PD-L1 targeting DNA vaccine for cancer immunotherapy

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 PD-L1. In particular, the present invention relates to said attenuated strain of Salmonella for use in the treatment of cancer.

Claims

1. A method of treating cancer in a cancer patient, comprising administering orally to the cancer patient an effective amount of an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising a eukaryotic expression cassette encoding PD-L1, wherein the PD-L1 is a protein comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1, wherein immunogenicity of the protein comprising the amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1 is reduced by less than 50% when compared to the immunogenicity of the protein comprising the amino acid sequence as set forth in SEQ ID NO: 1; and wherein the cancer comprises PD-L1-positive tumor cells and the immunogenicity of the protein is PD-L1-specific immunogenicity.

2. The method of claim 1, wherein the attenuated strain of Salmonella provides anti-cancer immunotherapy to the cancer patient.

3. The method of claim 1, wherein the method further comprises administering chemotherapy, radiotherapy or biological cancer therapy, wherein the attenuated strain of Salmonella is administered before, during and/or after the chemotherapy or the radiotherapy or the biological cancer therapy.

4. The method of claim 3, wherein the method further comprises administering biological cancer therapy, which comprises administering at least one further DNA vaccine encoding a tumor antigen and/or a tumor stroma antigen.

5. The method of claim 4, wherein the at least one further DNA vaccine comprises an attenuated strain of Salmonella typhi Ty21a comprising a eukaryotic expression cassette.

6. The method of claim 4, wherein the at least one further DNA vaccine encodes a tumor antigen selected from the group consisting of Wilms' Tumor Protein (WT1), Mesothelin (MSLN), carcinoembryonic antigen (CEA), and CMV pp65, and/or encodes a tumor stroma antigen selected from the group consisting of VEGFR-2 and human fibroblast activation protein (FAP).

7. The method of claim 6, wherein the at least one further DNA vaccine encodes a tumor antigen selected from the group consisting of Wilms' Tumor Protein (WT1) having the amino acid sequence as set forth in SEQ ID NO: 6, Mesothelin (MSLN) having the amino acid sequence as set forth in SEQ ID NO: 7, CEA having the amino acid sequence as set forth in SEQ ID NO: 8, CMV pp65 having the amino acid sequence as set forth in SEQ ID NO: 9, CMV pp65 having the amino acid sequence as set forth in SEQ ID NO: 10, and CMV pp65 having the amino acid sequence as set forth in SEQ ID NO: 11; and wherein the at least one further DNA vaccine encodes a tumor stroma antigen selected from the group consisting of VEGFR-2 having the amino acid sequence as set forth in SEQ ID NO: 12, and human fibroblast activation protein (FAP).

8. The method of claim 1, wherein the cancer is selected from lymphoma, leukemia, myeloma, lung cancer, non-small cell lung cancer (NSCLC), melanoma, renal cell cancer, ovarian cancer, glioblastoma, merkel cell carcinoma, bladder cancer, head and neck cancer, colorectal cancer, esophageal cancer, cervical cancer, gastric cancer, hepatocellular cancer, prostate cancer, breast cancer, pancreatic cancer, and thyroid cancer.

9. The method of claim 1, wherein a single dose of the attenuated strain of Salmonella comprises from about 10.sup.5 to about 10.sup.11 colony forming units (CFU) of the strain.

10. The method of claim 1, further comprising assessing the cancer patient's PD-L1 expression pattern and/or pre-immune response against the PD-L1 before and/or after the treatment with the attenuated strain of Salmonella.

11. The method of claim 1, wherein the PD-L1 comprises an amino acid sequence that shares at least 90% sequence identity with SEQ ID NO: 1.

12. The method of claim 8, wherein the cancer is glioblastoma.

13. The method of claim 6, wherein the at least one further DNA vaccine encodes VEGFR-2.

14. The method of claim 13, wherein the at least one further DNA vaccine comprises a S. typhi Ty21a encoding the VEGFR-2, wherein the VEGFR-2 comprises the amino acid sequence of SEQ ID NO: 12.

Description

SHORT DESCRIPTION OF FIGURES

(1) FIG. 1: Amino acid sequence of human full length PD-L1 (SEQ ID NO 1), which is encoded by PD-L1 cDNA contained in plasmid pVAX10.PD-L1h.

(2) FIG. 2: Amino acid sequence of a truncated form of human PD-L1 (SEQ ID NO 2) lacking the signaling peptide (MRIFAVFIFMTYWHLLNA; SEQ ID NO 19), which is encoded by PD-L1 cDNA contained in plasmid pVAX10.PD-L1ha.

(3) FIG. 3: Nucleic acid sequence (SEQ ID NO 3) contained in plasmid pVAX10.PD-L1h and encoding human full length PD-L1 of SEQ ID NO 1.

(4) FIG. 4: Nucleic acid sequence (SEQ ID NO 4) contained in plasmid pVAX10.PD-L1ha and encoding truncated human PD-L1 of SEQ ID NO 2.

(5) FIG. 5: 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 5).

(6) FIG. 6: Amino acid sequence of human WT1 encoded by WT1 cDNA contained in plasmid pVAX10.hWT1 (SEQ ID NO 6).

(7) FIG. 7: Amino acid sequence of human MSLN encoded by MSLN cDNA contained in plasmid pVAX10.hMSLN (SEQ ID NO 7).

(8) FIG. 8: Amino acid sequence of human CEA encoded by CEA cDNA contained in plasmid pVAX10.hCEA (SEQ ID NO 8).

(9) FIG. 9: Amino acid sequence of CMV pp65 encoded by CMV pp65 cDNA contained in plasmid pVAX10.CMVpp65_1 (SEQ ID NO 9).

(10) FIG. 10: Amino acid sequence of CMV pp65 encoded by CMV pp65 cDNA contained in plasmid pVAX10.CMVpp65_2 (SEQ ID NO 10).

(11) FIG. 11: Amino acid sequence of CMV pp65 encoded by CMV pp65 cDNA contained in plasmid pVAX10.CMVpp65_3 (SEQ ID NO 11).

(12) FIG. 12: Amino acid sequence of VEGFR-2 encoded by VEGFR-2 cDNA contained in plasmid pVAX10.VR2-1 (SEQ ID NO 12).

(13) FIG. 13: Amino acid sequence of truncated human PD-L1 (SEQ ID NO 13) comprising the extracellular domain (amino acids 19-238) and the signaling peptide (amino acids 1-18).

(14) FIG. 14: Nucleic acid sequence (SEQ ID NO 14) encoding the truncated human PD-L1 of SEQ ID NO 13.

(15) FIG. 15: Effects of the prophylactic administration of VXM10m and VXM10ma on the survival of C57BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia. Mice were vaccinated with (A) empty vector (1.6×10.sup.8 CFU); (B) VXM10m (1.8×10.sup.8 CFU); (C) VXM10m (1.0×10.sup.10 CFU); (D) VXM10ma (3.6×10.sup.8 CFU); or (E) VXM10ma (1.0×10.sup.10 CFU). The vertical arrow indicates tumor inoculation.

(16) FIG. 16: Effects of the prophylactic administration of VXM10m and VXM10ma on long-term survival of C57BL/6 mice after re-challenge with FBL-3 cells. Mice were vaccinated and treated as follows (A) empty vector (1.6×10.sup.8 CFU); (B) untreated (control re-challenge); (C) VXM10m (1.8×10.sup.8 CFU); (D) VXM10m (1.0×10.sup.10 CFU); (E) VXM10ma (3.6×10.sup.8 CFU); or (F) VXM10ma (1.0×10.sup.10 CFU). The vertical arrows indicate tumor inoculation.

(17) FIG. 17: Effects of the therapeutic administration of VXM10 m and VXM10ma on the survival of C57BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia. A) Schedule for the therapeutic vaccination with VXM10m and VXM10ma in the FBL-3 model. B) Mice were vaccinated with (A) empty vector (1.0×10.sup.9 CFU); (B) VXM10m (1.0×10.sup.9 CFU); or (C) VXM10ma (1.0×10.sup.9 CFU). The vertical arrow indicates tumor inoculation.

(18) FIG. 18: (A) Experimental design, and (B) anti-PD-L1 response in the sera of C57BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia, collected 79 days after the final vaccination (vaccination schedule: d1, d3, d5, d7, d14, d21; FBL-3 challenge d20) with VXM10 10.sup.8 CFU (square), VXM10 10.sup.10 CFU (circle), VXM10a 10.sup.8 CFU (triangle, tip down), VXM10a 10.sup.10 CFU (triangle, tip up), negative control (rectangle). The dashed line represents the cut-off value derived from the values of the negative control group (95% confidence). Soluble recombinant murine PD-L1 was used for immunization with CFA/IFA in the positive control group (cross).

(19) FIG. 19: (A) Experimental design, and (B) level of IFNγ (open symbols) and TNFα (closed symbols) secreted by splenocytes isolated from mice immunized with the empty vector (circles), VXM10 (squares) or VXM10a (triangles), and stimulated with a pool of 5 peptides derived from PD-L1, as measured in the culture supernatant by ELISA after 6 days of stimulation (mean of n=5).

EXAMPLES

Example 1: Assessment of the Antitumor Activity of VXM10m in C57BL/6 Mice Bearing Disseminated Syngeneic FBL-3 Erythroleukemia

(20) The aim of this study was to investigate the antitumor efficacy of two Salmonella based PD-L1 DNA vaccines in C57 BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia. VXM10m is Salmonella typhimurium aroA strain SL7207 transformed with expression plasmid pVAX10 encoding murine full-length native PD-L1 (with the nucleic acid sequence of SEQ ID NO 17). VXM10ma is Salmonella typhimurium aroA strain SL7207 transformed with expression plasmid pVAX10 encoding a truncated form of murine PD-L1 (with the nucleic acid sequence of SEQ ID NO 18), more specifically the N-terminus truncated by 17 amino acid residues.

(21) The treatment started the day of randomization that was considered as day 1 (D1). Thirty healthy male C57BL/6 mice, 4-6 weeks old, were randomized according to their body weight into 5 groups of 6 animals each. Animal allocation to treatment groups is summarized in Table 1. A statistical test (Student t test) was performed to test for homogeneity between the groups (data not shown).

(22) TABLE-US-00001 TABLE 1 Group No. Treatment # Animals Vaccine Dose Route Schedule 1 6 Empty vector 1.6 × 10.sup.8 p.o. d1, d3, d5, (VXM0m_empty) CFU/adm d7, d14, d21 2 6 VXM10m 1.8 × 10.sup.8 p.o. d1, d3, d5, (PD-L1 full-length) CFU/adm d7, d14, d21 3 6 VXM10m-HD 1.0 × 10.sup.10 p.o. d1, d3, d5, (high-dose) CFU/adm d7, d14, d21 4 6 VXM10ma 3.6 × 10.sup.8 p.o. d1, d3, d5, (PD-L1 truncated) CFU/adm d7, d14, d21 5 6 VXM10ma-HD 1.0 × 10.sup.10 p.o. d1, d3, d5, (high-dose) CFU/adm d7, d14, d21

(23) Group 1 was treated with the empty vector control (VXM0m_empty; S. typhimurium bacterial vector control harboring no exogenous expression plasmid). Groups 2 to 5 were treated with VXM10m or VXM10ma, at two different single doses.

(24) VXM0m-empty, VXM10m and VXM10ma were administered by oral gavage (per os, po) in 100 μl in final volumes per application. Regardless of animal groups, each animal received pre-dose application buffer po to neutralize acid in the stomach prior dosing (100 μl/animal/application). This buffer was produced 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 VXM0m-empty, VXM10m and VXM10ma.

(25) Prime vaccination started at day 1 and consisted of 4 administrations every second day (d1, 3, 5, 7). Prime vaccination was followed by two boost vaccinations at days 14 and 21.

(26) Tumors were induced in all animals by I.P. injection of 5.0×10.sup.6 FBL-3 cells in 500 μl RPMI 1640 on day 20. FBL-3 is a Friend leukemia virus-induced erythroleukemia cell line originated from C57BL/6 mice. This cell line expresses unique 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 naïve syngeneic mice results in tumor growth, suggesting that the FBL-3 tumor cells possess mechanisms of escaping immune recognition and destruction. Of note, PD-L1 was shown to be highly expressed on the FBL-3 cell line.

(27) Animal body weight, viability and animal behavior were monitored throughout the study.

(28) Survival of test animals is displayed in a Kaplan-Meier plot in FIG. 15.

Example 2: Effects of the Administration of VXM10m and VXM10ma on the Long-Term Survival of C57BL/6 Mice after Re-Challenge with FBL-3 Cells

(29) The aim of this study was to investigate the long-term effect of antitumor efficacy of two Salmonella based PD-L1 DNA vaccines in C57 BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia following re-challenge.

(30) The Study of Example 1 was continued and mice were receiving a second tumor induction dose on day 100. Further unvaccinated control animals (n=10) that were present in the study from the start of the experiment, but received no first tumor induction, were included as a control for the tumor re-challenge. Re-challenge was performed by tumor induction in all animal of Groups 2-5 (see Table 1) and the unvaccinated control animals by I.P. injection of 5.0×10.sup.6 FBL-3 cells in 500 μl RPMI 1640 on day 100.

(31) Animal body weight, viability and animal behavior were continuously monitored. Over the treatment phase, when compared with control group, administration of VXM10m and VXM10ma generated a potent memory T cell response against the leukemia, with 100% of long-term surviving mice also after re-challenge with FBL-3 cells. No vaccination-related toxicity or body weight loss was observed throughout the study. Survival of test animals is displayed in a Kaplan-Meier plot in FIG. 16.

Example 3: Assessment of the Therapeutic Antitumor Activity of VXM10m and VXM10ma in C57BL/6 Mice Bearing Disseminated Syngeneic FBL-3 Erythroleukemia

(32) The aim of this study was to investigate the antitumor efficacy of VXM10m and VXM10ma administered therapeutically in C57BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia.

(33) Tumors were induced in all animals by intraperitoneal (i.p.) injection of 5.0×10.sup.6 FBL-3 cells in 500 μl RPMI 1640 on day 0.

(34) The treatment started the day of randomization that was considered as day 1 (D1, after tumor injection). For the treatment groups sixteen healthy male C57BL/6 mice, 4-6 weeks old, were randomized according to their body weight into 2 groups of 8 animals each. A statistical test (Student t test) was performed to test for homogeneity between the groups (data not shown).

(35) VXM0m-empty, VXM10m and VXM10ma were administered by oral gavage (per os, po) at 1.0×10.sup.9 CFU in 100 μl as described in Example 1.

(36) One group of 8 mice was treated with the empty vector control (VXM0m_empty; S. typhimurium bacterial vector control harboring no exogenous expression plasmid). The other group of 8 mice were treated with VXM10m or VXM10ma, with a prime treatment 4 times on Days 1, 3, 5, 7 and 2 weekly boosts on Days 14 and 21 (FIG. 17A).

(37) Animal body weight, viability and animal behavior were monitored 3 times weekly during the prime-boost period and upon the peak immune response, i.e., from study day 0 to 28, and then twice weekly until the end of the study.

(38) Over the treatment phase, when compared with control group, oral administration of VXM10m and VXM10ma resulted a strong anti-tumor effect in the FBL-3 leukemia model, with 100% of surviving animals 80 days after leukemia challenge (FIG. 17B). No vaccination-related toxicity or body weight loss was observed throughout the study. Administration of the empty vector did not show any anti-cancer effect.

Example 4: Assessment of the Anti-PD-L1 Antibody Response Following Vaccination with VXM10m and VXM10ma in C57BL/6 Mice Bearing Disseminated Syngeneic FBL-3 Erythroleukemia

(39) The aim of this study was to investigate the anti-PD-L1 response to VXM10 or VXM10a administered at 10.sup.8 CFU and 10.sup.10 CFU at days 1, 3, 5 and 7 with a boost at days 14 and 21 in C57 BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia. Tumors were induced in all animals by I.P. injection of 5.0×10.sup.6 FBL-3 cells on day 20. Vaccination and tumor induction was performed basically as described in Example 1. Soluble recombinant murine PD-L1 was used for immunization with CFA/IFA in the positive control group.

(40) The systemic antibody response was evaluated by ELISA in the serum of animals vaccinated with either VXM10 or VXM10a, 79 days after the final vaccination on day 21 (FIG. 18A). Anti-PD-L1 antibodies were detected in a few animals vaccinated with VXM10 and VXM10a, and the response was more pronounced in the highest dose treatment groups, with 50% of the animals (3 out of 6) showing a signal-to-background ration above the cut-off value (FIG. 18B)

Example 5: Assessment of the T-Cell Response Against PD-L1 Following Vaccination with VXM10m and VXM10ma in C57BL/6 Mice

(41) The aim of this study was to investigate the T-cell response induced against PD-L1 epitopes in healthy C57 BL/6 mice (n=5 per group) immunized four times every other day (days 1, 3, 5 and 7) via oral route with 10.sup.10 CFU of either VXM10, VXM10a or the empty vector control (FIG. 19A).

(42) Ex vivo restimulation of the spenocytes was performed 10 days after the last immunization (day 17), using a pool of 5 immunogenic peptides derived from murine PD-L1. The content of the culture supernatant was tested for the presence of IFNγ and TNFα by ELISA after 6 days of in vitro stimulation.

(43) The level of TNFα, and to a lesser extend IFNγ, was significantly increased in the supernatant of spenocytes derived from animals vaccinated with VXM10a and stimulated with PD-L1 peptides (FIG. 19B). These data confirm that immunization with VXM10a induced a pool of T-cells specific for PD-L1.

Example 6: Assessment of the Antitumor Activity of VXM10mb in C57BL/6 Mice Bearing Disseminated Syngeneic FBL-3 Erythroleukemia

(44) The aim of this study is to investigate the long-term effect of antitumor efficacy of a third Salmonella based PD-L1 DNA vaccines in C57 BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia. VXM10mb is Salmonella typhimurium aroA strain SL7207 transformed with expression plasmid pVAX10 encoding a truncated form of murine PD-L1, more specifically the extracellular domain including the N-terminal signaling peptide (amino acid sequence SEQ ID NO 15; nucleic acid sequence SEQ ID NO 16). The Experiment is essentially performed as described in Examples 1 and 2.

Example 7: Assessment of the Therapeutic Antitumor Activity of VXM10mb in C57BL/6 Mice Bearing Disseminated Syngeneic FBL-3 Erythroleukemia

(45) The aim of this study is to investigate the antitumor efficacy of VXM10mb administered therapeutically in C57BL/6 mice bearing disseminated syngeneic FBL-3 erythroleukemia. The Experiment is essentially performed as described in Example 3.