Recombinant Mycobacterium as an Immunotherapeutic Agent for the Treatment of Cancer

Abstract

The invention relates to a recombinant Mycobacterium cell for use as an immunotherapeutic agent in the treatment of cancer, particularly in the treatment of solid tumors. More particularly, the invention relates to the immunotherapy of bladder carcinoma.

Claims

1. A recombinant Mycobacterium bovis cell which comprises a recombinant nucleic acid molecule encoding a fusion polypeptide comprising: (a) a domain capable of eliciting an immune response, and (b) a Listeria phagolysosomal escape domain in combination with a carrier suitable for use with an immunotherapeutic agent in the treatment of bladder carcinoma, in particular recurrent bladder carcinoma, wherein the individual to be treated has relapsed and/or has progressed after a first treatment for bladder carcinoma.

2. The cell of claim 1, wherein said cell is a urease-deficient cell.

3. The cell of claim 1, wherein said cell is a recombinant M. bovis BCG cell from strain Danish subtype Prague.

4. The cell of claim 1, wherein the domain capable of eliciting an immune response is selected from the group consisting of immunogenic peptides and polypeptides from M. bovis or M. tuberculosis.

5. The cell of claim 1, wherein the recombinant nucleic acid molecule does not comprise any functional selection marker.

6. The cell of claim 1, wherein the fusion polypeptide comprises (a) a domain capable of eliciting an immune response comprising the amino acid sequence from aa.41 to aa.51 in SEQ ID NO: 2, and (b) a Listeria phagolysosomal escape domain encoded by a nucleic acid molecule selected from (i) a nucleotide sequence comprising nucleotides 211-1722 as shown in SEQ ID NO: 1, (ii) a nucleotide sequence which encodes the same amino acid sequence as the sequence from (i), and (iii) a nucleotide sequence hybridising under stringent conditions with the sequence from (i) or (ii).

7. The cell of claim 1, wherein the carrier is suitable for local administration of the immunotherapeutic agent to the tumor site, particularly after surgery.

8. The cell of claim 1, wherein the carrier is suitable for vesicular instillation into the urinary bladder.

9. A method for the immunotherapy of bladder carcinoma in a subject in need thereof, comprising administering to said subject a recombinant Mycobacterium bovis cell comprising a recombinant nucleic acid molecule encoding a fusion polypeptide comprising: (a) a domain capable of eliciting an immune response, and (b) a Listeria phagolysosomal escape domain, wherein the subject to be treated has relapsed and/or has progressed after a first treatment for bladder carcinoma.

10. The method of claim 9, wherein said immunotherapy of bladder carcinoma produces focal and/or multifocal lymphocytic infiltration at the site of administration.

11. The method of claim 10, wherein said immunotherapy of bladder carcinoma produces for focal and/or multifocal tissue infiltration with CD4 and CD8 T cells.

12. The method of claim 10, wherein the focal and/or multifocal lymphocytic tissue infiltration is increased as compared to administration of native BCG.

13. The method of claim 9, wherein the recombinant Mycobacterium bovis cell is administered into the bladder according to a schedule involving weekly instillations during (i) an induction phase with, in particular 6 weekly instillations, (ii) a maintenance phase of at least one year, in particular a first maintenance phase after about 3 months with e.g. 3 weekly instillations, a second maintenance phase after about 6 months with e.g. 3 instillations and a third maintenance phase after about 12 months with e.g. 3 instillations.

14. The method of claim 9, wherein the recombinant Mycobacterium bovis cell is used at a dose of from about 10.sup.6 to 10.sup.10 CFU per administration.

15. The method of claim 9, further comprising a non-tumor site specific administration of the recombinant Mycobacterium bovis cell.

16. The method of claim 9, wherein the bladder carcinoma is non-invasive bladder carcinoma, particularly carcinoma in situ (T.sub.cis), non-invasive papillary carcinoma (T.sub.a), or a tumor invading subepithelial connective tissue (T.sub.1).

17. The method of claim 9, wherein the subject to be treated has received a first treatment for bladder carcinoma selected from the group consisting of cisplatin-based chemotherapy, cisplatin-based chemotherapy followed by surgical removal of the bladder or radiation therapy, concomitant chemotherapy and standard BCG.

18. The method of claim 17, wherein the subject to be treated has received standard BCG as a first treatment for bladder carcinoma.

19. The method of claim 9, wherein the subject to be treated is a smoker.

20. The method of claim 9, wherein the subject underwent cystectomy or another local treatment or systemic chemotherapy.

21. The method of claim 9, wherein the bladder carcinoma to be treated is non-muscle-invasive bladder cancer (NMIBC).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0086] FIG. 1. CONSORT 2010 diagram of SAKK 06/14. BCG: Bacillus Calmette-Guerin. ITT: Intention to treat. FAS: Full analysis set (population). PP: Per-protocol (population).

[0087] .sup.1 includes also the 2 exclusions from FAS.

[0088] FIG. 2: Kaplan Meier plot for time to recurrence in the bladder for the FAS (median follow up 2.9 years).

[0089] FIGS. 3A and 3B: Immunohistochemistry for lymphocyte sub-typing in the urinary bladder tissue revealed the highest incidence of focal and multifocal lymphocytic infiltration with CD4 and CD8 positive cells in groups 2 and 3 treated once with 2×106 or 2×108 CFU rBCG/animal by intravesical instillation, whereas animals of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). FIG. 3A shows focal and multifocal lymphocytic infiltration after administration of rBCG. FIG. 3B shows only diffuse and singular infiltration after administration of BCG medac (200× magnification).

[0090] FIGS. 4A and 4B: Immunohistochemistry for lymphocyte sub-typing in the urinary bladder tissue revealed the highest incidence of multifocal lymphocytic infiltration with CD4 and CD8 positive cells in group 2 treated with 2×106 CFU rBCG/animal by 6 intravesical instillations, followed by group 3 treated with 2×108 CFU rBCG/animal by 6 intravesical instillations, whereas animals of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). FIG. 4A shows focal and multifocal lymphocytic infiltration after administration of rBCG. FIG. 4B shows only diffuse and singular infiltration after administration of BCG medac (200× magnification).

TABLE-US-00004 TABLE 1 FAS Variable n (%) T classification T1 18 (45.0) T1/CIS 6 (15.0) Ta 6 (15.0) Ta/CIS 5 (12.5) CIS 5 (12.5) 1973 WHO grading Grade 2 5 (12.5) Grade 3 34 (85.0) Unknown 1 (2.5) 2004 WHO grading Low-grade urothelial carcinoma 4 (10.0) High-grade urothelial carcinoma 35 (87.5) Unknown 1 (2.5) EORTC progression score 0 1 (2.5) 1-6 7 (17.5)  7-13 26 (65.0) 14-23 6 (15.0) Median (min, max) 9 (0.0 -18) EORTC recurrence 1-4 35 (87.5) 5-9 5 (12.5) Median (min, max) 3 (1-7)

TABLE-US-00005 TABLE 2 FAS Variable n (%) Sex Female 4 (10.0) Male 36 (90.0) WHO performance score 0 35 (87.5) 1 5 (12.5) Smoking status Current smoker 10 (25.0) Former smoker 11 (27.5) Non-smoker 18 (45.0) Not available 1 (2.5) BCG maintenance performed during previous therapy No 26 (65.0) Yes 14 (35.0) T classification for recurrence T1 7 (17.5) T1/CIS 5 (12.5) Ta 6 (15.0) Ta/CIS 5 (12.5) CIS 17 (42.5) 1973 WHO grading of recurrence Grade 2 3 (7.5) Grade 3 37 (92.5) 2004 WHO grading of recurrence for study inclusion High-grade urothelial carcinoma 40 (100.0) EORTC progression score  7-13 12 (30.0) 14-23 28 (70.0) Median (min, max) 16 (7, 20) EORTC recurrence score 1-4 4 (10.0) 5-9 30 (75.0) 10-17 6 (15.0) Median (min, max) 8 (4, 11) Baseline PPD test No reaction 31 (77.5%) Positive 7 (17.5%) Missing 2 (5.0%)

TABLE-US-00006 TABLE 3 no yes/former Smoking (n = 18) (n = 21) p Recurrence-free rate in 53.1 45.5 0.652 the bladder at 60 weeks [27.8, 73.2] [22.4, 66.1] in % [95% CI] no yes CIS (n = 13) (n = 27) p Recurrence-free rate in 61.5 42.4 0.289 the bladder at 60 weeks [30.8, 81.8] [22.0, 61.5] in % [95% CI] Previous BCG no yes maintenance (n = 26) (n = 14) p Recurrence-free rate in 54.3 38.4 0.371 the bladder at 60 weeks [32.9, 71.6] [12.2, 64.6] in % [95% CI] BCG unresponsive no yes (FDA definition) (n = 24) (n = 16) p Recurrence-free rate in 63.6% 24.0% 0.022 the bladder at 60 weeks [40.3%, 79.9%]  [5.9%, 48.8%] in % [95% CI] no PPD− to PPD+ PPD conversion (n = 15) (n = 5) p Recurrence-free rate in 62.9% 80.0% 0.505 the bladder at 60 weeks [32.3%, 82.6%] [20.4%, 96.9%] in % [95% CI]

TABLE-US-00007 TABLE 4 Treatment n (%) Cystectomy 13* (32.5) BCG (upper tract) 2 (5.0) Systemic chemotherapy 3 (7.5) Intravesical chemotherapy 6* (15.0)

TABLE-US-00008 TABLE 5 Grade 1 Grade 2 Grade 3 AE n (%) n (%) n (%) Vertigo 1 (2.4) Gastrointestinal disorders 2 (4.8) Fatigue 2 (4.8) 3 (7.1) Fever 2 (4.8) 1 (2.4) Frequency, urgency  7 (16.7)  5 (11.9) Malaise 1 (2.4) BCG induced systemic inflammatory 1 (2.4) reaction Cold 1 (2.4) GU infection 14 (33.3) 2 (4.8) Alanine aminotransferase increased 1 (2.4) Neuralgia 1 (2.4) Hematuria 2 (4.8) Macrohematuria 1 (2.4) Urinary retention 1 (2.4) Urinary tract obstruction 1 (2.4) 1 (2.4) Urinary tract pain  6 (14.3) 1 (2.4) Vaginal pain 1 (2.4) Skin affection 3 (7.1) 2 (4.8) Thromboembolic event 1 (2.4)