WT1 TARGETING DNA VACCINE FOR COMBINATION THERAPY

Abstract

The present invention relates to an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding Wilms' Tumor Protein (WT1), for use in the treatment of cancer, wherein the treatment further comprises the administration of at least one checkpoint inhibitor, particularly selected from at least one antibody against PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3, and LAG-3. The present invention further relates to a pharmaceutical composition comprising an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding WT1 for use in the treatment of cancer, wherein the treatment further comprises the administration of at least one checkpoint inhibitor, particularly selected from at least one antibody against PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3, and LAG-3.

Claims

1. A method for treating cancer in a subject, comprising administering an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding Wilms' Tumor Protein (WT1), wherein the method further comprises the administration of at least one checkpoint inhibitor.

2. The method according to claim 1, wherein the checkpoint inhibitor is selected from at least one antibody against PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3, and LAG-3.

3. The method according to claim 1, wherein the attenuated strain of Salmonella is of the species Salmonella enterica, particularly wherein the attenuated strain of Salmonella is Salmonella typhi Ty21a.

4. The method according to claim 1, wherein the expression cassette is a eukaryotic expression cassette, particularly wherein the expression cassette comprises a CMV promoter.

5. The method according to claim 1, wherein (a) WT1 is selected from the group consisting of human WTI having the amino acid sequence as found in SEQ ID NO 4 and a protein that shares at least about 80% sequence identity therewith, (b) wherein WT1 is truncated, more particularly wherein the zinc finger domain of WT1 is deleted, or (c) wherein WT1 is selected from the group consisting of WT1 having the amino acid sequence as found in SEQ ID NO 1 and a protein that shares at least about 80% sequence identity therewith.

6. The method according to claim 1, wherein the DNA molecule comprises the kanamycin antibiotic resistance gene, the pMB1 ori, and a CMV promoter, particularly wherein the DNA molecule comprises the DNA sequence as found in SEQ ID NO 2.

7. The method according to claim 1, wherein the attenuated strain of Salmonella is administered simultaneously with, prior to or after said at least one checkpoint inhibitor.

8. The method according to claim 1, wherein the treatment is accompanied by chemotherapy, radiotherapy or biological cancer therapy, particularly wherein the attenuated strain of Salmonella is administered before, during or after the chemotherapy or the radiotherapy treatment cycle or the biological cancer therapy, or before and during the chemotherapy or the radiotherapy treatment cycle or the biological cancer therapy.

9. The method according to claim 8, wherein the biological cancer therapy comprises administration of one or more further attenuated strain(s) of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding a tumor antigen and/or a tumor stroma antigen, particularly wherein said one or more further attenuated strain(s) of Salmonella is/are Salmonella typhi Ty21a comprising a eukaryotic expression cassette

10. The method according to claim 9, wherein said tumor antigen is selected from the group consisting of Mesothelin (MSLN), CEA, and CMV pp65 and/or said tumor stroma antigen is selected from the group consisting of VEGF receptor protein and human fibroblast activation protein (FAP).

11. The method according to claim 10, wherein said tumor antigen is selected from the group consisting of MSLN having the amino acid sequence as found in SEQ ID NO 5 and a protein that shares at least about 80% sequence identity therewith, CEA having the amino acid sequence as found in SEQ ID NO 6 and a protein that shares at least about 80% sequence identity therewith, CMV pp65 having the amino acid sequence as found in SEQ ID NO 7 and a protein that shares at least about 80% sequence identity therewith, CMV pp65 having the amino acid sequence as found in SEQ ID NO 8 and a protein that shares at least about 80% sequence identity therewith, and CMV pp65 having the amino acid sequence as found in SEQ ID NO 9 and a protein that shares at least about 80% sequence identity therewith, and/or wherein said tumor stroma antigen is selected from the group consisting of a VEGFR-2 having the amino acid sequence as found in SEQ ID NO 10 and a protein that shares at least about 80% sequence identity therewith, and human fibroblast activation protein (FAP).

12. The method according to claim 1, wherein the attenuated strain of Salmonella is administered orally.

13. The method according to claim 1, wherein the cancer is selected from leukemia, particularly from acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), from multiple myeloma, and from solid tumors, particularly 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.

14. The method according to claim 1, wherein the single dose of the attenuated strain of Salmonella 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 according to claim 1, wherein the treatment is individualized cancer immunotherapy comprising the step of assessing the WT1 expression and/or the pre-immune response against WT1 in a patient.

16. A method for treating cancer in a subject, comprising administering a pharmaceutical composition comprising an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding WT1, wherein the method further comprises the administration of at least one checkpoint inhibitor, particularly selected from an antibody against PD-1, PD-L1, CTLA-4, IDO, OX-40, GITR, TIM-3, and LAG-3.

17. The method according to claim 16, wherein the attenuated strain of Salmonella is Salmonella typhi Ty21a, wherein the expression cassette is a eukaryotic expression cassette, particularly comprising a CMV promoter, and wherein WT1 is selected from the group consisting of human WT1 having the amino acid sequence as found in SEQ ID NO 1 and a protein that shares at least about 80% sequence identity therewith, particularly wherein human WT1 has the amino acid sequence as found in SEQ ID NO 1.

Description

SHORT DESCRIPTION OF FIGURES AND TABLES

[0096] FIG. 1: Amino acid sequence of human WT1, wherein the zinc finger domain is deleted (SEQ ID NO 1), which is encoded by WT1 cDNA contained in plasmid pVAX10.hWT1

[0097] FIG. 2: Nucleic acid sequence comprised in empty expression vector pVAX10 (sequence of expression vector pVAX10 without the portion of the multiple cloning site which is located between the restriction sites NheI and XhoI (SEQ ID NO 2).

[0098] FIG. 3: Nucleic acid sequence contained in plasmid pVAX10.hWT1 and encoding human WT1 of SEQ ID NO 1

[0099] FIG. 4: Amino acid sequence of human full length WT1, UniProt ref P19544-7 (SEQ ID NO 4)

[0100] FIG. 5: Amino acid sequence of human MSLN encoded by MSLN cDNA contained in plasmid pVAX10.hMSLN (SEQ ID NO 5)

[0101] FIG. 6: Amino acid sequence of human CEA encoded by CEA cDNA contained in plasmid pVAX10.hCEA (SEQ ID NO 6)

[0102] FIG. 7: Amino acid sequence of CMV pp65 encoded by CMV pp65 cDNA contained in plasmid pVAX10.CMVpp65_1 (SEQ ID NO 7)

[0103] FIG. 8: Amino acid sequence of CMV pp65 encoded by CMV pp65 cDNA contained in plasmid pVAX10.CMVpp65_2 (SEQ ID NO 8)

[0104] FIG. 9: Amino acid sequence of CMV pp65 encoded by CMV pp65 cDNA contained in plasmid pVAX10.CMVpp65_3 (SEQ ID NO 9)

[0105] FIG. 10: Amino acid sequence of VEGFR-2 encoded by VEGFR-2 cDNA contained in plasmid pVAX10.VR2-1 (SEQ ID NO 10)

[0106] FIG. 11: Plasmid map of pVAX10.hWT1

[0107] FIG. 12: Effects of the combined administration of VXM06m with anti-CTLA-4 on the survival of C57BL/6 mice bearing disseminated FBL-3

[0108] FIG. 13: Effects of the combined administration of VXM06m with anti-PD-L1 on the survival of C57BL/6 mice bearing disseminated FBL-3

EXAMPLES

[0109] Assessment of the Antitumor Activity of VXM06m in Combination with PD-L1 and CTLA-4 Checkpoint Inhibitor Blockade:

[0110] The aim of this study was to investigate the antitumor activity of VXM06m in combination with PD-L1 and CTLA-4 checkpoint inhibitor blockade in a syngeneic tumor model of leukemia induced by the mouse cell line FBL-3 intraperitoneally implanted in C57BL/6 mice.

[0111] 60 male C57/BL6 mice, 4-6 weeks old, with an average weight of about 20 g/mouse, were randomized into 4 groups of 15 animals each.

[0112] Group 1 (control group): The mice (n=15) were treated with the empty vector VXM0m_empty (S. typhimurium bacterial vector control harboring no exogenous expression plasmid) at days, 1, 3, 5, 7, 14 and 22. At day 20, FBL-3 tumor cells were implanted in mice by I.P. route.

[0113] Group 2: The mice (n=15) were treated with VXM06m (S. typhimurium containing pVAX10.mWT1 coding for truncated murine WT1) at a dose of 10.sup.8 CFU/application at days, 1, 3, 5, 7, 14 and 22. Meanwhile, the mice were treated with 100 μg/application of anti-PD-L1 monoclonal antibody (BP0101, In Vivo Plus anti mouse PD-L1, Clone 10F.9G2, BioXCell) at days 24 and 29 by I.P. route. At day 20, FBL-3 tumor cells were implanted in mice by I.P. route.

[0114] Group 3: The mice (n=15) were treated with VXM06m at a dose of 10.sup.8 CFU/application at days, 1, 3, 5, 7, 14 and 22. Meanwhile, the mice were treated with 100 μg/application of anti-CTLA-4 monoclonal antibody (Anti CTLA-4 antibody, clone UC10-4F10, BioXCell) at days 11 and 18, by I.P. route. At day 20, FBL-3 tumor cells were implanted in mice by I.P. route.

[0115] Group 4 (control group): The mice (n=15) were treated with the empty vector VXM0m_empty (10.sup.8 CFU/application) at days, 1, 3, 5, 7, 14 and 22 and with anti-CTLA-4 (100 μg/dose) at days 11 and 18. At day 20, FBL-3 tumor cells were implanted in mice by I.P. route.

[0116] FBL-3 is a Friend leukemia virus-induced erythroleukemia cell line originated from C57BL/6 mice. This cell line expresses unique TSTA (Tumor Specific Transplantation Antigens) that can be recognized by the immune system. Priming syngeneic mice with FBL-3 tumor cells leads to the subsequent rejection of future live tumor challenges. Although FBL-3 is immunogenic, injection of live FBL-3 tumor cells into naive syngeneic mice results in tumor growth, suggesting that the FBL-3 tumor cells possess mechanisms to escaping immune recognition and destruction. Importantly, FBL-3 has been shown to overexpress Wilms tumor 1 (WT1).

[0117] Mice survival time was carefully monitored throughout the whole study and is illustrated in FIGS. 12 and 13.

[0118] One mouse death was observed in group 1 treated with empty vector on day 7 probably due to the accidental perforation of the esophagus the day of vaccine administration. [0119] On Day 23 one mouse was dead in group 4. [0120] On Day 25 one mouse was dead in group 4. [0121] On day 29; 5 mice of each group were sacrificed and spleens were collected. [0122] On day 30, one mouse was dead in group 2. [0123] On day 39, one mouse was dead in group 1. [0124] On day 58, 3 mice were dead in group 1. [0125] On day 65, 2 mice were dead in group 1. [0126] On day 74, 2 mice were dead in group 1. [0127] On day 83, 2 mice were dead in group 1 & 4. [0128] On day 90, one mouse was dead in group 4.

[0129] Thus, after the first tumor cell challenge at day 20, animal mortality was observed only in groups 1, 2 and 4, and for the other groups, all animals were healthy and no tumor development was observed.

[0130] These results clearly indicate that the combination of VXMO6m with either of the checkpoint inhibitors anti-CTLA-4 and anti-PD-L1 is highly effective in the FBL-3 model, generating a rapid and sustained anti-tumor effect. The VXMO6m plus anti-CTLA-4 combination led to 100% survival at the end of study (day 196).

[0131] In contrast, treatment with empty vector did not show anti-tumor effect, with a mean survival of 45 days after tumor challenge and 0% recovery. Treatment with anti-CTLA-4 led to 40% death at the end of study.