VEGFR-2 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 a VEGF receptor protein, for use in the treatment of cancer, wherein the treatment further comprises the administration of at least one further anti-cancer agent. 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 a VEGF receptor protein, wherein the pharmaceutical composition further comprises at least one further attenuated strain of Salmonella comprising at least one copy of a further DNA molecule comprising a further expression cassette encoding a tumor antigen or a tumor stroma antigen.

Claims

1. A method of treating a human patient against cancer comprising orally administering to the patient a composition comprising a therapeutically effective dose of an attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an eukaryotic expression cassette encoding a vascular endothelial growth factor receptor-2 (VEGFR-2) comprising the amino acid sequence of SEQ ID NO: 1, wherein the treatment further comprises administering an antibody against CTLA4, wherein the attenuated strain of Salmonella is Salmonella typhi Ty21a, wherein the therapeutically effective dose of the attenuated strain of the Salmonella comprises about 10.sup.6 to about 10.sup.9 colony forming units (CFU) and wherein the cancer is a solid tumor.

2. The method of claim 1, wherein the DNA molecule comprises a kanamycin antibiotic resistance gene, a pMB1 ori, and a CMV promoter.

3. The method of claim 1, wherein the attenuated Salmonella typhi Ty21a is administered simultaneously with or prior to the administration of the antibody against CTLA4.

4. The method of claim 1, wherein the treatment is accompanied by chemotherapy or radiotherapy.

5. The method of claim 1, wherein the method further comprises assessing the expression pattern of a tumor antigen of said cancer in said patient and/or assessing the pre-immune response against said tumor antigen in said patient.

6. The method of claim 1, wherein the DNA molecule comprises the DNA sequence as set forth in SEQ ID NO: 2.

7. The method of claim 1, wherein the cancer is colon carcinoma or melanoma.

8. The method of claim 1, wherein the oral administration comprises a single administration or multiple administrations of the composition.

Description

SHORT DESCRIPTION OF FIGURES AND TABLES

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

(2) 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).

(3) FIG. 3: Amino acid sequence of truncated human WT-1 encoded by WT-1 cDNA contained in plasmid pVAX10.hWT1 (SEQ ID NO 3)

(4) FIG. 4: Amino acid sequence of human MSLN encoded by MSLN cDNA contained in plasmid pVAX10.hMSLN (SEQ ID NO 4)

(5) FIG. 5: Amino acid sequence of human CEA encoded by CEA cDNA contained in plasmid pVAX10.hCEA (SEQ ID NO 5)

(6) FIG. 6: Amino acid sequence of CMV pp65 encoded by CMV pp65 cDNA contained in plasmid pVAX10.CMVpp65_1 (SEQ ID NO 6)

(7) FIG. 7: Amino acid sequence of CMV pp65 encoded by CMV pp65 cDNA contained in plasmid pVAX10.CMVpp65_2 (SEQ ID NO 7)

(8) FIG. 8: Amino acid sequence of CMV pp65 encoded by CMV pp65 cDNA contained in plasmid pVAX10.CMVpp65_3 (SEQ ID NO 8)

(9) FIG. 9: Nucleic acid sequence contained in plasmid pVAX10.VR2-1 and encoding human VEGFR-2 of SEQ ID NO 1

(10) FIG. 10: Nucleic acid sequence contained in plasmid pVAX10.hWT1 and encoding human WT-1 of SEQ ID NO 3

(11) FIG. 11: Nucleic acid sequence contained in plasmid pVAX10.hMSLN and encoding human MSLN of SEQ ID NO 4

(12) FIG. 12: Nucleic acid sequence contained in plasmid pVAX10.hCEA and encoding human CEA of SEQ ID NO 5

(13) FIG. 13: Nucleic acid sequence contained in plasmid pVAX10.CMVpp65_1 and encoding CMV pp65 of SEQ ID NO 6

(14) FIG. 14: Nucleic acid sequence contained in plasmid pVAX10.CMVpp65_2 and encoding CMV pp65 of SEQ ID NO 7

(15) FIG. 15: Nucleic acid sequence contained in plasmid pVAX10.CMVpp65_3 and encoding CMV pp65 of SEQ ID NO 8

(16) FIG. 16: Plasmid map of pVAX10.VR2-1

(17) FIG. 17: Plasmid map of pVAX10.hWT1

(18) FIG. 18: Plasmid map of pVAX10.hMSLN

(19) FIG. 19: Plasmid map of pVAX10.hCEA

(20) FIG. 20: Plasmid map of pVAX10.CMVpp65_1

(21) FIG. 21: Plasmid map of pVAX10.CMVpp65_2

(22) FIG. 22: Plasmid map of pVAX10.CMVpp65_3

(23) FIG. 23: Effects of the combined administration of VXM01 and anti-CTLA4 in a MC38 mouse tumor model-tumor growth

(24) FIG. 24: Effects of the combined administration of VXM01 and anti-CTLA4 in a MC38 mouse tumor model-survival

(25) FIG. 25: Effects of the combined administration of VXM01 and anti-CTLA4 in B16 mouse tumor model-survival

(26) FIG. 26: Treatment schedule Example 3

(27) FIG. 27: Effect of VXM01 treatment either with or without cyclophosphamide on tumor size [mm.sup.3] on day 30. Each dot represents the result of the tumor of one animal.

(28) FIG. 28: Percentages of VEGFR-2-specific CD8.sup.+ cells in spleens of BALB/C mice bearing subcutaneous CT26 colon tumor cells. Each dot represents the results of one spleen. The results are given in total % of 3 pooled VEGFR2 pentamers.

(29) FIG. 29: Anti-CD3 immunohistochemistry staining of tumor samples from animals treated with the empty vector and VXM01, respectively. CD3 positive cells appear in brown color (see arrow for example); 200 magnification.

(30) FIG. 30: Quantification of immune cell infiltrates and PD-L1 mean fold induction in tumor samples from animals treated with VXM01 or VXM01 plus cyclophosphamide as compared to animals treated with the empty vector control. Data are derived from absolute cell count/tissue area [mm.sup.2]; 200 magnification.

(31) FIG. 31: Percentages of VEGFR-2- and CEA-specific CD8.sup.+ cells in spleens of healthy mice treated with mice bearing subcutaneous CT26 colon tumor cells. Each dot represents the results of one spleen. The results are given in total % of 2 pooled VEGFR2 pentamers.

EXAMPLES

Example 1: MC38 Colon Carcinoma Anti-CTLA4 Combination Study

(32) Four groups of C57/Bl6/6J mice (n=6 each) were challenged with a subcutaneous administration of 510.sup.5 MC38 tumor cells on Day 0 of the study.

(33) The animals were treated with VXM01mlow (Salmonella typhimurium carrying a murine VEGFR-2-encoding eukaryotic expression cassette, manufactured by Richter-Helm BioLogics, Hannover, Germany) alone at a dose of 10.sup.8 CFU via oral gavage on Day 1, Day 1, Day 4, and Day 6 (n=6), or with VXM01mlow at the same dose, route of administration, and administration scheme plus the murine anti-CTLA4 antibody on Day 12, 14, 16, and 18 (n=6), or with the murine antiCTLA4-antibody on Day 12, 14, 16, and 18 alone (n=6), or without treatment (n=6, control).

(34) Tumor growth was measured using a micro-caliper. Animals were sacrificed as soon as tumor volume reached 1500 mm.sup.3 for animal welfare reasons.

(35) Survival of test animals was recorded once daily.

(36) Tumor growth is graphically depicted in FIG. 23.

(37) Survival of test animals is displayed in a Kaplan-Meier plot in FIG. 24.

Example 2: B16-F10 Melanoma Anti-CTLA4 Combination Study

(38) Four groups of C57/Bl6/6J mice (n=6 each) were challenged with an intravenous administration of 210.sup.5 B16-F10 tumor cells on Day 0 of the study.

(39) The animals were treated with VXM01mlow (Salmonella typhimurium carrying a murine VEGFR-2-encoding eukaryotic expression cassette, manufactured by Richter-Helm BioLogics, Hannover, Germany) alone at a dose of 10.sup.8 CFU via oral gavage on Day 5, Day 3, Day 0, and Day 2 (n=6), or with VXM01mlow at the same dose, route of administration, and administration scheme plus the murine anti-CTLA4 antibody on Day 8, 10, 12, and 14 (n=6), or with the murine antiCTLA4-antibody on Day 8, 10, 12, and 14 alone (n=6), or without treatment (n=6, control).

(40) Survival of test animals was recorded once daily.

(41) Survival of test animals is displayed in a Kaplan-Meier plot in FIG. 25.

Example 3: Antitumor Activity of VMX01 Vaccine in CT26 Murine Tumor Model

(42) The aim of this study was to evaluate the antitumor activity of VXM01 with or without cyclophosphamide in BALB/C mice bearing subcutaneous CT26 colon tumors, and to characterize the immune responses elicited by the treatments in spleen and tumor.

(43) Control VXM0m-empty (S. typhimurium vector control with no expression plasmid) and VXM01mlow (Salmonella typhimurium carrying a murine VEGFR-2-encoding eukaryotic expression cassette) were administered at 10.sup.8 CFU/adm by oral gavage (per os, PO) via a gavage tube. Regardless of animal groups, each animal received pre-dose application buffer PO to neutralize acid in the stomach prior dosing (100 l/animal/application). This buffer was composed by dissolution of 2.6 g sodium hydrogen carbonate, 1.7 g L-ascorbic acid and 0.2 g lactose monohydrate in 100 ml of drinking water and was applied within 30 min prior application of VXM0m-empty or VXM01mlow.

(44) Cyclophosphamide was injected at 100 mg/kg/adm into the peritoneal cavity of mice (intraperitoneally, IP). The IP injection volume did not exceed 10 ml/kg and was calculated according to the most recent body weight of mice.

(45) The treatment started at day 0 (D0), one day after randomization that was considered as day 1 (D1). 33 healthy female BALB/C (BALB/CByJ) mice, 6 weeks old, were randomized according to their body weight into 4 groups of 11 animals each using Vivo Manager software (Biosystemes, Couternon, France). A statistical test (analysis of variance) was performed to test for homogeneity between groups.

(46) The treatment schedule was as follows:

(47) Group 1: The animals from group 1 received a total of 6 PO administrations of VXM0m-empty on D1, D3, D5, D7, D14 and D21.

(48) Group 2: The animals of group 2 received a total of 6 PO administrations of VXM01mlow on D1, D3, D5, D7, D14 and D21.

(49) Group 3: The animals of group 3 received one single IP injection of cyclophosphamide on D0 and a total of 6 PO administrations of VXM01mlow on D1, D3, D5, D7, D14 and D21.

(50) The treatment schedule is summarized in Table 1 and FIG. 26.

(51) TABLE-US-00001 TABLE 1 Treatment Schedule No. Treatment Group Animals Treatment Dose Route Schedule 1* 11 Empty vector 10.sup.8 CFU/adm PO D1, D3, D5, D7, D14 and D21 2* 11 VXM01mlow 10.sup.8 CFU/adm PO D1, D3, D5, D7, D14 and D21 3* 11 VXM01mlow 10.sup.8 CFU/adm PO D1, D3, D5, D7, D14 and D21 Cyclophosphamide 100 mg/kg/adm IP D0 TOTAL 33 *Each animal received pre-dose application buffer per os (PO) to neutralize acid in the stomach prior dosing

(52) Tumors were induced by subcutaneous injection of 110.sup.6 of CT26 cells in 200 l of RPMI 1640 into the right flank of the test animals on day 8 (D8).

(53) On the day of termination (D30, i.e. 22 days after tumor inoculation), tumors from all mice were collected and tumor size was measured.

(54) The results are graphically depicted in FIG. 27. Tumor size was significantly decreased in animals treated with either VXM01 alone or VXM01 plus cyclophosphamide as compared to the empty vector control. Tumor size reduction was most pronounced in the animals treated with both cyclophosphamide and the VXM01 vaccine.

(55) On the day of termination, spleens were collected from all mice (11 samples per group) and placed individually into tubes containing chilled PBS (2-8 C.). Immunomonitoring of VEGFR-2 specific T-cell responses using flow cytometry with pentamers was performed.

(56) For this purpose, the spleen samples were washed with PBS and subsequently homogenized by plunging them through a 100 m nylon cell strainer. During homogenization, the strainer was rinsed several times with cool sterile PBS. The samples were centrifuged at 1500 rpm for 10 minutes at 2-8 C., the supernatant was discarded and the cell pellet was resuspended in 2 ml ACK red blood cell lysis buffer (1 ml buffer per spleen). The cells were incubated in the lysis buffer for 1 min at RT. Then, PBS was added to 40 ml to stop the lysis and the cell suspension was sieved through a fresh strainer (40 m) and the flow through was collected in a new 50 ml tube. After centrifugation at 1500 rpm for 10 min at 2-8 C. the supernatant was discarded and the pellet was resuspended in 5 ml II-2 supplemented DMEM medium. The cells were incubated overnight at 37 C. and 5% CO.sub.2.

(57) Prior to pentamer staining, a live/dead (L/D) staining using the Live Dead (L/D) Fixable Yellow Dead Cell Stain Kit by Invitrogen was performed according to the manufacturer's instructions, in order to exclude dead cells by gating on negative population.

(58) Pentamer staining was performed using Pro5 Recombinant MHC Pentamers by Proimmune, Oxford, UK, according to the manufacturer's instructions.

(59) The following KDR (VEGFR-2) pentamers were used:

(60) TABLE-US-00002 H-2Kd-SYQYGTMQTL KDR-STL (SEQIDNO:16) H-2Kd-KYLSYPAPDI KDR-KDI (SEQIDNO:17) H-2Kd-RFVPDGNRI KDR-RRI (SEQIDNO:18) H-2Kd-TYQSIMYIV KDR-TIV (SEQIDNO:19) H-2Kd-DFLTLEHLI KDR-DLI (SEQIDNO:20)

(61) The results of the pentamer staining are shown in FIG. 28.

(62) The number of VEGFR-2 specific CD8.sup.+ cytotoxic T-cells was significantly increased in animals treated with either VXM01 alone or VXM01 plus cyclophosphamide as compared to the empty vector control. The cyclophosphamide treatment together with VXM01 significantly increased the KDR pentamer response as compared to the response obtained with the vaccine VXM01 alone.

(63) Tumors from 5 mice in each group were analyzed by immunohistochemistry (IHC).

(64) For that purpose, the tumors were fixed in 10% neutral buffered formalin for 24 h to 48 h, transferred into ethanol and then embedded in paraffin. The embedded samples were subjected to immunohistochemical staining. The results are graphically depicted in FIGS. 29 and 30.

(65) The mean number of T-cells per unit of tissue are was found to be increased in the tumors of mice treated with either VXM01 alone or VXM01 plus cyclophosphamide as compared to the empty vector control. CD3.sup.+ and CD8.sup.+ cell populations were found to be increased approximately three-fold in the tumor samples of mice treated with VXM01 plus cyclophosphamide and approximately two-fold in tumor samples of mice treated with VXM01 alone. Also the CD4.sup.+ T-cell population was increased in VXM01 vaccine treated animals with and without cyclophosphamide pretreatment, with a 1.7 fold increase in the mean number of CD4.sup.+ cells/tissue area in both vaccine groups as compared to the empty vector control.

(66) Furthermore, the number of PD-1 positive immune cells was increased by a factor of 2.0 and 2.1 and the tumor was enriched in PD-L1-expressing cells' upon treatment with VXM01 either as single agent or in combination with cyclophosphamide, clearly indicating that VXM01 treatment might increase the susceptibility of tumors towards the treatment with anti-PD-1 and anti-PD-L1 checkpoint inhibitors.

Example 4: VXM01/VXM08 Combination Study in Healthy C56BL/6J Mice

(67) The aim of this study was to evaluate the capability of VXM01mlow and VXM08hm to trigger an immune response in healthy mice.

(68) Control VXM0m-empty (S. typhimurium vector control with no expression plasmid), vaccine VXM01mlow (Salmonella typhimurium harboring a murine VEGFR-2-encoding eukaryotic expression cassette) and vaccine VXM08hm (Salmonella typhimurium harboring a human CEA encoding eukaryotic expression cassette) were administered at 10.sup.8 CFU/adm in 50 l per application by oral gavage (per os, PO) via a gavage tube. Regardless of animal groups, each animal received pre-dose application buffer PO to neutralize acid in the stomach prior dosing (50 l/animal/application prior to single vaccine administration; 100 l/animal/application prior to combined administration of VXM01 and VXM08). This buffer was composed by dissolution of 2.6 g sodium hydrogen carbonate, 1.7 g L-ascorbic acid and 0.2 g lactose monohydrate in 100 ml of drinking water and was applied within 30 min prior application of VXM0m-empty, VXM01mlow and/or VXM08hm.

(69) 40 healthy female C57BL/6J mice, 6-7 weeks old, were randomized on day 0 (D0) according to their body weight into 5 groups of 8 animals each using Vivo Manager software (Biosystemes, Couternon, France). A statistical test (analysis of variance) was performed to test for homogeneity between groups.

(70) The treatment schedule was as follows:

(71) Group 1: The animals from group 1 received a total of 6 PO administrations of VXM0m-empty on D1, D3, D5, D7, D14 and D21.

(72) Group 2: The animals of group 2 received a total of 6 PO administrations of VXM01mlow on D1, D3, D5, D7, D14 and D21.

(73) Group 3: The animals of group 3 received a total of 6 PO administrations of VXM08hm on D2, D4, D6, D8, D15 and D22.

(74) Group 4: The animals of group 4 received a total of 6 PO administrations of VXM01mlow on D2, D4, D6, D8, D15 and D22 and a total of 6 PO administrations of VXM08hm on D2, D4, D6, D8, D15 and D22.

(75) Group 5: The animals of group 5 received a total of 6 PO administrations of VXM01mlow on D1, D3, D5, D7, D14 and D21 and a total of 6 PO administrations of VXM08hm on D2, D4, D6, D8, D15 and D22.

(76) The treatment schedule is summarized in Table 2.

(77) TABLE-US-00003 No. Dose Volume Group Mice Treatment (CFU/adm) (l) Route Treatment Schedule 1 8 Empty vector 50 PO Prime: D1, D3, D5, D7 Boost: D14, D21 2 8 VXM01mlow 10.sup.8 50 PO Prime: D1, D3, D5, D7 Boost: D14, D21 3 8 VXM08hm 10.sup.8 50 PO Prime: D2, D4, D6, D8 Boost: D15, D22 4 8 VXM01mlow 10.sup.8 50 PO Prime: D2, D4, D6, D8 Boost: D15 and D22 VXM08hm* 10.sup.8 50 PO Prime: D2, D4, D6, D8 (concomitant) Boost: D15, D22 5 8 VXM01mlow 10.sup.8 50 PO Prime: D1, D3, D5, D7 Boost: D14, D21 VXM08hm 10.sup.8 50 PO Prime: D2, D4, D6, D8 (alternate days) Boost: D15, D22 *VXM08 was administered just after VMX01, at the same day of application

(78) On the day of termination (i.e. D29), spleens were collected from all mice (8 samples per group) and placed individually into tubes containing chilled PBS (2-8 C.). Immunomonitoring of VEGFR-2 and CEA specific T-cell responses using flow cytometry with pentamers was performed. Pentamer analysis including preceding live/dead staining was performed as described in Example 3.

(79) The following KDR (VEGFR-2) pentamers were used as a pool mix at same ratio:

(80) TABLE-US-00004 H-2Db-VILTNPISM KDR2 (SEQIDNO:21) H-2Db-FSNSTNDILI KDR3 (SEQIDNO:22)

(81) The following CEA pentamers were used as a pool mix at same ratio:

(82) TABLE-US-00005 H-2Db-CGIQNSVSA CEA-CSA-Penta (SEQIDNO:23) H-2Db-LQLSNGNRTL CEA-LTL-Penta (SEQIDNO:24) H-2Db-CGIQNKLSV CEA-CSV-Penta (SEQIDNO:25)

(83) The results of the pentamer staining are shown in FIG. 31. The mean frequency of VEGFR-2 (KDR) specific CD8.sup.+ T-cells was 1.71, 4.36 and 2.76-fold higher in mice treated with VXM01mlow, VXM01mlow/VXM08hm (concomitant) and VXM01mlow/VXM08hm (alternate days) respectively than in the control group. Mice treated with VXM01mlow/VXM08hm either concomitantly or on alternate days showed a higher frequency of VEGFR-2 specific CD8.sup.+ T-cells as compared to mice treated with VXM01mlow alone. Although not statistically significant, the synergy was slightly higher when VXM01mlow and VXM08hm vaccine had been applied concomitantly, i.e. the same day as compared to the alternate day regimen.

(84) The mean frequency of CEA-specific CD8.sup.+ T-cells was 1.29, 2.23 and 1.95-fold higher in mice treated with VXM08hm, VXM01mlowNXM08hm (concomitant) and VXM01mlowNXM08hm (alternate days) respectively than in the control group. Mice treated with VXM01mlow/VXM08hm either concomitantly or on alternate days showed a higher frequency of CEA specific CD8.sup.+ T-cells as compared to mice treated with VXM08hm alone. Although not statistically significant, the synergy was slightly higher when VXM01mlow and VXM08hm vaccine had been applied concomitantly, i.e. the same day as compared to the alternate day regimen.