse oligonucleotides were phosphorylated.

(18) The reaction setup is summarized in the following Table 1:

(19) TABLE-US-00001 Volume Ingredient 10 l Primer mix 10 l 10x T4 Polynucleotide kinase (PNK) buffer 10 l ATP (25 mM) 68 l Sterile water 2 l T4 PNK (10 U/l)

(20) The reaction mixture was incubated for 1 hour at 37 C. in a water bath. Then the T4 polynucleotide kinase was inactivated by a five-minute heat step at 95 C. and afterwards immediately cooled on ice until further treatment.

(21) 12.5 L of the kinase mixture was used directly for ligation (20 U Taq DNA ligase, 35 l reaction volume (New England Biolabs, M0208S).

(22) The denaturation step at 95 C. was followed by progressive cooling (1 C./min). During this process, the complementary oligonucleotides assemble to a double strand DNA. The process was performed in a thermocycler (Personal Cycler, Biometra). By addition of the thermostable Taq DNA ligase, the 5-PO.sub.4-end of the oligonucleotides were linked with the free 3-OH-end of the following oligonucleotide. By repeated denaturation and renaturation steps, mismatched oligonucleotides were released. At the end of the program, the mixture was cooled at 4 C.

(23) Amplification of the Ligation Products by PCR:

(24) 5 l of the obtained ligation products (about 1.1 kb) were amplified by PCR in a 50 l volume with flanking primers as depicted in the following Table 2:

(25) TABLE-US-00002 no step temperature time No of cycles 1 denaturation 95 C. 5 min 1 2 denaturation 95 C. 30 sec 3 annealing 57 C. 30 sec {close oversize brace} 30x 4 elongation 72 C. 90 sec 5 extra-elongation 72 C. 5 min 1 6 cooling 4 C. 1

(26) The PCR mixture contained the following components: 0.2-0.5 M of each primer, 20 mM Tris-CI, pH 8.8, 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 2 mM MgSO.sub.4, 0.1% Triton X-100, 25 mM dNTP (each), 2 U Vent.sub.R polymerase.

(27) The in vitro synthesized DNA fragments (human and murine truncated WT1; about 1.1 kb each) were cloned into the pVAX10 backbone via NheI/XhoI (the VEGFR-2 coding region of recombinant plasmid pVAX10.VR2-1 was replaced by truncated human or murine WT1). 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 plasmids were designated pVAX10.mWT1 and pVAX10.hWT1.

Example 2: Transformation of Attenuated Salmonella Strains with the Recombinant Plasmids

(28) S. typhi Ty 21a was transformed with plasmid pVAX10.hWT1. S. typhimurium SL7207 (aroA.sup.) was transformed with plasmid pVAX10.mWT1. The transformation was performed by electroporation.

(29) Preparation of Competent Salmonella Cells:

(30) 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).

(31) Transformation of Competent Salmonella Cells:

(32) 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.hWT1 for competent S. typhi Ty21a cells and pVAX10.mWT1 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, 20s), 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.

(33) Plasmid Preparation of Recombinant Salmonella Clones:

(34) 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.

(35) 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.

(36) Complete Sequencing of Recombinant Plasmid DNA Isolated from Salmonella:

(37) 3 ml of liquid LB-Kan medium (ACF soy peptone) were inoculated with one colony of recombinant Salmonella (S. typhi Ty21a harboring pVAX10.hWT1 and S. typhimurium SL7207 harboring pVAX10.mWT1) 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 VXM06 (S. typhi Ty21a harboring plasmid pVAX10.hWT1) and VXM06m (S. typhimurium SL7207 harboring plasmid pVAX10. mWT1).

Example 3: Assessing Antitumor Activity of VXM06 and VXM06m in Syngeneic Leukemia Mouse Model

(38) The efficacy of VXM06 and VXM06m was assessed in a syngeneic leukemia C57/BL6J mouse model over a period of 43 days (with dose administration on alternate days for 4 occasions followed by leukemia cell inoculation at day 17). Two groups, each comprising 10 male mice (n=10) either received VXM06 (S. typhi Ty21a containing pVAX10.hWT1 coding for truncated human WT1) or VXM06m (S. typhimurium containing pVAX10.mWT1 coding for truncated murine WT1) at doses of 10.sup.10 CFU/occasion. One similarly constituted control group received VXM0m_empty (S. typhimurium vector control with no expression plasmid) at the same dose as the treated groups. During the study, body weight, mortality, and survival investigations were undertaken. Prior to this main study, a pilot study over 14 days in male C57616 mice (n=5 per group) without vaccination was performed using 5.010.sup.6 and 3.010.sup.7 FBL-3 cells, after which the first cell concentration was judged as optimal for the main vaccination study.

(39) DNA Vaccines:

(40) VXM06, VXM06m, and the S. typhimurium empty vector (VXM0m_empty) without expression plasmid were stored at 80 C. until use. Vials being used during vaccination were not frozen again but discarded afterwards. The DNA vaccines under investigation are characterized in the following Table 3:

(41) TABLE-US-00003 Test item Batch Concentration Quantity VXM0m_empty VXM01m-e.1-01/2010 10.sup.11 CFU/ml 0.7 ml/vial (10 vials) VXM06m VXM06m-031.1-01/2012 10.sup.11 CFU/ml 0.7 ml/vial (10 vials) VXM06 VXM06-031.1-01/2012 10.sup.11 CFU/ml 0.7 ml/vial (10 vials)
Route of Drug Administration:

(42) 100 l of VXM0m_empty, VXM06m and VXM06 were applied per animal and application. After thawing of test items, application took place within 30 min. All test substances were administered by oral gavage (per os, P.O) via cannula with an injection volume of 100 l/mouse.

(43) Regardless of animal groups, each animal received pre-dose application buffer to neutralize acid medium in the stomach prior to dosing (100 l/animal/application for all dose groups). This buffer contained 2.6 g sodium hydrogen carbonate, 1.7 g L-ascorbic acid, 0.2 g lactose monohydrate and 100 ml of drinking water. Pre-dose applications were performed up to 30 minutes prior to application of the test items.

(44) Cell Culture:

(45) The WT1 overexpressing mouse leukemia cells FBL-3 required passaging to ensure viability and to attain the required amount of cells.

(46) The exponentially growing tumor cells were collected, mixed with trypan blue (at the recommended dilution 1:1) for viability determination, and manually counted using a counting chamber under optical microscope. FBL-3 cells were washed and resuspended in serum-free RPMI medium for injections into C57BL/6 mice.

(47) Vaccination and Tumor Cell Inoculation:

(48) Cell injection conditions for intraperitoneal (I.P.) injection: cell viability 97%; 5.010.sup.6 cells/500 l/mouse.

(49) Animals (30 C57BL/6 mice, 4-6 weeks, male, 20 g each, Charles River, France) were numbered, given a unique animal identification ear notch mark; the body weight was measured twice a week.

(50) Ten mice each (n=10 male) were vaccinated with VXM0m_empty, VXM06m, and VMX06. Vaccination was carried out by oral gavage of 10.sup.10 CFU/application at days 1, 3, 5, and 7. At day 17, FBL-3 tumor cells were inoculated in mice by I.P. route. The experimental design is summarized in the following Table 4:

(51) TABLE-US-00004 Tumor Dose Admin. Group Animals cells Treatment (CFU/adm) route Treatment schedule 1 10 5 10.sup.6 VXM0m_empty 10.sup.10 (in 100 l) PO Q2D 4 (D1, D3, D5, D7) 2 10 5 10.sup.6 VXM06m 10.sup.10 (in 100 l) PO Q2D 4 (D1, D3, D5, D7) 3 10 5 10.sup.6 VXM96 10.sup.10 (in 100 l) PO Q2D 4 (D1, D3, D5, D7)
Results:

(52) Survival data for the three treatment groups are listed in the following Table 5:

(53) TABLE-US-00005 Group 1; Group 2; Group 3; VXM0m_empty VXM06m VXM06 Day Survival Survival Survival 3 10 10 10 1 10 10 10 5 9 9 10 7 9 9 10 12 9 9 10 15 9 9 10 19 9 8 10 22 9 8 10 26 9 8 10 29 5 7 9 33 0 4 4 36 0 1 3 40 0 1 1 43 0 0 0

(54) A daily clinical examination of all animals was performed: behavior, signs of suffering (cachexia, becoming, and difficulties moving or feeding).

(55) The number of dead animals after tumor challenge are listed in the following Table 6:

(56) TABLE-US-00006 Time Survival Dead treatment 1 9 0 VXM0m_empty 12 6 3 VXM0m_empty 15 2 4 VXM0m_empty 16 0 2 VXM0m_empty 1 8 0 VXM06m 12 7 1 VXM06m 14 6 1 VXM06m 16 4 2 VXM06m 19 1 3 VXM06m 26 0 1 VXM06m 1 10 0 VXM06 12 9 1 VXM06 14 8 1 VXM06 15 6 2 VXM06 16 4 2 VXM06 19 3 1 VXM06 v21 2 1 VXM06 22 1 1 VXM06 26 0 1 VXM06

(57) FIG. 4 depicts Kaplan-Meier survival curves of mice bearing FBL-3 leukemia treated with VXM0m_empty, VXM06m and VXM06. Mice treated with VXM06m and VXM06 survived longer compared to the control mice (up to 26 days). About 40% the mice that received VXM06m and VXM06 survived longer than those treated with VXM0m_empty. Mean and median survival of the three test groups are depicted in the following Table 7:

(58) TABLE-US-00007 VXM0m_empty VMX06m VXM06 9 8 10 Mice Survival time 1 12 12 12 2 12 14 14 3 12 16 15 4 15 16 15 5 15 19 16 6 15 19 16 7 15 19 19 8 16 26 21 9 16 22 10 26 Median 15 17.5 16 Mean 14.2 17.6 17.6 SD 1.6 4.0 4.1 SEM 0.5 1.4 1.3 Var. 2.6 15.7 16.6

(59) FIG. 5 depicts the mean survival of mice bearing FBL-3 leukemia treated with VXM0m_empty, VXM06m and VXM06. Mice treated with VXM06m and VXM06 survived longer compared to the control mice.

(60) This study demonstrated the effectiveness of the constructs VXM06 and VXM06m in targeting WT1 overexpressing leukemia cells in a mouse model. The control mice treated with empty vector survived for up to 16 days after tumor cell challenge (see FIG. 4). In contrast, the mice treated with VXM06m or with VXM06 showed prolonged survival compared to the mice vaccinated with the control vector VXM0m_empty (up to 26 days for both test items). About 40% of the mice that received VXM06m or VXM06 survived longer than those in the control group treated with VXM0m_empty.

(61) In summary, VXM06m and VXM06 showed a pharmacodynamic effect on the survival of test animals in this syngeneic C57616 leukemia mouse model. A similar pharmacodynamic effect of the two compounds VXM06m and VXM06 compared to empty vector was observed. These results show that these vaccines were able to trigger a response against WT1 in an immune-competent mouse leukemia model resulting in longer survival of animals compared to animals treated with empty vector.