VEGFR-2 targeting immunotherapy approach

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 cancer immunotherapy, wherein the cancer is characterized by VEGF receptor protein expressing cancer cells. The present invention further 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 cancer immunotherapy, wherein the cancer is characterized by VEGF receptor protein expressing cancer cells, and wherein the cancer is selected from the group consisting of glioblastoma, carcinoid cancer, kidney cancer, particularly renal cell carcinoma, thyroid cancer, lung cancer, particularly Non-Small Cell Lung Cancer (NSCLC), breast cancer, ovarian cancer, prostate cancer, gastrointestinal cancer, particularly colorectal cancer, more particularly colon cancer, and skin cancer, particularly melanoma. The present invention further 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 cancer immunotherapy in a patient comprising at least one VEGF receptor protein expressing cancer cell.

Claims

1. A method of treating cancer in a human patient comprising: determining that the patient has a cancer comprising VEGFR-2 expressing cancer cells or has at least one cancer cell expressing VEGFR-2, and orally administering to the patient having the cancer comprising VEGFR-2 expressing cancer cells or the at least one cancer cell expressing VEGFR-2, a therapeutically effective dose of a live attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising a eukaryotic expression cassette encoding VEGFR-2, wherein the live attenuated strain of Salmonella is Salmonella typhi Ty21a, and wherein the VEGFR-2 comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 1.

2. The method of claim 1, wherein the determining comprises assessment of VEGFR-2 mRNA expression in cancer cells from the patient.

3. The method of claim 1, wherein the determining comprises assessment of VEGFR-2 protein expression in cancer cells from the patient.

4. The method of claim 1, wherein the cancer is selected from the group consisting of glioblastoma, carcinoid cancer, kidney cancer, renal cell carcinoma, thyroid cancer, lung cancer, Non-Small Cell Lung Cancer (NSCLC), breast cancer, ovarian cancer, prostate cancer, gastrointestinal cancer, colorectal cancer, colon cancer, skin cancer, and melanoma.

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

6. The method of claim 1, further comprising administering chemotherapy, radiotherapy or biological cancer therapy, wherein the attenuated strain of Salmonella typhi Ty21a is administered before, during or after the chemotherapy or the radiotherapy treatment or the biological cancer therapy, or before and during the chemotherapy or the radiotherapy treatment or the biological cancer therapy.

7. The method of claim 6, wherein the biological cancer therapy comprises administration of at least one further DNA vaccine encoding a tumor antigen and/or a tumor stroma antigen.

8. The method of claim 7, wherein said tumor antigen encoded by said at least one further DNA vaccine is selected from the group consisting of human Wilms' Tumor Protein (WT1), human Mesothelin (MSLN), human CEA and CMV pp65.

9. The method of claim 8, wherein said tumor antigen is selected from the group consisting of human Wilms' Tumor Protein (WT1) comprising the amino acid sequence as set forth in SEQ ID NO 3, a protein that has at least 80% sequence identity with SEQ ID NO: 3, human Mesothelin (MSLN) comprising the amino acid sequence as set forth in SEQ ID NO 4, a protein that has at least 80% sequence identity with SEQ ID NO: 4, human CEA comprising the amino acid sequence as set forth in SEQ ID NO 5, a protein that has at least 80% sequence identity with SEQ ID NO: 5, CMV pp65 comprising the amino acid sequence as set forth in SEQ ID NO 6, a protein that has at least 80% sequence identity with SEQ ID NO: 6, CMV pp65 comprising the amino acid sequence as set forth in SEQ ID NO 7, a protein that has at least 80% sequence identity with SEQ ID NO: 7, and CMV pp65 comprising the amino acid sequence as set forth in SEQ ID NO 8, and a protein that has at least 80% sequence identity with SEQ ID NO: 8.

10. The method of claim 7, wherein said tumor stroma antigen encoded by said at least one further DNA vaccine is human fibroblast activation protein (FAP).

11. The method of claim 1, wherein a single therapeutically effective dose of the Salmonella typhi Ty21a comprises from about 10.sup.5 to about 10.sup.11 colony forming units (CFU).

12. The method of claim 1, wherein the method comprises assessing the expression pattern of and/or the pre-immune response against the VEGFR-2 in the patient.

13. The method of claim 5, wherein the DNA molecule comprises the sequence of SEQ ID NO: 2.

14. The method of claim 7, wherein the at least one further DNA vaccine encoding the tumor antigen and/or the tumor stroma antigen comprises a live attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising an expression cassette encoding the tumor antigen and/or the tumor stroma antigen.

15. The method of claim 11, wherein the single dose of the Salmonella typhi Ty21a comprises from about 10.sup.6 to about 10.sup.9 CFU.

16. The method of claim 11, wherein the single dose of the Salmonella typhi Ty21a comprises from about 10.sup.6 to about 10.sup.8 CFU.

17. The method of claim 1, wherein the cancer is glioblastoma.

18. The method of claim 1, wherein the cancer is colorectal cancer or colon cancer.

19. The method of claim 1, wherein the cancer is kidney cancer or renal cell carcinoma.

20. The method of claim 1, wherein the cancer is lung cancer or Non-Small Cell Lung Cancer (NSCLC).

21. A method of treating cancer in a human patient comprising: determining that the patient has at least one cancer cell expressing VEGFR-2, and orally administering to the patient having said cancer cell, a therapeutically effective dose of a live attenuated strain of Salmonella comprising at least one copy of a DNA molecule comprising a eukaryotic expression cassette encoding VEGFR-2, wherein the live attenuated strain of Salmonella is Salmonella typhi Ty21a, and wherein the VEGFR-2 comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 1.

Description

SHORT DESCRIPTION OF FIGURES

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

(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 (zinc-finder domain deleted) 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: Plasmid map of pVAX10.VR2-1

(10) FIGS. 10A-E: Brain MRI images of patient 2605: baseline (FIG. 10A), day 35—before reoperation (FIG. 10B), week 12 after reoperation (FIG. 10C), week 20 after reoperation (FIG. 10D), and week 76 after reoperation (FIG. 10E).

EXAMPLES

Example 1 VXM01 Treatment of Patients with Operable Recurrence of Glioblastoma

(11) The aim of this study was to examine safety, tolerability, immune and biomarker response to VEGFR-2 encoding DNA vaccine VXM01.

(12) The study was conducted in patients with operable recurrence of a glioblastoma who have failed at least one standard treatment that must have included radiochemotherapy with temozolomide. All patients received DNA vaccine VXM01 as an add-on to their standard therapy.

(13) The study consisted of a screening period, a treatment and observation period up to month 3, a tumor follow-up from month 3 to month 12 and a boosting treatment period between week 8 and week 48 during the tumor follow-up period. After study end, patients are followed up for up to 2 years.

(14) The treatment and observation period included one oral administration of VXM01 each on day 1, 3, 5 and 7 and reoperation at 5±1 weeks after inclusion. In the boosting treatment period VXM01 was administered in oral 4-weekly single boosting doses at weeks 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48.

(15) VXM01 was administered orally at single doses of 10.sup.6 and 10.sup.7 colony forming units (CFU)/ml.

(16) Five out of nine glioblastoma patients showed a favorable course of disease.

(17) Patient 2605:

(18) Patient 2605 is a 55-year-old female patient with recurrent glioblastoma WHO grade IV. Previous cancer treatment included a first operation of glioblastoma and a first line radiochemotherapy with Gy 60 followed by 75 mg/m.sup.2 temozolomide.

(19) The patient was treated with VXM01 at a dose of 10.sup.6 CFU. VXM01 treatment was started with 4 initial administrations on study day 1, 3, 5 and 7 and continued after the routine operation on day 35 with 4-weekly boosting administrations starting on week 8. At week 10, lomustine/etoposide chemotherapy was started on top of VXM01.

(20) The tumor reference target lesion at the screening visit was 25×10 mm. Tumor size development is summarized in Table 1:

(21) TABLE-US-00001 TABLE 1 Target Lesion Tumor Diameter 1 [mm] Tumor Diameter 2 [mm] Baseline 25 10 Day 10 28 13 Day 21 27 13 Day 35 25 12 Week 12 0 0 Week 20 0 0 Week 36 0 0 Week 52 0 0 Week 60 0 0 Week 76 0 0

(22) The respective MRI images at baseline at day 35, week 12, week 20 and week 76 are depicted in FIGS. 10A-E.

(23) The tumor size tended to decrease between study day 10 and the routine operation on day 35 from 28×13 mm to 25×12 mm. According to RANO criteria, this was assessed as stable disease (SD). At week 12, 7 weeks after the routine reoperation on day 35, the assessment according the RANO criteria was progressive disease (PD) due to the occurrence of a new non-target lesion. After the operation, there was no visible “target lesion” on the MRI report week 12. Lomustine/etoposide chemotherapy was started on top of VXM01. At week 20 (i.e. 15 weeks after reoperation), the tumor was assessed as stable disease (SD) according to RANO criteria. At week 36, lomustine/etoposide chemotherapy was stopped and patient was continued to be treated with VXM01 every 4 weeks and treatment has not been stopped until filing of this application.

(24) The Karnofsky Index was 100% on screening and 90% at week 12.

(25) Immunohistochemistry staining of the primary tumor sample collected pre-study revealed that the tumor cells of this patient expressed VEGFR-2. In the recurrent tumor sample on day 35, after treatment with VXM01, the tumor cells were shown not to express VEGFR-2.

(26) In tumor tissue immunohistochemistry CD8+ T-cells increased in the recurrent tumor after VXM01 treatment compared to primary tumor by factor 2.3.