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 method for the immunotherapy of bladder carcinoma in a human subject, comprising a) obtaining a urease C-deficient recombinant Mycobacterium (M.) bovis Bacillus Calmette-Guérin (BCG) cell from strain Danish subtype Prague as an immunotherapeutic agent comprising a recombinant nucleic acid molecule encoding a fusion polypeptide comprising: (i) a domain capable of eliciting an immune response, and (ii) a Listeria phagolysosomal escape domain listeriolysin (Hly), wherein said urease C-deficient recombinant Mycobacterium (M.) bovis Bacillus Calmette-Guérin (BCG) cell is provided in lyophilized form, b) reconstituting said urease C-deficient recombinant Mycobacterium (M.) bovis Bacillus Calmette-Guérin (BCG) cell in a liquid carrier, and c) administering said reconstituted urease C-deficient recombinant Mycobacterium (M.) bovis Bacillus Calmette-Guérin (BCG) cell by vesicular instillation into a subject's urinary bladder.

2. The method of claim 1, wherein the domain capable of eliciting an immune response is selected from immunogenic peptides or polypeptides from M. bovis or M. tuberculosis.

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

4. The method 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 hybridizing under stringent conditions with a full length complement of the sequence from (i) or (ii), wherein said stringent conditions comprise washing for one hour with 0.2× or 1× SSC and 0.1% SDS at a temperature of 55-68° C.

5. The method of claim 1, wherein the bladder carcinoma is selected from the group consisting of non-invasive bladder carcinoma, non-invasive papillary carcinoma (Ta), and a tumor invading subepithelial connective tissue (T1).

6. The method of claim 5, wherein said non-invasive bladder carcinoma is carcinoma in situ (Tcis).

7. The method of claim 1, wherein the immunotherapeutic agent is administered after surgery.

8. The method of claim 1 wherein said human subject is a patient with newly diagnosed or recurrent bladder carcinoma who has not been treated previously with standard BCG.

9. The method of claim 1 wherein said human subject is a patient with recurrent bladder carcinoma who has been treated previously with standard BCG.

10. The method of claim 1, wherein the immunotherapeutic agent is administered into the human subject's urinary bladder according to a schedule involving weekly instillations during an induction phase, a first maintenance phase after about 3 months, a second maintenance phase after about 6 months and a third maintenance phase after about 12 months.

11. The method of claim 10, wherein the immunotherapeutic agent is administered into the human subject's urinary bladder according to a schedule involving weekly instillations during an induction phase with 6 weekly instillations, a first maintenance phase after about 3 months with 3 weekly instillations, a second maintenance phase after about 6 months with 3 instillations and a third maintenance phase after about 12 months with 3 instillations.

12. The method of claim 1, wherein the immunotherapeutic agent is administered at a dose of from about 10.sup.6 to 10.sup.10 CFU per administration.

13. The method of claim 1, wherein the immunotherapy is combined with a non-tumor site specific administration of the recombinant M. bovis cell.

14. The method of claim 1 wherein focal and/or multifocal lymphocytic infiltration at the site of administration is obtained.

15. The method of claim 14 wherein focal and/or multifocal tissue infiltration with CD4 and CD8 T cells is obtained.

16. The method of claim 4, wherein said stringent conditions comprise washing for one hour with 0.2×SSC and 0.1% SDS at a temperature of 55-68° C.

17. A method for the immunotherapy of bladder carcinoma in a human subject, comprising administering to said subject a urease C-deficient recombinant Mycobacterium (M.) bovis Bacillus Calmette-Guérin (BCG) cell from strain Danish subtype Prague as an immunotherapeutic agent 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 listeriolysin (Hly), wherein said immunotherapeutic agent is provided in lyophilized form and reconstituted with a liquid carrier prior to administration by vesicular instillation into the urinary bladder wherein the immunotherapeutic agent is administered into the human subject's urinary bladder according to a schedule involving weekly instillations during an induction phase, a first maintenance phase after about 3 months, a second maintenance phase after about 6 months and a third maintenance phase after about 12 months.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A and FIG. 1B—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 2X10.sup.6 or 2X10.sup.8 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. 1A shows focal and multifocal lymphocytic infiltration after administration of rBCG. FIG. 1B shows only diffuse and singular infiltration after administration of BCG medac (200X magnification).

(2) FIG. 2A and FIG. 2B—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 2X10.sup.6 CFU rBCG/animal by 6 intravesical instillations, followed by group 3 treated with 2X10.sup.8 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. 2A shows focal and multifocal lymphocytic infiltration after administration of rBCG. FIG. 2B shows only diffuse and singular infiltration after administration of BCG medac (200X magnification).

EXAMPLE 1 SINGLE DOSE TOXICITY STUDY OF RECOMBINANT BCG (RBCG) IN RATS FOLLOWING INTRAVESICULAR INSTILLATION

(3) 1.1 Conduct of Study

(4) TABLE-US-00001 Test item rBCG lyophilized (rBCG Danish subtype Prague ΔUrec::Hly + w/o functional selection marker gene) Approximate viable 5.41 × 10.sup.8 CFU/vial counts Reference item BCG medac Approximate viable counts 2 × 10.sup.8 to 2 × 10.sup.9 CFU/vial Test species/Strain/Stock Rat/CD ®/Crl:CD(SD) Breeder Charles River Laboratories, Research Models, and Services, Germany GmbH Sandhofer Weg 7 97633 Sulzfeld, Germany Number and sex of 23 female animals; animals 3 animals for group 1; 5 animals for groups 2 to 5. Dose regime Group 1: Control (diluent) Group 2: ~2 × 10.sup.6 CFU rBCG (lyophilized)/animal Group 3: ~2 × 10.sup.8 CFU rBCG (lyophilized)/animal Group 4: ~2 × 10.sup.6 CFU rBCG (frozen w/o cryoprotectant)/ animal Group 5: ~2 × 10.sup.6 CFU BCG medac/animal Route of administration Intravesical instillation in the bladder Frequency of administration Single dose on test day 1. Administration volume 500 μL/animal Duration of study 12 adaptation days 4 in-life test weeks 28 incubation days

(5) 1.2 Results

(6) TABLE-US-00002 Mortality None of the animals died prematurely. Clinical signs No changes of behaviour, external appearance or condition of faeces were observed for any animal at any treatment. Body weight The body weight of all animals of all dose groups was in the normal range throughout the course of the study. Food and drinking The food intake of all animals of all dose water consumption groups was in the normal range throughout the course of the study. The visual appraisal of the drinking water consumption did not reveal any test or reference item-related influence. IL-2 levels The IL-2 levels in urine and serum of all animals of all groups were below the lower limit of quantification. Macroscopic post mortem No test or reference item-related changes findings were noted. Organ weights No test or reference item-related changes were noted. CFU counts rBCG (groups 2 to 4) vs. control (group 1) No test-item related CFU counts were noted for the examined organs and the blood of the animals treated once with an intravesical instillation of 2 × 10.sup.6 or 2 × 10.sup.8 CFU rBCG (lyophilized)/animal, or of 2 × 10.sup.6 CFU rBCG (frozen)/animal. In particular, no CFU counts at all were noted for the urinary bladder four weeks after instillation of the test item, indicating a rapid clearance of the administered mycobacteria from the site of instillation. No differences were noted between the animals treated with 2 × 10.sup.6 or 2 × 10.sup.8 CFU rBCG (lyophilized)/animal or with 2 × 10.sup.6 CFU rBCG (frozen)/animal and the control animals. BCG medac (group 5) vs. control (group 1) No reference-item related CFU counts were noted for the examined organs and the blood of the animals treated once with an intravesical instillation of 2 × 10.sup.6 BCG medac/animal. In particular, no CFU counts at all were noted for the urinary bladder four weeks after instillation of the reference item, indicating a rapid clearance of the administered mycobacteria from the site of instillation. No differences were noted between the animals treated with 2 × 10.sup.6 BCG medac/animal and the control animals. rBCG (groups 2 to 4) vs. BCG medac (group 5) No difference in CFU counts was noted for the examined organs and the blood of the animals treated once with an intravesical instillation of 2 × 10.sup.6 or 2 × 10.sup.8 CFU rBCG (lyophilized)/animal, or of 2 × 10.sup.6 CFU rBCG (frozen)/animal compared to the reference group treated in the same way with 2 × 10.sup.6 CFU BCG medac/animal. Immunohistochemistry 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 × 10.sup.6 or 2 × 10.sup.8 CFU rBCG (lyophilized)/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). Nearly all animals (12 of 13) of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). In contrast nearly all animals (9 of 10) of groups 2 and 3 contained CD4 and CD8 positive lymphocytes in focal or multifocal lymphocyte infiltrates in addition to the diffuse infiltration. Neutrophilic granulocytes could rarely be detected in any slides examined.

(7) In conclusion, no signs of toxicity were noted for the animals treated with the test item, the reference item or the dilutent. There were no differences in the systemic spread of mycobacteria in blood and organs between rBCG and BCG medac for the treatment by intravesical instillation. No test-item related CFU counts were noted for the examined organs and the blood of the animals treated once with an intravesical instillation of rBCG compared to the control animals. In particular, no CFU counts at all were noted for the urinary bladder 4 weeks after instillation of the test item, indicating a rapid clearance of the administered mycobacteria from the site of instillation.

(8) 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 2×10.sup.6 or 2×10.sup.8 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). Nearly all animals (12 of 13) of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). In contrast, nearly all animals (9 of 10) of groups 2 and 3 contained CD4 and CD8 positive lymphocytes in focal or multifocal lymphocyte infiltrates in addition to the diffuse infiltration. FIG. 1A shows focal and multifocal lymphocytic infiltration after administration of rBCG. FIG. 1B shows only diffuse and singular infiltration after administration of BCG medac (200× magnification).

EXAMPLE 2 REPEATED DOSE TOXICITY STUDY OF RECOMBINANT BCG (RBCG) IN RATS FOLLOWING INTRAVESICULAR INSTILLATION

(9) 2.1 Conduct of Study

(10) TABLE-US-00003 Test item rBCG lyophilized (rBCG Danish subtype Prague ΔUrec::Hly + w/o functional selection marker gene) Approximate viable counts 5.41 × 10.sup.8 CFU/vial Reference item BCG medac Approximate viable counts 2 × 10.sup.8 to 2 × 10.sup.9 CFU/vial Test species/Strain/Stock Rat/CD ®/Crl:CD(SD) Breeder Charles River Laboratories, Research Models, and Services, Germany GmbH Sandhofer Weg 7 97633 Sulzfeld, Germany Number and sex of animals 23 female animals; 3 animals for group 1; 5 animals for groups 2 to 5. Dose regime Group 1: Control (diluent) Group 2: ~2 × 10.sup.6 CFU rBCG (lyophilized)/animal Group 3: ~2 × 10.sup.8 CFU rBCG (lyophilized)/animal Group 4: ~2 × 10.sup.6 CFU rBCG (frozen w/o cryoprotectant)/ animal Group 5: ~2 × 10.sup.6 CFU BCG medac/ animal Route of administration Intravesical instillation in the bladder Frequency of administration Repeated administration; once weekly on test days 1, 8, 15, 22, 29, and 36. Administration volume 500 μL/animal Duration of study 21 adaptaion days 9 in-life test weeks 28 incubation days

(11) 2.2 Results

(12) TABLE-US-00004 Mortality None of the animals died prematurely. Clinical signs No changes of behaviour, external appearance or condition of faeces were observed for any animal at any treatment. Body weight The body weight of all animals of all dose groups was in the normal range throughout the course of the study. Food and drinking The food intake of all animals of all dose water consumption groups was in the normal range throughout the course of the study. The visual appraisal of the drinking water consumption did not reveal any test or reference item-related influence. IL-2 levels The IL-2 levels in urine and serum of all animals of all groups were below the lower limit of quantification. Delayed type hyper- No delayed type hypersensitivity was noted. sensitivity (DTH assay) Macroscopic post mortem No test or reference item-related changes findings were noted. Organ weights No test or reference item-related changes were noted. CFU counts rBCG (groups 2 to 4) vs. control (group 1) No test-item related CFU counts were noted for the examined organs and the blood of the animals treated six times with an intravesical instillation of 2 × 10.sup.6 or 2 × 10.sup.8 CFU rBCG (lyophilized)/animal, or of 2 × 10.sup.6 CFU rBCG (frozen/w/o cryoprotectant)/animal. In particular, no CFU counts at all were noted for the urinary bladder four weeks after the last instillation of the test item, indicating a rapid clearance of the administered mycobacteria from the site of instillation. No differences were noted between the animals treated with 2 × 10.sup.6 or 2 × 10.sup.8 CFU rBCG (lyophilized)/animal or with 2 × 10.sup.6 CFU rBCG (frozen)/animal and the control animals. BCG medac (group 5) vs. control (group 1) No reference-item related CFU counts were noted for the examined organs and the blood of the animals treated six times with an intravesical instillation of 2 × 10.sup.6 BCG medac/animal. In particular, no CFU counts at all were noted for the urinary bladder four weeks after the last instillation of the reference item, indicating a rapid clearance of the administered mycobacteria from the site of instillation. No differences were noted between the animals treated with 2 × 10.sup.6 BCG medac/animal and the control animals. rBCG (groups 2 to 4) vs. BCG medac (group 5) No difference in CFU counts was noted for the examined organs and the blood of the animals treated six times with an intravesical instillation of 2 × 10.sup.6 or 2 × 10.sup.8 CFU rBCG (lyophilized)/ animal, or of 2 × 10.sup.6 CFU rBCG (frozen)/animal compared to the reference group treated in the same way with 2 × 10.sup.6 CFU BCG medac/animal. Immunohistochemistry 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 × 10.sup.6 CFU rBCG/animal by 6 intravesical instillations, followed by group 3 treated with 2 × 10.sup.8 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). Neutrophilic granulocytes could rarely be detected in any slides examined.

(13) In conclusion, no signs of toxicity were noted for the animals treated with the test item, the reference item or the diluent. There were no differences in the systemic spread of mycobacteria in blood and organs between rBCG and BCG medac for the treatment by intravesical instillation. No test-item related CFU counts were noted for the examined organs and the blood of the animals treated six times with an intravesical instillation of rBCG compared to the control animals. In particular, no CFU counts at all were noted for the urinary bladder 4 weeks after the last instillation of the test item, indicating a rapid clearance of the administered mycobacteria from the site of instillation.

(14) 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 2×10.sup.6 CFU rBCG/animal by 6 intravesical instillations, followed by group 3 treated with 2×10.sup.8 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. 2A shows focal and multifocal lymphocytic infiltration after administration of rBCG. FIG. 2B shows only diffuse and singular infiltration after administration of BCG medac (200× magnification).

EXAMPLE 3 PHASE I/II OPEN LABEL CLINICAL TRIAL ASSESSING SAFETY AND EFFICACY OF INTRAVESICAL INSTILLATION OF RECOMBINANT BCG (RBCG) IN HUMAN PATIENTS WITH RECURRENT NON-MUSCLE INVASIVE BLADDER CANCER AFTER STANDARD BCG THERAPY

(15) 3.1 Clinical Protocol

(16) The recombinant BCG (as defined above) is currently applied in clinics as part of a Phase I/II clinical trial by instillation into bladder. The clinical trial phase I/II aims at assessing safety and efficacy of intravesical instillation of rBCG in human patients with recurrent non-muscle invasive bladder cancer after standard BCG therapy. rBCG is administered into bladder in 15 weekly instillations (induction phase: instillation 1-6, maintenance 3 months: instillation 7-9, maintenance 6 months: instillation 10-12, maintenance 12 months: instillation 13-15).

(17) The primary endpoint of the phase I is dose limiting toxicity (DLT) of intravesical rBCG instillations in patients with recurrence after standard BCG therapy in non-muscle invasive bladder cancer. The DLT period corresponds to 3 instillations plus 1 week and covers acute toxicities induced by treatment. Patients are treated in two cohorts of three, following the rules of a 3+3 design (dose de-escalation rules: if patients treated at dose level 1 show signs of DLT, dose of instilled rBCG will be reduced to level −1, which is 10 times lower than level 1).

(18) The dose levels are as follows: Dose level 1: 1−19.2×10.sup.8 CFUs of rBCG Dose level −1: 1−19.2×10.sup.7 CFUs of rBCG

(19) 3.2 Current Status

(20) The clinical trial currently is recruiting patients in trial sites in Switzerland (Basel, Geneva, Chur, Bern, Bellinzona, St. Gallen) within the frame of the Phase I.

(21) The DLT period for the first three patients (cohort 1) was completed on 23 Feb. 2016. Safety data, including DLT data, from this cohort has been collected by the sponsor and delivered to the Independent Safety Data Committee (ISDC). Since no DLT has been observed, the next three patients (second cohort) were allowed to be enrolled simultaneously and the recombinant BCG dose was maintained at dose level 1. An approval from the ISDC was received and all parties involved in the trial have been informed about this on 9 Mar. 2016 by the sponsor. In addition, no tumor recurrence has been observed in this cohort of patients at the time of evaluation.

(22) Currently recruitment of the second cohort is ongoing, with the first patient already enrolled in this cohort.