WO2001056387A1 - Methode de traitement par immunotherapie destinee a des patients souffrant d'un cancer superficiel de la vessie et pour lesquels au moins un traitement therapeutique par immunostimulation n'a pas fonctionne - Google Patents

Methode de traitement par immunotherapie destinee a des patients souffrant d'un cancer superficiel de la vessie et pour lesquels au moins un traitement therapeutique par immunostimulation n'a pas fonctionne Download PDF

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WO2001056387A1
WO2001056387A1 PCT/US2001/002827 US0102827W WO0156387A1 WO 2001056387 A1 WO2001056387 A1 WO 2001056387A1 US 0102827 W US0102827 W US 0102827W WO 0156387 A1 WO0156387 A1 WO 0156387A1
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bcg
treatment
interleukin
patient
cytokine
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Michael A. O'donnell
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Donnell Michael A O
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins

Definitions

  • the present invention is concerned with therapeutic treatments for bladder cancers; and is particularly directed to immunotherapeutic treatments for persons afflicted with superficial bladder cancers and upper urinary tract (ureters and renal pelvic region) cancers in the human body.
  • Carcinoma of the bladder is the fourth leading solid malignant disease among men and the eighth among women in the United States [American Cancer Society, 1998, Cancer Facts and Figuresl.
  • the majority of bladder cancers (75 to 80%) are present initially as superficial tumors and over 90% are transitional cell in origin [Droller, M.J., "Transitional cell carcinoma: upper tracts and bladder”. Campbell's Urology. W.B. Saunders, 1986].
  • Transitional cell carcinoma (TCC) also affects the upper urinary tract (ureter and renal pelvis) but with an overall incidence rate of 2- 4% . However, this rate rises to 10% in the setting of advanced bladder cancer; and rises to up to a 20% rate after successful BCG therapy of aggressive superficial bladder cancer.
  • Transitional cell carcinomas (the majority of cases present as superficial tumors) most often appear as papillary growths, but higher-grade lesions are often sessile and ulcerated. Grading is based on histologic architecture, size, pleomorphism, mitotic rate, and hyperchromatism. The frequency of cancer recurrence and progression is strongly correlated with grade, the grades being general categories of how well the tumor is differentiated (as shown by Table A). It will be recognized and appreciated that tumor grading is an approximation and estimate at best.
  • Tumor Grade 1 well differentiated cells
  • Tumor Grade 4 relatively undifferentiated cells
  • Carcinoma in-situ is recognizable as a flat, nonpapillary, anaplastic epithelium and may occur focally or diffusely, but it is most often found in association with papillary bladder cancers. Its presence identifies a patient at increased risk of recurrence and progression.
  • Adenocarcinomas and squamous cell cancers account for approximately 2% and 7% (respectively) of all bladder cancers detected in the USA. The latter is often associated with schistosomiasis, vesical calculi, or chronic catheter use.
  • Bladder cancer staging is another scheme for characterization of tumors. Bladder cancer staging is based on the extent of bladder wall penetration and the presence of either regional or distant metastases. The TNM classification of the American Joint Cancer Committee for bladder cancer staging is shown by Table B below. By definition and staging criterion, superficial bladder cancers include any of the TO, Tis, Ta and TI stages.
  • Table B TNM staging system for bladder cancer
  • bladder cancer The natural progression history of bladder cancer is based on two separate but related processes: tumor recurrence; and progression to higher stage disease. Both are related to tumor grade and stage.
  • tumor recurrence the process of bladder cancers
  • TI the process of bladder cancers
  • Lymph node metastases and progression are uncommon in such patients when they are properly treated; and 10 year survival is excellent at 81 %.
  • Patients with superficial cancers are typically treated with complete transurethral resection and the selective use of intravesical therapy. The latter is used to prevent or to delay recurrence.
  • Patients who present with large, high-grade, recurrent Ta lesions, those with TI cancers, and those with carcinoma in-situ are deemed to be appropriate candidates for intravesical therapy.
  • T2, T3 Patients with more invasive (T2, T3), but still localized, cancers are at risk of both nodal metastases and progression; and these persons require more aggressive surgery, irradiation, or the combination of chemotherapy and selective surgery or irradiation due to the much higher risk of tumor progression compared to patients with lower-stage lesions. Patients with evidence of lymph node or distant metastases typically undergo systemic chemotherapy initially.
  • CIS carcinoma in-situ
  • Relatively non-aggressive bladder tumors are confined to the mucosa (Stage Ta) and well-moderately differentiated (Grade 1-2); but other characteristics such as size, multiplicity, and recurrence rate may also lead to progression in 5-15% of these cases. Recurrence rates are similar among both aggressive and non-aggressive subtypes.
  • Immunotherapeutic or chemotherapeutic agents can be and most commonly are delivered directly into the bladder by a urethral catheter. These agents can be used to eradicate existing disease or to reduce the likelihood of recurrence in those who have undergone complete transurethral resection. Such therapy is more effective in the latter situation. Most of the agents are administered weekly for 6-12 weeks. The use of maintenance therapy after the initial induction regimen may be beneficial in some cases. Efficacy may be increased by prolonging contact time.
  • the common agents include thiotepa, mitomycin, doxorubicin, and BCG, the last of these being the most effective agent when compared with the others.
  • the known side effects of intravesical chemotherapy include irritative voiding symptoms and hemorrhagic cystitis, but systemic effects are rare.
  • IL-2 T-cell proliferation enhancement of cytotoxicity; rescue T cell from anergy and apoptosis of T cells and neutrophils
  • IL- 6 Co-stimulation, upregulation of IL-2 receptor on T cells; T cell, macrophage, urothelium, PMN inhibition or stimulation of tumor cell growth; induction of IL-1, IL-8, TNF
  • IL-8 Chemotaxis of T lymphocytes and neutrophils T cell, urothelium, macrophage, PMN IL-10 TH2 polariser; suppresses TNF, ICAM-1, B7 and MHC T cell, macrophage, urothelium? induction
  • IL-12 TH1 polariser induction of T cell cytotoxicity, rescue T cell, macrophage from anergy and costimuation
  • IL-18 Enhances ⁇ interferon production Urothelium?, macrophage GM-CSF Costimulation and enhancement of T cell cytotoxicity; Macrophage, T cell, urothelium, PMN rescue of macrophages from apoptosis; maturation of dendritic cellst interferon- ⁇ Stimulates IL-12 production; anti-angiogenic ⁇ , induces T cell, macrophage
  • ICAM-1, B7, MHC I and II, CD40, FAS cytostatic and cytotoxic TNF Co-stimulation and enhancement of T-cell cytotoxicity
  • T cell, macrophage, PMN, urothelium cytostatic and cytotoxic induces MHC class I and II
  • ICAM-1 ICAM-1, FAS, IL-6, IL-8; and angiogenic ⁇
  • T-helper lymphocyte Production of cytokines (IL-2, interferon ⁇ , TNF, IL- 12); bystander lysis of tumor cells; help to BCG- In-vitro phenomenon activated killer cells Determine genetic resistance to therapy
  • BCG-activated killer cells Tumor specific destruction through Nrampl alleles Macrophage Antigen presentation!); bystander tumor destruction; help First and most abundant to BCG-activated killer cells infiltrate;suppress/augment side-effects?
  • Neutrophil Cytokine production e.g., IL-8, IL-12, GM CSF, interferon alpha, MlP ⁇ and ⁇ , TNF, IP-10)
  • FAS TNF-like receptor
  • ICAM intercellular adhesion molecule
  • IL interleukin
  • IP interferon gamma inducible protein
  • LFA leukocyte function-associated molecule
  • MHC major histocompatility complex
  • MIF migration inhibition factor
  • Intravesical BCG therapy is commonly applied as an adjuvant treatment to prophylax against tumor recurrence; and as a therapeutic treatment to eliminate residual small volume disease and CIS.
  • Immunotherapy with BCG has consistently resulted in initial complete response rates of 55-65% for papillary tumors and 70- 75 % for CIS [Morales et aL, ⁇ Urol. 125: 649 (1981); Lamm et aL , IL Eur, ⁇ Med. 325: 1205 (1991)].
  • Long-term studies have documented benefits in terms of decreased recurrence rate, decreased progression rate, reduced cystectomy rate, and even improved survival [Herr et al.. ⁇ Urol. 135: 265 (1986)].
  • intravesical therapy with cytotoxic chemotherapeutic agents provides an estimated benefit of between 7-16% without any documented evidence of prolonged efficacy [Herr et al.. ⁇ Urol. 138: 1363 (1987)].
  • the large preponderance of controlled trials randomizing patients with superficial bladder cancer to BCG treatment or chemotherapy have shown strong statistically significant responses that in the aggregate are roughly 3 fold greater for BCG [Traynelis et al.. Urol. Annual. Vol. 8, 113 (1994)].
  • initial treatment with BCG is currently regarded as the most effective intravesical agent for the treatment of superficial transitional cell carcinoma (TCC) of the bladder. Failures of BCG Intravesical Treatment
  • BCG failure x 1 Institution of a single re- induction course after an initial failure (i.e., BCG failure x 1) has been shown to decrease the total failure rate to 30-40%, but further re-treatments (i.e., BCG failure x 2, etc.) results in disappointing net responses of under 20% [Catalona et al.. J Urol. 137: 220 (1987)]. Furthermore, the delay caused by additional minimally effective re-treatments is associated with a proportional increased risk of life- threatening disease advancement. Among initial BCG successes, 30-50% of patients gradually fail with recurrent bladder cancer within 5 years [Lamm et al. , NL Engl. . Med. 325: 1205 (1991)]. Long-term studies suggest a lasting benefit in as few as 20% of the original patient group by 10-15 years [Lamm et al.. J. Urol.157: 2134 (1997)].
  • Torti et aL 16 Papillary transitional cell Ca Recombinant interferon- ⁇ 2b 50-1,000 MU 4 Complete (25) 17.5+ (mean) 19 Ca in-situ/severe dysplasia 50-1,000 MU 6 Complete (32) 21 + (mean)
  • Table F Interferon combination therapy for transitional cell carcinoma prophylaxis and treatment
  • Pavone-Macaluso 62 Recombinant interferon- ⁇ 2b + 5-10 MU/30-50 mg. 59 24 and Serretta et al. epirubiciii
  • IFN-a interferon-alpha
  • a first aspect is provided by an immunotherapeutic method for treating a living patient afflicted with superficial bladder cancer, said patient having failed at least one immunostimulatory therapeutic treatment attempt previously, said immunotherapeutic method comprising the steps of: choosing the bladder for immunotreatment; initiating at least one treatment occasion for the patient comprised of (i) introducing an effective quantity of at least one live
  • Mycobacterium species to the bladder of the patient, said Mycobacterium species being one selected from the group consisting of a recombinant DNA Mycobacterial strain and Mycobacterium bovis BCG.
  • cytokine being at least one selected from the group consisting of any type or isoform of inter feron-alpha, interferon-beta, inter feron-gamma, inter leukin-1, interleukin-2, interleukin-3, interleukin-12, interleukin-15, and interleukin-18; and then allowing said Mycobacterium species and cytokine to act in combination in the bladder as an immunotherapeutic treatment for a preset period of time.
  • a second aspect of the present invention is an immunotherapeutic method for treating a living patient afflicted with upper urinary tract cancer, said immunotherapeutic method comprising the steps of: choosing an anatomic site in the upper urinary tract region for immunotreatment ; initiating at least one treatment occasion for the patient comprised of (i) introducing an effective quantity of at least one live Mycobacterium species to the chosen anatomic site in the upper urinary tract of the patient, said Mycobacterium species being one selected from the group consisting of a recombinant DNA Mycobacterial strain and Mycobacterium bovis BCG.
  • cytokine being at least one selected from the group consisting of any type or isoform of inter feron-alpha, interferon-beta, interferon-gamma, interleukin- 1, interleukin-2, interleukin-3, interleukin-12, interleukin-15, and interleukin-18; and then allowing said Mycobacterium species and cytokine to act in combination at the chosen anatomic site in the upper urinary tract as an immunotherapeutic treatment for a preset period of time.
  • a third aspect of the present invention provides an immunotherapeutic method for treating a living patient afflicted with superficial bladder cancer, said patient having failed at least one cytokine-included treatment attempt previously, said immunotherapeutic method comprising the steps of: identifying the cytokine administered previously to the patient in the failed immunostimulatory treatment; choosing the bladder for immunotreatment; initiating at least one treatment occasion for the patient comprised of (i) introducing an effective quantity of at least one live Mycobacterium species in the bladder of the patient, said Mycobacterium species being one selected from the group consisting of a recombinant DNA Mycobacterial strain and Mycobacterium bovis BCG. and
  • a fourth aspect of the present methodology provides a primary immunotherapeutic method for treating a living patient afflicted with superficial bladder cancer or upper urinary tract cancer, said patient not having received any immunostimulatory agents previously as a cancer treatment regimen, said immunotherapeutic method comprising the steps of: choosing an anatomic site in the body of the patient for immunotreatment; initiating not less than one treatment occasion for the patient comprised of (i) introducing an effective quantity of at least one live Mycobacterium species to the chosen anatomic site in the body of the patient, said Mycobacterium species being one selected from the group consisting of a recombinant DNA Mycobacterial strain, a substantially non-pathogenic species Mycobacterium and Mycobacterium bovis BCG. and
  • Fig. 1 is a depiction of the cytokine network evoked during conventional
  • BCG immunotherapy of superficial bladder cancer depicting the role of proximal stimulating and inhibiting cytokines that lead to IFN- ⁇ production.
  • the density of the arrows roughly correlates to the importance of each individual cytokine.
  • Figs. 2A and 2B are graphs representing the kinetics of cytokine production from mouse splenocytes (2 A) or human PBMCs (2B) after BCG stimulation. Values are expressed as a percentage of maximum measured amount. In both cases IFN- ⁇ is among the latest cytokines expressed.
  • Figs. 3 A and 3B show the dependence of IFN- ⁇ induced by BCG-stimulation of mouse splenocytes (3 A) or human PBMCs (3B) on proximal cytokines by selective antibody neutralization.
  • Fig. 4 is a graph showing that the multiple cytokines induced from mouse spleen cells after BCG stimulation can cooperate to recapitulate IFN- ⁇ amplification.
  • Purified recombinant murine cytokines were added to spleen cells in roughly the same concentrations as they are endogenous produced following BCG stimulation. *s depict the addition of IL-12 and/or IL-12 + IL-2 which are the most active agents in this system.
  • Fig. 5 is a graph illustrating the endogenous production of urinary cytokines (IFN- ⁇ , IL-2, IL-10, and IL-12) in a patient receiving weekly intravesical BCG therapy for superficial bladder cancer.
  • Fig. 6 depicts the IFN- ⁇ production of human PBMCs stimulated in-vitro with BCG plus a single exogenous cytokine as indicated. Based on IFN- ⁇ production, cytokines were placed in functional groups including strong inhibitors, weak inhibitors, neutral, weak stimulators, and strong stimulators. The far right graph uses a y-axis scale roughly 300X that of the left graph.
  • Controls included the lectins phytohemagglutinin (PHA), concanavalin A (CON A) and phorbol myristate acetate (PMA)/Ionomycin (IM). Near identical results were obtained from PBMCs from a separate donor.
  • PHA phytohemagglutinin
  • CON A concanavalin A
  • PMA phorbol myristate acetate
  • IM Ionomycin
  • Figs. 7 A and 7B demonstrate enhanced IFN- ⁇ production from human PBMCs stimulated with the synergistic multicytokine combination of IL-2 + IL-12 + IFN- ⁇ with or without BCG.
  • Fig. 7A shows how even extremely low concentrations of this multicytokine mixture retains strong stimulation potential with BCG.
  • Fig. 7B shows how the BCG dose can be dramatically decreased 1000-fold in the presence of the multicytokine mixture and retain high activity.
  • Fig. 8 is a graph showing the induction of IFN- ⁇ from murine splenocytes by different cytokine-secreting rBCGs. Enhanced activity is manifested for GMCSF rBCG, IL2 rBCG, and IFN- ⁇ rBCG over a wide does range of BCG.
  • Fig. 9 illustrates the augmented IFN- ⁇ stimulatory properties of rBCG secreting human rIL-2 or rIFN- ⁇ . Mixtures of the 2 rBCGs is further stimulatory and maintains high activity relative to control wild type (wt) BCG even when diluted down 1000-fold.
  • Fig. 10 is a graph showing the tumor burden in a mouse model bearing a syngeneic transplantable murine transitional cell carcinoma MB-49. Animals that were administered neutralizing antibody to IFN- ⁇ showed enhanced tumor growth relative to control antibody IgG or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Fig. 11 indicates the value of urinary IFN- ⁇ alone and the IFN- ⁇ /IL-10 ratio in predicting cancer response (resp) or non-response(non resp) to conventional BCG monotherapy.
  • Fig. 12 is a graph showing the induction effects on IFN- ⁇ of single cytokines, multiple cytokines, BCG and BCG plus single cytokines using the murine in-vitro splenocyte system.
  • Figs. 13 A and 13B are graphs illustrating the inductive effect on IFN- ⁇ of single cytokines, multiple cytokines, BCG and BCG plus single or multiple cytokines using the human in-vitro PBMC system.
  • PBMCs from either immuno- protive (13A) or BCG-presensitized (13B) patients are responsive to BCG plus cytokine(s).
  • Figs. 14A and 14B demonstrate that IFN- ⁇ in the murine system behaves markedly differently than in the human system by decreasing IFN- ⁇ production from BCG-stimulated splenocytes while increasing IL-10 production from the same.
  • Fig. 15 shows that BCG plus IL-12 administered directly in- vivo into the mouse bladder substantially increases the urinary production of IFN- ⁇ .
  • Fig. 16 demonstrates that combination immunotherapy of BCG plus IL-12 reduces tumor growth more efficiently than either agent alone in the syngeneic, in- vivo, subcutaneous MB-49 mouse bladder cancer model.
  • Figs. 17 A and 17B show the in-vitro PBMC and in- vivo urinary IFN- ⁇ responses of patient MN who had originally failed prior BCG plus IFN- ⁇ intravesical therapy.
  • In-vitro testing revealed combination BCG plus IL-12 would be most effective, an approach subsequently verified by in- vivo urinary cytokine monitoring.
  • Fig. 18 shows two graphs illustrating the induction of IFN- ⁇ in human PBMCs by two different cytokine-secreting rBCGs.
  • Fig. 19 is a graph showing improved survival for bladder cancer bearing mice treated with rBCG expressing the interferon-inducible protein IP-10.
  • Fig. 20 shows the cancer free survival for 60 human bladder cancer patients after clinical treatment with BCG plus IFN- ⁇ in combination.
  • Fig. 21 shows the cancer free survival for a subgroup of 38 failed BCG bladder cancer patients after clinical treatment with BCG plus IFN- ⁇ in combination.
  • Figs. 22A and 22B are graphs showing the in-vitro PBMC response of patient MN to varying doses of BCG combined with IL-12 (22A) or varying doses of IL-12 combined with BCG (22B). Neither agent by itself is active while both together are profoundly stimulatory for IFN- ⁇ production. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is a broad immunotherapeutic methodology for treating different forms and types of cancer in the bladder and upper urinary tract of a living human.
  • These treatable cancers are specifically the various tumors and neoplasms constituting superficial bladder cancers; and the different tumors and neoplasms found in the ureter and renal pelvis regions, the upper tract, of the human urinary system.
  • a particular benefit and advantage provided by the instant methodology is its focus and emphasis - a therapeutic treatment system and regimen for those bladder cancer patients who have already undergone one or more treatment attempts unsuccessfully; and presently have no medical recourse or course of treatment alternatives short of more radical methods for controlling, much less eliminating, their tumor.
  • the present innovations not only does provide substantial new treatment means for effective control and elimination of upper tract and superficial bladder cancers; this invention also provides a hope for the desperate and hopeless patient.
  • the invention is an immunotherapeutic treatment method specifically for patients with superficial bladder cancers (and transitional cell carcinomas in particular) who have undergone at least one immunostimulatory treatment therapeutic attempt (with or without prior surgery), but still have failed to achieve a disease-free state of meaningful duration.
  • Such persons collectively comprise "failed immunostimulatory therapeutic treatment patients"; and such failures of immunostimulatory therapeutic regimen include the individual categories of the failed BCG patients, the failed cytokine patients, and the failed BCG + cytokine patients, as each of these patient failure categories are defined and described hereinafter.
  • this first therapeutic use and medical application is directed to those persons who have not been successfully treated for superficial bladder cancer using the conventionally known intravesical therapeutic treatment protocols and agents; and now are left without an effective treatment alternative aside from radical surgery or removal of the bladder.
  • the second major use and application of the invention is as the initial therapeutic treatment procedure for treating cancers, and transitional cell carcinomas in particular, of the upper tracts (anatomically, the ureter and renal pelvis) in the urinary system.
  • the present treatment method is uniquely able to provide a regimen of immunotherapeutic induction treatment to the upper urinary tracts such that a remission occurs and a disease-free state eventually results in the ureter and renal pelvis areas.
  • cancers of the upper tracts regions are medically different and anatomically distinguished from superficial carcinomas of the bladder itself.
  • the present treatment method can be effectively used as an initial induction treatment regimen for patients having bladder cancer, particularly transitional cell carcinomas, who have not previously had any immunostimulatory intravesical treatment or other immunotherapeutic regimen (surgery, chemotherapy, or irradiation treatments notwithstanding).
  • Such patients are deemed collectively "immuno-pristine patients" (or “naive") in view of their lack of exposure to immunostimulatory agents.
  • the present methodology relies upon a novel underlying mechanism of action model; and provides a meaningful alternative to the conventional modes of immunotherapy for bladder cancer patients as a first or initial regimen of therapeutic treatment.
  • rBCG Recombinant Mycobacteria species having an altered DNA content and presenting attenuated cell characteristics similar to M. bovis BCG which are suitable for immunotherapy purposes.
  • Such recombinant Mycobacterial cells typically comprise at least one foreign or heterologous DNA sequence; and can include DNA coding for one or more biologically active cytokines which are expressed intracellularly and exported (secreted) extracellularly.
  • BCG or M-. bovis BCG Mycobacterium bovis Bacillus Calmette-Guerin species; a live attenuated bacterial strain.
  • BCG A collective designation identifying either or both rBCG and/or M. bovis BCG.
  • Initial induction treatment A first course of intravesical immunotherapy used as an initial therapeutic treatment for a previously immunologically untreated patient diagnosed with bladder cancer, the "naive" or irnmuno-pristine patient.
  • Second induction treatment A second, followup, immunotherapeutic course of intravesical therapeutic treatment used as a second treatment regimen for a bladder cancer patient after the initial induction treatment has been completed or found to be unsuccessful or unsuitable.
  • Third induction treatment A third, followup, immunotherapeutic course of intravesical therapeutic treatment used as a third treatment regimen for a bladder cancer patient after the initial and second induction treatments have been completed or found to be either unsuccessful or unsuitable.
  • Maintenance treatment A course of intravesical therapeutic treatment instituted AFTER a disease-free status state has been achieved through one or more induction treatments. Accordingly, the purpose of maintenance treatment is to maintain the disease-free state by repetitive "boosters" of the therapeutic regimen.
  • rCytokine An exogenous cytokine expressed in-situ by a recombinant
  • Mycobacterial or other live species carrying a heterologous DNA segment coding for a cytokine of choice carrying a heterologous DNA segment coding for a cytokine of choice.
  • Cytokine A discrete biologically active polypeptide typically expressed by blood mononuclear cells which can be either endogenous or exogenous, or a rCytokine.
  • Immuno-pristine patient A bladder cancer patient who has not received any immunotherapy as a therapeutic regimen; but may have undergone chemotherapy, surgery or irradiation treatments. Also termed a "naive" patient.
  • BCG patient A bladder cancer patient who has received at least one induction treatment with BCG as the therapeutic regimen.
  • Cytokine patient A bladder cancer patient who has received at least one induction treatment with any cytokine or combination of cytokines without BCG as the therapeutic regimen.
  • BCG + cytokine patient A bladder cancer patient who has received at least one induction treatment with BCG and a cytokine in combination as a therapeutic regimen.
  • Classes Type(s) BCG treatment regimen Failures are characterized by
  • Relapsed patients intermediate relapse regression or prophylactic effect; Recurrence of tumor between 1-2 followed by a recurrence of years' time post-treatment tumor growth late relapse Recurrence of tumor after 2 years' time post-treatment
  • Relapsed patients intermediate relapse regression or prophylactic effect; Recurrence of tumor between 1-2 followed by a recurrence of years' time post-treatment tumor growth late relapse Recurrence of tumor after 2 years' time post-treatment
  • a cytokine which may have included among others IL-2, IL- 12, TNF, GMCSF, IFN- ⁇ , ⁇ or ⁇ or any combination thereof.
  • Classes Type(s) treatment regimen Failures are characterized by
  • Relapsed patients intermediate relapse regression or prophylactic effect; Recurrence of tumor between 1-2 followed by a recurrence of years' time post-treatment tumor growth late relapse Recurrence of tumor after 2 years' time post-treatment
  • IFN ⁇ or ⁇ BCG + IL-2, BCG + IL-12.
  • the category of "failed BCG patients” includes many different kinds of patients suffering from superficial bladder carcinoma - all of whom have received at least an initial induction treatment with BCG or M. bovis BCG as the immunotherapeutic regimen and have not responded well nor achieved a disease-free state of meaningful duration.
  • the separate and distinct category of "failed cytokine patients” includes all the different persons suffering from superficial bladder cancer who received an initial induction treatment using a cytokine as the mode of immunostimulation, but did not respond well nor reach a disease-free state of duration as a consequence of the therapeutic regimen.
  • the third discrete category of "failed BCG + cytokine patients" is also recognized as constituting all the refractory, relapsed or intolerant patients who did not respond favorably and did not reach a disease-free state of meaningful duration after receiving the combination therapy. All of these persons who failed treatment previously did not have an immunotherapeutic alternative or followup course of inductive treatment which could be expected to be medically effective.
  • the broad aspects and proper usage of the methodology described herein now provides an alternative course of immunotherapeutic treatment for all persons in each of these three categories of failed patients.
  • cytokine network model has been formulated as a result of extensive experimental testing in which additions of stimulatory cytokines or functional subtractions of inhibitory cytokines profoundly synergize the induction of IFN- ⁇ by BCG. This is shown by Fig. 1.
  • the width of the arrows indicates the relative importance of each cytokine contributing to IFN- ⁇ release.
  • a substantial body of data has also been collected from in-vitro and in-vivo experiments in mice implicating a critical role for Thl responses and, in particular, the induction of IFN- ⁇ as part of BCG anti-tumor activity.
  • IFN- ⁇ is a highly regulated distal product of multiple interacting proximal cytokines induced by immune cell stimulation with BCG.
  • BCG was found to induce multiple cytokines in-vitro, in-vivo and clinically including: IL-1, IL-2, IL-6, IL-8, IL-10, IL-12, TNF- ⁇ , GMCSF, IP-10, and IFN- ⁇ .
  • IL-4 was only detectable using highly sensitive RNA PCR methods. Kinetic analysis revealed that these cytokines are produced in a sequential manner. See Figs. 2 A and 2B. While there are some clear differences between the murine splenocyte and human PBMC assays, in both cases IFN- ⁇ is among the last cytokines produced.
  • proximal cytokines leading to IFN- ⁇ that is indirectly regulated by the timely appearance of down-modulatory IL-10.
  • the dependence of IFN- ⁇ on these proximal cytokines was verified by antibody neutralization in culture (see Figs. 3 A and 3B) and the ability to recapitulate progressively higher IFN- ⁇ production by adding purified combinations of proximal cytokines to murine splenocytes that mimic their natural appearance following BCG therapy (Fig. 4).
  • Adherent cells primarily macrophages - a known source of IL-12
  • CD4 + or CD8 + cells were found to be essential for significant IFN- ⁇ production.
  • IFN- ⁇ increases during BCG rechallenge as the result of immune memory and serves as a natural accelerator during sequentially administered BCG as it is currently applied therapeutically for bladder cancer.
  • select urinary cytokines were analyzed for the first 12 hours following each weekly treatment in several patients. The result in one such patient as examplified by Fig. 5. From this analysis it is apparent that IFN- ⁇ , IL-2, and IL-12 (all Thl cytokines) rise closely in parallel. IL-10 is displaced roughly 4 weeks after the rise in IFN- ⁇ suggesting it has a functional rheostatic role in quenching the IFN- ⁇ cascade.
  • the IFN- ⁇ amplification pathway can be markedly enhanced by the addition of dose-limiting proximal cytokines, certain exogenous cytokines, and antibodies to IL-10.
  • mice significant enhancement of BCG-induced IFN- ⁇ release was seen with additions of GMCSF, IL-2, IL-12, and/or IL-18.
  • the highest degree of amplification with single cytokines in mice was achieved by IL-18 (350 X) followed by IL-12 (100 X) while IL-2 and GMCSF provoked only 5-10 X increases.
  • half maximal levels of stimulation for IL-18 and IL-12 were achieved with under 10 pg/ml while 20-50 fold higher concentrations of IL-2 or GMCSF were required.
  • IFN- ⁇ 5-10 fold increases of IFN- ⁇ could be achieved when IL-18 and IL-12 (both at 10 pg/ml) were added together. Similar degrees of synergy with IL-2 plus IL-12 could be obtained but required 20-50 fold higher amounts of IL-2.
  • anti-IL-10 antibody to either murine or human immune cell cultures also enhanced IFN- ⁇ as much as the addition of the weakly stimulatory cytokines, reinforcing IL-10 's role as a functional down-modulator of BCG induced IFN- ⁇ and suggesting a further means by which the Thl pathway may be promoted.
  • the failure of IL-10 neutralization to recapitulate the extremely high levels of IFN- ⁇ (up to 1000-fold) achieved with the strong stimulators demonstrates the complexity of this synergy mechanism.
  • Cytokine rBCGs [BCG bacterial cells able to express a recombinant DNA coding for an active form of a specific cytokine sequence] expressing the appropriate murine or human co-stimulatory cytokines show the expected substantial up-regulation of IFN- ⁇ during in-vitro testing. Cytokine rBCGs expressing murine GMCSF, IL-2, and IFN- ⁇ were each able to substantially augment IFN- ⁇ production while the control non-expressing rBCG (261) and rBCG expressing IL-6 were not significantly different from wild type BCG, as shown by Fig. 8.
  • IFN- ⁇ secreted by mIFN- ⁇ rBCG activated a positive feedback loop resulting in production of over 10 ng/ml amounts of endogenous IFN- ⁇ .
  • rBCGs expressing single hIL-2 or hlFN- ⁇ have been produced which show ⁇ 3 fold greater activity than wt BCG during in-vitro testing. When simply mixed together, they are roughly 10 times as potent. More important, this combination retains substantial potency (6 fold higher than full dose wt BCG; 18 fold higher than an equivalent dose of wt BCG) even when reduced 1000 fold in concentration, as shown by Fig. 9. Furthermore, with this degree of dilution, both IL-2 and IFN- ⁇ are calculated to be below 1 pg/ml.
  • the persistence of synergy at this low free cytokine concentration provides strong support for the existence of paracrine stimulation in which the rBCG cytokine microenvironment stimulates neighboring immune cells. This represents a distinctive advantage of cytokine secreting rBCG over exogenous cytokines delivered with BCG, a situation which may allow very low doses of rBCG to be used with a concomitant increase in relatively safety. 4) IFN- ⁇ is associated with the BCG anti-tumor mechanism.
  • IFN- ⁇ has a direct and profound effect on murine (MB49 and MBT-2) and human bladder cancer cell lines by suppressing growth; by inducing cytokine expression from the tumor; and by upregulating MHC class I & II antigens, the apoptotic Fas receptor, the ICAM adhesion molecules, and the TNF- ⁇ receptor.
  • Neutralizing antibodies to IFN- ⁇ also accelerate the in-vivo growth of the MB49 bladder cancer in mice (see Fig. 10).
  • Fig. 11 shows that patients expressing high amounts of urinary IFN- ⁇ (Thl amplitude) or IFN- ⁇ /IL-10 ratios (Thl polarity) are more likely to be bladder cancer responders (closed symbols) than non-responders (open symbols). Together these observations suggest that IFN- ⁇ is a good marker of effective BCG therapy and a necessary component in the mechanistic process.
  • the Mycobacterium Species utilizes at least one species or strain of Mycobacterium to be delivered on one or multiple treatment occasions as a component part of the prepared combination immunostimulant.
  • the particular species of choice used as a component may be selected from among bovis BCG. an attenuated non-pathogenic (for humans) strain of Mycobacterium. and/or a recombinant DNA strain of Mycobacterium. Each of these will be described in detail hereinafter.
  • Mycobacteria Owing to their impact as major human pathogens and as a result of their profound immunostimulatory properties, Mycobacteria have long been intensively studied. In the early 1920s, an attenuated Mycobacterium. Mycobacterium (M.) bovis Bacille Calmette-Guerin (M. bovis BCG or BCG) was isolated for use as a vaccine against tuberculosis [Calmette et al.. Acad. Natl. Med. (Paris) SI: 1 SI -196 (1924), reviewed in Collins, F.M.. Bacterial Vaccines (R. Germanier, ed.), Academic Press, pp. 373-418 (1984)].
  • M. bovis Bacille Calmette-Guerin M. bovis BCG or BCG
  • BCG is among the most widely used human vaccines [Luelmo, F.. Am. Rev. Respir. Pis. 125: 70-72 (1982); Fine, P.E.M., Reviews of Infectious Diseases II (supp. 2), 5353-5359 (1989)].
  • Bacille Calmette-Guerin is a non-virulent strain of M. bovis and has been used as a live vaccine for more than 50 years. In the past 35 years, it has been administered to over 2.5 billion people, with remarkably few adverse effects (e.g. , estimated mortality of 60/billion). BCG has been found in numerous studies to have protective efficacy against tuberculosis. It is also the species of choice most preferred for use in intravesical immunotherapy of superficial bladder cancers in humans and animals [See for example: Alexandroff et al.. Lancet 353: 1689-1694 (1999) and the references cited therein].
  • strains of Mycobacterium exist and are deemed to be suitable for use in this treatment system.
  • Such strains include ⁇ . smegmatis. M. phlei. and M. piscium. respectively in that naturally-existing forms; and attenuated live mutant species of these.
  • Mycobacteria has led to the identification of many of the major antigens that are targets of the immune response to infection by Mycobacteria [Kaufman, S.H.E., Immunol. Today U: 129-136 (1990); Young, R.A., Ann. Rev. Immunol. &: 401- 420 (1990); Young et aL, Academic Press Ltd., London, pp. 1-35, 1990; Young et aL, Mol. Microbiol. ⁇ 5: 133-145 (1992)] and to an improved understanding of the molecular mechanisms involved in resistance to anti-Mycobacterial antibiotics [Zhang et al.. Nature 358: 591-593 (1992); Telenti et al..
  • Genome mapping and sequencing projects are providing valuable information about the M. tuberculosis and M_ leprae genomes that will facilitate further study of the biology of these pathogens [Young and Cole, J. Bacteriol. 175: 1-6 (1993)].
  • BCG BCG receptor mediated endothelial growth factor
  • chemokines such as interleukin-8 (which serve to attract leukocytes to a local site), pro-inflammatory cytokines (granulocyte-macrophage colony-stimulating- factor, tumor necrosis factor ⁇ , interleukin- 6), and the upregulation of adhesion- molecule expression (intracellular adhesion molecule 1), which promotes effector cell-tumor cell interactions.
  • chemokines such as interleukin-8 (which serve to attract leukocytes to a local site), pro-inflammatory cytokines (granulocyte-macrophage colony-stimulating- factor, tumor necrosis factor ⁇ , interleukin- 6), and the upregulation of adhesion- molecule expression (intracellular adhesion molecule 1), which promotes effector cell-tumor cell interactions.
  • a marked infiltration of the bladder wall occurs, which is characterized by the presence of T lymphocytes, macrophages, and neutrophils in the urine, as well as further induction on tumor of intercellular adhesion molecule 1 , MHC class I and II molecules, and reversion of cytology from positive to negative. It is believed that the leukocyte infiltrate secretes numerous cytokines, including those that are the hallmark of activated T cells and natural-killer cells (such as interleukin 2 and interferon gamma), which are typically observed. Maximum levels of cytokine secretion, cellular influx, intercellular adhesion molecule 1, and MHC expression and clinical response are usually attained by the fifth and sixth instillation.
  • BCG-induced/activated T lymphocytes are considered to be the most important elements of the anti tumor response; and both CD4 and CD8 T lymphocytes are essential for the successful treatment of bladder cancer. Also, BCG-activated killer cells are unique in origin and ability to distinguish between normal and tumor cells. Whether BCG-activated killer cells represent a mixture of two different populations (CD8 and CD56) or a single population that coexpresses markers of T cells and natural killer cells remains unknown. After BCG treatment on multiple occasions has ended, the activity of the immune system will gradually subside. This is reflected by a decline in infiltrating leukocytes, MHC expression on epithelium, and cytokine levels.
  • the present immunotherapeutic treatment system utilizes at least one preselected active cytokine in combination with the Mycobacterial species collectively as a combination immunostimulatory agent.
  • the chosen active cytokine(s) may be a prepared-in-advance, discrete substance co-administered with the M- species and introduced at the proper anatomic site in the bladder or upper urinary tract.
  • the selected active cytokine is a recombinant cytokine expressed in-situ by an introduced recombinant Mycobacterial species or by a genetically modified host cell expressing in-situ at least one exogenous DNA sequence encoding the cytokine of choice. Any or all of these alternative formats for introducing the selected active cytokine to the tumor in the living patient's body is suitable for use when practicing the present methodology.
  • Cytokines by broad definition, are factors (such as a lymphokine or monokine) produced by mononuclear cells that affect other cells. Cytokines, often acting in serial sequence, serve as endogenous signals between cells which, in conjunction with antigens, rapidly amply both the localized and systemic host defenses and defense mechanisms involving macrophages, lymphocytes, and other activated or naturally-occurring cell types. Among the well characterized cytokines produced and detected locally are those listed by Table C previously herein.
  • the present bladder cancer treatment method and system purposely causes and requires an introduction of at least one cytokine - and preferably as many as ten different families of cytokines - into the patient's bladder or upper urinary tract (ureter and renal pelvis regions) concurrently and in combination with each therapeutic administration of a Mycobacterial species on each treatment occasion.
  • cytokines conventionally known to exist or able to be produced are suitable for use in the present cancer treatment methodology.
  • there are major differences in the functional properties and clinical value among the conventionally known cytokines differences which cause them to be ranked as superior or inferior as well as useful or non-useful within an ordered system of clinical worth.
  • interferon-alpha includes by definition the subtypes interferon- ⁇ 2b and interferon- ⁇ 2a, the synthetic interferon ⁇ con-1, and leukocyte derived "natural" interferon- ⁇ .
  • Table 1 provides information not only on the magnitude of stimulation but also on the probability of achieving substantial stimulation when treating heterogenous cancer patients. For instance, in-vitro experiments show the combination of BCG with IFN alpha or beta + IL-2 + IL-12 achieves over 75 % maximal stimulation in almost all patient instances whereas BCG, with single IFN-a or -b results in -80% of patients having at least a 4-fold amplification with -50% reaching within 75% of maximal stimulation. By contrast, the addition of IL-18 to BCG amplifies IFN-g in less than 1/2 of patients and by less than 3 fold in all. TNF- ⁇ is neutral in almost all cases with rare minor improvements of less than 100% . IL-10 is inhibitory in all cases tested, the extent of which, however, depends on the basal amount of IFN-g induced by BCG alone.
  • the "Most Preferred/Most Active” group of 6 specific cytokines and the “Weakly Active” group of 4 specific cytokines may be used singly or in multiple blends in conjunction with the Mycobacterial species of choice for immunotherapeutic purposes.
  • the members of the "Inactive/Neutral" rank, the "Weakly Inhibitory” group and the “Strongly Inhibitory” set are individually, cumulatively and collectively to be avoided clinically and are not to be administered to the patient at any time or used on any immunotherapeutic treatment occasion.
  • the failed patients in each of these categories each received a cytokine alone or in combination with BCG as a therapeutic treatment for their superficial bladder cancer; and, equally important, such prior treatment attempts comprising a cytokine all failed to result in a disease-free state for the patient.
  • the present methodology provides a clear and unequivocal cytokine selection process, which is: pick and use a different single cytokine or a blended plurality of cytokines from among those in the allowable listing. A second trial or reduction with that particular regimen is generally to be avoided; however, re-administration of that particular cytokine of the prior failed treatment may be given in conjunction with other members of the Most Preferred/Most Active group.
  • any of the 5 other cytokines in this grouping may be chosen as used alone or with the others in blends of 2-6 cytokines in admixture.
  • the selection criterion for a cytokine of choice is similar regarding the four cytokine members constituting the "Weakly Active" grouping. Should the cytokine used previously for the patient in the prior failed treatment attempt be one of these, either of the two other members of this grouping may be employed presently, alone or as a blending of the others. Also, the entire membership constituting the "Most Preferred/Most Active" grouping also remains available for use, singly or as a blend of up to six different individual cytokines.
  • a host cell has been genetically modified to carry and express in-situ at least one exogenous DNA sequence encoding a single cytokine or a series of different cytokines.
  • the technology for recombinant formats is well documented and described both in the scientific literature as well as in mature patents.
  • various recombinant DNA Mycobacterial strains have been developed to include and express heterogenous DNA sequences coding for one or more specific cytokines. Describing such recombinant Mycobacterial species able to express such cytokines in-situ and in-vivo are the following, each of which is expressly incorporated by reference herein: U.S. Patent Nos. 5,776,465; 5,591,632; 5,866,403; 5,854,055; 5,840,855; 5,830,475; 5,504,005; 5,807,723; and the references cited within each of these individually.
  • This prepared expression vector is a plasmid which is used to transform the desired Mycobacterial strain; and will serve for expression and secretion in-situ of a recombinant human form of IFN- ⁇ wherever and whenever the recombinant Mycobacterial strain is introduced at a chosen anatomic site in the human body.
  • T influenza virus hemagglutinin epitope tag sequence
  • the Complete Immunotherapeutic Formulation is at a minimum: a first format comprising a non-pathogenic strain of Mycobacterium (such as ML. bovis BCG) in admixture with at least one cytokine selected from the group consisting of IL-1, IL-2, IL-3, IL-12, IL-15, IL-18, IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ ; and a second format comprising a recombinant Mycobacterium species carrying and expressing in-situ at least one exogenous DNA sequence (in integrated or non-integrated form) encoding not less than one cytokine selected from the same selective grouping. Mixtures of recombinant Mycobacterium species are likewise permitted.
  • the live Mycobacterial cells will be combined with a synthesized and/or purified cytokine(s) of choice in quantitative ratios as described hereinafter by the illustrative treatment protocols.
  • the viable Mycobacterium cells and the cytokine(s) of choice may be given concurrently, but as individual preparations; but preferably are combined in admixture and co-administered as a blended mixture to the prechosen anatomic site in the patient's urinary system as a therapeutic agent.
  • the recombinant Mycobacterium strain (having been genetically modified in advance to carry and include a DNA sequence encoding at least one preselected cytokine) is itself the cellular vehicle and the prepared formulation for immunotherapeutic treatment of the patient.
  • the live Mycobacteria Upon introduction of the viable recombinant Mycobacterial cells to the bladder or the upper urinary tract of the patient, the live Mycobacteria will express and secrete the selected cytokine(s) of choice in-situ at the anatomic site as an active substance.
  • a concomitant in-situ introduction of the chosen cytokine is caused which is concurrent with the administration of the live Mycobacteria.
  • the introduction of recombinant cells also intrinsically causes and achieves a concurrent introduction of the selected cytokine in due course of time.
  • Routings And Manner Of Administration Administration of the immunotherapeutic agent(s) into the bladder is generally accomplished by the atraumatic insertion of a catheter into the bladder.
  • the liquid therapeutic agent(s) usually in a volume between 25-100 ccs are then delivered through the catheter into the bladder under low pressure either by gravity or gentle pressure upon which the catheter is generally immediately removed allowing the liquid medication to remain inside.
  • the immunotherapeutic is then retained for 1-3 hours before evacuation by physiologic voiding or catheter reinsertion if voiding is impaired.
  • sequential weekly or biweekly administration is given totalling 5-12 (but most usually 6) separate administrations. Maintenance treatment may be given according to various schedules but most often as a 3-dose weekly miniseries at intervals of 3-6 months or as a once monthly administration.
  • Administration of the immunotherapeutic agent(s) into the upper tract may be accomplished via either an antegrade or retrograde fashion. In the former case, it is delivered via a percutaneous tube pre-inserted into the renal pelvis. In the latter case, a stent may be placed in a temporary or more durable manner to provide access from below into the renal pelvis. The agent(s) are then administered under low pressure, such as by gravity drip, at a slow continuous rate, usually over 1-4 hours.
  • the frequency of sequential administration for induction or maintenance treatment is similar to that described above for intravesical administration.
  • Group I is constituted of superficial bladder cancer patients who, individually and collectively, are immuno-pristine and have not previously received any immunostimulatory treatment for their cancer, particularly no induction of BCG or any other Mycobacterial species.
  • Group JJ is constituted of superficial bladder cancer patients, each of whom has previously undergone at least one immunostimulatory treatment with BCG on a prior occasion, but has failed to respond adequately to such treatment. These patients comprise the category of "failed BCG patients" as defined herein.
  • Group III is constituted of three different subgroupings of human patients:
  • Group Ilia comprises patients who have previously received cytokine monotherapy as a stimulatory agent for treatment of superficial bladder cancer, but have failed to achieve a disease-free state of any duration. This Group Ilia thus forms the "failed cytokine patients" category as described and defined herein.
  • Group Illb is constituted of patients afflicted with superficial bladder cancer, each of whom has previously received BCG + a cytokine as a therapeutic treatment on at least one prior occasion - but each of which has failed to achieve a disease-free state.
  • This Group Illb collectively includes the "failed BCG + cytokine patient" category as defined and described herein.
  • Group IIIc is constituted of patients suffering from cancer of the upper urinary tract, whether or not they have received prior BCG or prior cytokine monotherapy before. It should be appreciated that immunotherapy has hitherto not been conventionally used in this patient category.
  • Induction treatments shown by Table 3 consist of one of three representative regimens based on the patient's prior BCG exposure, his BCG tolerance, and his tumor location.
  • prior cytokine failure patients (Group Ilia) are treated with 6 weeks of 1/lOth standard dose BCG plus 100 MU of IFN-a- 2B.
  • Patients failing a prior induction cycle of combination BCG plus a cytokine may receive a 2nd induction cycle if clinically appropriate. Patients with upper tract transitional cell carcinoma regardless of prior therapy would also be treated with the same regimen (Group IIIc). If treatment intolerance occurs in any group during the induction period, the patient may optionally be given a 2- week rest followed by re-initiation of treatments at a BCG dose of roughly 1/3 that of the prior dose. Similar 2-week delays are permitted for repeat episodes of intolerance. Further, BCG dose reduction by 1/3 intervals are used as necessary.
  • Intravesical therapy is delivered weekly via a temporarily placed foley catheter for a total of 6 induction treatments for patients with bladder TCC.
  • a small (usually 4 French) temporary external stent is placed cystoscopically from the bladder into the mid renal pelvis when possible.
  • treatment may be given through a percutaneous nephrostomy tube or other similar device to deliver material into the renal pelvis. All patients are asked to reduce fluid consumption the night and morning before the treatment to facilitate holding the 50 cc of liquid medication for approximately 2 hours and/or minimizing excess dilution. They then void into the toilet. Excessive fluid consumption for the day of the treatment is discouraged.
  • Standard cystoscopic and cytological evaluations are performed at roughly 3- month intervals during the first 2 years although 6-month intervals may be appropriate during the second year for patients with less aggressive disease. Progression at any time to muscle invasive disease or metastatic disease require this treatment to be terminated. Recurrence of disease which is surgically resectable and/or intravesical therapy amenable after the first induction course may be offered a second course of induction therapy beginning with an even lower dose of BCG (1/lOth standard dose) and a higher dose of IFN-a (100 MU) or an alternate BCG plus cytokine regimen. However, the decision to re-treat is left to the clinician's judgment. In general, patients with non-aggressive bladder tumor (mucosal, low- intermediate grade) will be more suited for re-treatment while only the exceptional patient with recurrent aggressive disease (Stage TI, grade 3, or CIS) should be considered.
  • bladder cancer In the United States most patients with bladder cancer are over 60 years of age, and many are smokers. The age-adjusted incidence of bladder cancer is approximately 17 per 100,000. Bladder cancer is 3.7 times more common among men than women and approximately 2-fold higher in white men than black men and 3-fold higher in white men than Hispanic men. The incidence of upper tract TCC is roughly 4% that of bladder cancer but is 3-5 times higher in patients with advanced bladder cancer, those with long duration disease, or patients successfully treated with BCG for aggressive superficial bladder cancer. Approximately 70-80% of cases present initially as superficial disease (Stage Ta, TI, and CIS). Roughly 25-30% of superficial cases are of the aggressive subtype (Stage TI, grade 3, CIS).
  • Patients may be of either sex and any race and have superficial TCC only.
  • patients with superficial TCC of the bladder are eligible for this treatment protocol, given the vacuum of acceptable and efficacious treatments for BCG failure patients and those with upper tract disease, it is likely that these persons will be over-represented.
  • Pre-Exclusion Criteria 1) Females who are pregnant or breast feeding.
  • Papillary or solid TCC resection, bladder biopsy, or TURP within the previous 7 days or evidence of gross hematuria within the previous 2 days to minimize the chance of inadvertent BCG intravasation into the bloodstream.
  • Unstable cardiac disease including angina, ischemia, arrhythmia, and/or congestive heart failure that might be precipitated by occasional fevers and chills caused by the treatment.
  • Post-Exclusion Criteria After onset of therapy, a patient should be excluded from further treatment for any of the following reasons: 1) Progressive disease to stage T2 or higher at any evaluation point.
  • Bacillus Calmette-Guerin (BCG) is widely regarded as the most effective intravesical agent to prophylax against recurrent superficial bladder cancer, treat residual papillary tumor, and treat carcinoma in-situ (CIS). Mild-moderate toxicity is experienced by many patients consisting of irritative bladder symptoms (frequent and painful urination lasting less than 3 days), blood in the urine, fever, fatigue, or flu-like symptoms lasting less than 3 days. Serious but infrequent ( ⁇ 5% incidence) reactions include the following: skin rash, arthritis, urinary tract infection, shrinkage of the bladder, obstruction of the kidney urinary tube (ureter), inflammation of the prostate gland and testicles, inflammation of the liver, and kidney abscess.
  • BCG can cause severe systemic infection. Such an occurrence may require hospital admission, intravenous antibiotics and up to 3-6 months of oral anti-tuberculosis medication. In extreme cases such severe infections could result in breathing difficulties, shock, or even death.
  • Interferon-alpha - Intrinsic Risks Interferon-alpha - Intrinsic Risks: Interferon-alpha (IFN-a) by itself when instilled directly into the bladder has generally been very well tolerated. Occasionally mild cystitis, chills, and transient fever or flu-like symptoms may occur in the minority ( ⁇ 20%) of patients. Dose- limiting toxicity is not reached even at the highest dose of 1000 MU/week, well below the 50-100 MU/week dose in this clinical protocol.
  • BCG dose reduction allows more patients to tolerate this therapy.
  • maintenance therapy is still controversial, variably practiced, and adds to local toxicity, it should not be an absolute requirement.
  • BCG serious BCG infection
  • the drugs should be delivered by gravity instillation to prevent high pressure entry into the bloodstream.
  • BCG dose reduction and/or treatment delay may be employed.
  • symptoms suggestive of serious BCG infection such as fever over 103 F with shaking chills, patients are instructed to return to their medical center for evaluation. Any patient suspected of BCG sepsis is admitted to the hospital for monitoring and initiation of appropriate supportive and specific anti-Mycobacterial drug therapy. Further BCG treatment will be withdrawn.
  • MV261 BCG a Pasteur strain previously transfected with the kanamycin resistance plasmid pMV261 (36) was used in the in-vitro experiments. This strain had been shown to possess very similar immunostimulatory properties to that of commercial lyophilized BCG preparations.
  • This BCG strain was routinely kept at 37°C in 7H9 Middlebrook broth (Difco, Detroit, MI) supplemented with 10% albumin dextrose concentrate (5% BSA, 2% dextrose and 0.85% NaCl), 0.05 % Tween 80 (Sigma, St. Louis, MO), and 30 ⁇ g of kanamycin/ml.
  • One unit of absorbance at 600 nm for the BCG culture was calculated as 2.5 X 10 7 CFU.
  • lyophilized preparations of BCG (TheraCys; Connaught Pasteur Merieux, Ontario, Canada) was used.
  • PBMCs were prepared from buffy coat leukocytes purified on Ficoll-Paque (Pharmacia, Uppsala, Sweden). Viability by trypan blue exclusion usually exceeded 95%. PBMCs were suspended in RPMI 1640 medium containing 10% FCS and 30 ⁇ grml of kanamycin, and incubated at 37°C in a humidified 5 % CO 2 incubator at a density of 8 X 10 5 cells/200 ⁇ l/well in 96-well tissue culture plates in the presence or absence of designated doses of BCG, IFN- ⁇ 2B, or both.
  • an IFN- ⁇ 2B concentration of 1 X 10 5 IU/ml was selected to approximate the concentration applied clinically into the bladder.
  • human recombinant cytokines IL-12 (Genetics Institute, Cambridge, MA) and IL-10 (PharMingen, San Diego, CA) or neutralizing Abs: goat anti-human IL-12 (R&D Systems, Minneapolis, MN), mouse anti-human IL-10 (R&D Systems), and rabbit anti-human IFN- ⁇ (Pepro Tech, Rocky Hill, NJ) were further used to determine the immune pathway of IFN- ⁇ action.
  • the plates were incubated for 72 h and then frozen at -70°C until cytokine ELISA assays were performed.
  • Urine samples were stabilized during patient collection with a concentrated buffer containing 2 M Tris-HCl (pH 7.6), 5% BSA, 0.1 % sodium azide, and four protease inhibitors (aprotinin, pepstatin and leupeptin at 0.01 mg/ml for each and 4-(2-amino ethyl) benzenesulfonyl fluoride (AEBSF) at 0.1 mg/ml; all purchased from Sigma).
  • Tris-HCl pH 7.6
  • BSA 5% BSA
  • AEBSF 4-(2-amino ethyl) benzenesulfonyl fluoride
  • ELISA reagents including recombinant human cytokines and paired monoclonal capture and detecting Abs for the cytokines were obtained from Endogen (Cambridge, MA) for IFN- ⁇ , from Genetics Institute for IL-12, and from PharMingen for TNF- ⁇ , IL-6, and IL-10. Samples of conditioned PBMC cultures and urine collections were assayed by ELISA using a sandwich format according to the manufacturer's instructions. Cytokine concentrations were calculated in standard mass/volume format using standard curves derived from purified recombinant cytokine standards. For all of the above measured cytokines, one IU is equal to -50-100 pg of purified cytokine. Cytokines
  • Paired human and murine IFN- ⁇ Abs and a human IL-2 ELISA kit were obtained from Endogen (Boston, MA).
  • Hayashibara (Okayama, Japan) supplied polyclonal neutralizing Ab to murine IL-18, while polyclonal neutralizing Ab to human IL-18 was purchased from R&D Systems (Minneapolis, MN).
  • mice Female C57BL/6J mice were obtained at 6-8 weeks of age from The Jackson
  • Murine spleens were removed under sterile conditions, minced, filtered through a fine nylon mesh, and placed in ACK lysing buffer (0.15 M NB ⁇ Cl, 1.0 mM KHCO 3 , and 0. ImM Na ⁇ DTA, pH 7.4) to remove RBC. Pooled cells from usually three to five animals were then resuspended in complete RPMI 1640 medium containing kanamycin (30 ⁇ g/ml) and transferred at a final concentration of 1-4 X 10 6 cells/ml, depending on the experiment, to 24- or 96-well tissue culture plates containing the appropriate stimulus to be tested. Viability by trypan blue exclusion always exceeded 90%.
  • Human PBMCs were prepared from buffy coat leukocytes purified on Ficoll-Paque (Pharmacia, Uppsala, Sweden) and cultured under similar conditions as mouse splenocyte cultures. Supernatants were harvested after 72 h of stimulation unless otherwise stated and were frozen at -7CPC before batch testing in cytokine ELISAs.
  • mice were anesthetized by the i.p. administration of ketamine/xylazine/ acepromazine stock solution at a dose of 0.2 ml/10 g of body weight.
  • the stock solution was prepared by combining 1.5 ml of ketamine with 0.75 ml of xylazine and 0.5 ml of acepromazine (all products from J.A. Webster), and the resulting 2.75-ml volume was then mixed with 35.75 ml of sterile water and kept at room temperature before use. Under these conditions mice remain asleep for -1-2 h.
  • Bladders were catheterized with a 24-gauge Teflon i.v. cannula lubricated in glycerol. After aspiration of all remaining urine, 0.1 ml of drug was administered. Drug was retained by maintaining the cannula within the bladder with a 1-ml Tb syringe attached. After 1 h the cannula was removed, and mice were allowed to void normally. Mice were placed in metabolic cages overnight (15 h) with ample water but no solid food.
  • Urine was collected in a recovery tube on ice containing 0.1 ml/mouse of a 10X urine stabilizer solution (2 M Tris-HCl (pH 7.6), 5% BSA, 0.1 % sodium azide, plus 1/2 COMPLETE protease inhibitor tablet (Boehringer- Mannheim, Germany)). Mice produced an average of 0.5-1.0 ml of urine/mouse during this time. After collection the urine was spun to remove any solid debris and was stored at -70°C until batch ELISA cytokine measurements were performed.
  • 10X urine stabilizer solution 2 M Tris-HCl (pH 7.6), 5% BSA, 0.1 % sodium azide, plus 1/2 COMPLETE protease inhibitor tablet (Boehringer- Mannheim, Germany)
  • IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IL-2, IL-12 and IL-15 are also profoundly co-stimulatory alone and in combination. This is shown by the data of Figs. 6, 13A and 13B.
  • IFN- ⁇ works in part by reducing IL-10 production and increasing IL-12 production leading to a net change in favor of Thl cytokine polarity.
  • murine IFN- ⁇ is a Th2 polarizer in the murine system (Figs. 14A and 14B) and again highlights important differences between man and mouse and the absolute need to do parallel studies.
  • rBCG expressing the appropriate murine or human co-stimulatory cytokines show the expected substantial up-regulation of IFN- ⁇ during in-vitro testing. This is evidenced by the data of Fig. 8. rBCGs expressing murine GMCSF, IL-2, and IFN- ⁇ were able to substantially augment IFN- ⁇ production while the control non- expressing rBCG (261) and rBCG expressing IL-6 were not significantly different from wild type BCG.
  • cytokine rBCG The ability to adequately provide proof of principle that appropriate cytokine rBCG will be better agents for bladder cancer immunotherapy has been hampered somewhat by lack of the most active cytokine rBCGs (e.g., IL-12 rBCG and IL-18 rBCG).
  • IL-12 plus BCG is synergistic in the murine bladder tumor model (Figs. 15 and 16).
  • IP-10 secreting cytokine rBCG IP-10 is both a T cell chemokine as well as an anti- angiogenic cytokine that is induced by IFN- ⁇ does result in tumor growth delay in- vitro (Fig. 19).
  • the experiments also confirm the general principle that cytokines synergistic for IFN- ⁇ are superadditive in anti-tumor immunity. Multiple mixtures of cytokine rBCGs provide the greatest in-vivo activity. These cumulative results reveal that the best anti-tumor results may require multiple expression of synergistic cytokines from rBCG.
  • This human clinical trial included naive, untreated human patients and failed BCG patients to evaluate the immunotherapeutic efficacy and value of BCG + IFN- ⁇ as a treatment regimen.
  • One category tested were naive, human patients suffering from superficial bladder or upper urinary tract cancers.
  • the second category were failed BCG patients - those persons who previously received BCG alone as an immunostimulatory therapy on one or more occasions previously, but failed to achieve a disease-free state of any meaningful duration.
  • Table El The general patient characteristics of these persons is given by Table El below. Table E2
  • the initial therapy for very high-risk patients is a full dose BCG + IFN- ⁇ , 50 MU (million units);
  • the salvage therapy for patients who have previously failed BCG is 1/3 dose BCG + IFN- ⁇ , 50 MU;
  • the salvage therapy for BCG intolerant patients is 1/10 BCG + IFN- ⁇ , 100 MU;
  • Another clinical evaluation trial performed utilized a different patient category: a human afflicted with superficial bladder cancer whom had previously received BCG plus IFN- ⁇ - but nevertheless failed to achieve a disease-free state of meaningful duration.
  • the "failed BCG + a cytokine patient" in this clinical trial presented the following characteristics.
  • Clinical Hx (i) 76 year old male patient ("MN") with Stage TI Grade 3 TCC in bladder diverticulum s/p transurethral surgery then partial Cystectomy - no cancer found; (ii) Recurrent Stage TI Grade 3 at 3 months;
  • This patient MN then underwent a new regimen of BCG plus IL-12 treatment. Specifically, his clinical history was: (a) he received 4 treatments of low- dose BCG (1/3-1/lOth dose) plus rhTL-12 intravesically; subsequently (b) his cytology went from positive to atypical after treatment but developed metastasis to liver from a different cancer (likely bowel).
  • patient MN responded well to BCG plus IL-12 treatment despite having an inadequate response to BCG + IFN ⁇ .
  • This is shown by the in- vitro PBMC response of the patient as shown by Figs. 17 A and 22A/B respectively.
  • patient MN's urinary production of IFN-gamma mass was shown as dramatically increasing as a result of the BCG plus IL-12 immunotherapeutic treatment.
  • This urinary IFN-gamma mass data is shown by Fig. 17B.
  • Combination low-dose BCG plus interferon-alpha showed great success in patients with upper tract TCC.
  • Five of 5 patients with a total of 7 upper tracts affected by CIS manifested by positive cytologies have had complete responses to therapy with ongoing remissions at (23 + , 20+), 13 + , (11 + , 6+), 6+ and 5 + months, respectively.
  • the patient with recurrent low-grade papillary TCC despite laser ablation is disease free after 2 courses of reduced BCG plus interferon- alpha.
  • combination therapy was administered in a retrograde fashion through a temporary, small, atraumatic, 4 French external ureteral stent placed cystoscopically.
  • BCG dose ranged from 1/3, 1/10, 1/30, or 1/ 100th of the standard bladder dose in 50 cc of physiologic saline combined with 100 MU of IFN- ⁇ administered over 2 hours by gravity flow.
  • Full dose BCG 81 mg/50 ml is too viscous to flow by gravity through a 4 French ureteral stent.
  • Treatments were administered sequentially usually weekly or biweekly for a total of 6-8 treatments during the induction cycle. Additional maintenance cycles of 3 sequential treatments have also been given to some.

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Abstract

L'invention concerne une méthode immunothérapeutique large permettant de traiter un patient vivant souffrant d'un cancer superficiel de la vessie; une tentative précédente de traitement par immunostimulation n'ayant pas fonctionné chez ce patient. L'invention concerne également une méthode de traitement par immunothérapie de premier recours permettant de traiter, chez un patient vivant souffrant d'un cancer des voies urinaires supérieures, les régions de l'urètre et du bassinet du rein, une zone anatomique pour laquelle il n'existait encore aucun traitement contre le cancer. La méthode décrite dans l'invention consiste à introduire une quantité efficace d'au moins une espèce Mycobacterium viable dans un site anatomique choisi simultanément et en combinaison avec au moins un cytokine sélectif choisi parmi le groupe comprenant tout type ou isoforme d'interféron-alpha, d'interféron-beta, d'interféron-gamma, d'interleukine-1, d'interleukine-2, d'interleukine-3, d'interleukine-12, d'interleukine-15, et d'interleukine-18.
PCT/US2001/002827 2000-02-01 2001-01-29 Methode de traitement par immunotherapie destinee a des patients souffrant d'un cancer superficiel de la vessie et pour lesquels au moins un traitement therapeutique par immunostimulation n'a pas fonctionne WO2001056387A1 (fr)

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WO2005077411A2 (fr) * 2004-02-10 2005-08-25 Innate Pharma Composition et procede pour le traitement de carcinome
AU2011200580B2 (en) * 2004-06-07 2012-09-27 Qu Biologics Inc. Bacterial compositions for the treatment of cancer
US10086066B2 (en) 2004-06-07 2018-10-02 Qu Biologics Inc. Tissue targeted antigenic activation of the immune response to treat cancers
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US8034359B2 (en) 2004-06-07 2011-10-11 Qu Biologics Inc. Tissue targeted antigenic activation of the immune response to cancers
US9775896B2 (en) 2004-06-07 2017-10-03 Qu Biologics Inc. Tissue targeted antigenic activation of the immune response to treat cancers
WO2005120560A1 (fr) * 2004-06-07 2005-12-22 Harold David Gunn Compositions bacteriennes pour le traitement d'un cancer
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US9320787B2 (en) 2004-06-07 2016-04-26 Qu Biologics Inc. Tissue targeted antigenic activation of the immune response to treat cancers
WO2007126950A1 (fr) * 2006-03-31 2007-11-08 Schering Corporation Dose faible parentérale d'interférons de type 1 pour cancer de la vessie
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EP2059253A4 (fr) * 2006-09-14 2011-09-14 Univ Pennsylvania Modulation de lymphocytes t régulateurs par l'il-18 humaine
US8679471B2 (en) 2006-09-14 2014-03-25 The Trustees Of The Univesity Of Pennsylvania Modulation of regulatory T cells by human IL-18
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CN107412759A (zh) * 2006-10-27 2017-12-01 Qu生物制药公司 用于治疗癌症的免疫应答的组织定向性抗原激活
WO2008049231A1 (fr) * 2006-10-27 2008-05-02 Harold David Gunn Activation antigénique de la réponse immunitaire ciblée sur des tissus pour traiter des cancers
US10251946B2 (en) 2014-05-02 2019-04-09 Qu Biologics Inc. Anti-microbial immunomodulation
US10946083B2 (en) 2014-05-02 2021-03-16 Qu Biologies Inc. Anti-microbial immunomodulation
US11819543B2 (en) 2014-05-02 2023-11-21 Qu Biologics Inc. Anti-microbial immunomodulation
US20240252591A1 (en) * 2014-06-30 2024-08-01 Altor Bioscience, Llc Compositions comprising il-15n72d-based molecules and bacillus calmette-guerin (bcg)
US12097244B2 (en) 2014-06-30 2024-09-24 Altor BioScience, LLC. Methods of treating a neoplasia with IL-15-based molecules and CTLA-4 antibody
WO2019075452A1 (fr) * 2017-10-13 2019-04-18 Evelo Biosciences, Inc. Identification de bactéries pour la cancérothérapie

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