CANCER IMMUNE-BASED THERAPY

Abstract

The present disclosure provides a novel therapeutic vaccine approach that triggers a therapeutic antitumor response. The inventive approach is to selectively eliminate the carbohydrate sequences from tumor cells without affecting the tumor associated protein epitopes.

Claims

1. A composition comprising: a cancer cell lysate, wherein the cancer cell lysate comprises modified cancer cells that are characterized by having at least 50% of the carbohydrate functional groups thereon disrupted in comparison to a normal cancer cell from the same type of cancer as the modified cancer cell; and at least one additional component selected from the group consisting of pharmaceutically-acceptable carriers, adjuvants, diluents, preservatives, antibiotics, and combinations thereof.

2. The composition of claim 1, wherein said additional component is an adjuvant.

3. The composition of claim 3, wherein said adjuvant is AS04.

4. The composition of claim 1, wherein the disrupted carbohydrate functional groups are no longer able to function as a glycan shield.

5. The composition of claim 1, wherein the quantity of modified cancer cells is between 500,000 and 100,000,000.

6. The composition of claim 1, wherein the modified cancer cells are from a solid cancer.

7. The composition of claim 1, wherein the modified cancer cells are from a liquid cancer.

8. The composition of claim 1, wherein the modified cancer cells are from a Stage I, Stage II, Stage III, or Stage 4 cancer.

9. The composition of claim 1, further comprising a different cancer therapy or treatment.

10. The composition of claim 1, wherein the modified cancer cells are all from the same individual.

11. A method of treating cancer in a patient comprising the steps of: obtaining a sample of cancer cells; subjecting said cancer cells to a deglycosylating process wherein the cancer cells have at least 50% of the carbohydrate functional groups thereon disrupted in comparison to a normal cancer cell from the same type of cancer that has not been subjected to a deglycosylating process, thereby resulting in deglycosylated cancer cells; lysing said deglycosylated cancer cells; producing a cancer cell lysate from said lysed deglycosylated cancer cells; and administering said cancer cell lysate to the patient.

12. The method of claim 11, wherein the cancer cell lysate is combined with at least one additional component selected from the group consisting of pharmaceutically-acceptable carriers, adjuvants, diluents, preservatives, antibiotics, and combinations thereof prior to administration to the patient.

13. The method of claim 12, wherein said at least one additional component is an adjuvant.

14. The method of claim 13, wherein said adjuvant is AS04.

15. The method of claim 11, wherein the sample of cancer cells comprises between 500,000 and 100,000,000 cancer cells.

16. The method of claim 11, wherein said lysing is performed through a series of freeze/thaw cycles.

17. The method of claim 11, wherein the deglycosylating process comprises incubating said cancer cells with at least one deglycosylating agent.

18. The method of claim 11, wherein the cancer cells are from a solid cancer or from a liquid cancer.

19. The method of claim 11, further comprising the step of administering a different cancer therapy or treatment.

20. The method of claim 11, wherein said administering step comprises intradermal administration.

21. The method of claim 11, wherein said administering results at least one outcome selected from the group consisting of tumor growth impairment of at least 5%, tumors shrinking at least 5%, at least a 5% reduction in the incidence of tumor markers, at least a 5% increase in life expectancy, and combinations thereof.

Description

DRAWINGS

[0139] The drawings described herein are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0140] FIG. 1 is an x-ray showing metastatic nodule identified throughout the lung fields.

[0141] FIG. 2 is an x-ray showing the metastatic nodules identified in FIG. 1 had progressed.

[0142] FIG. 3 is an x-ray showing no metastatic disease clearly visible after treatment.

[0143] Corresponding reference numerals indicate corresponding parts throughout the several views of drawings.

DETAILED DESCRIPTION

[0144] The following description is merely exemplary in nature and is in no way intended to limit the present teachings, application, of uses. Throughout this specification, like reference numerals will be used to refer to like elements.

[0145] The disclosure teaches a novel therapeutic vaccine approach that can trigger a therapeutic antitumor response in a cancer patient or a healthy subject. The inventive approach is to selectively eliminate the carbohydrate sequences from tumor cells without affecting the tumor associated protein epitopes. The disclosure further teaches that the evidence that in humans, the expression of Lewis.sup.y, SLEX and other carbohydrate sequences increases dramatically on tumor cells near the end stage of cancer, suggests that these TACAs help to make them invulnerable to the immune response. Thus the loss of these TACAs will de-shield these tumor cells from companion dogs, revealing immunogenic molecules that will trigger a therapeutic antitumor response.

[0146] More specifically, the disclosure teaches that first, a suitable method may have to be generated to isolate tumor cells from other normal cells within the tumor. There are numerous ways to perform this type of isolation, which will likely be specific for the type of cancer cells that are targets. Once such tumor cells are isolated, it may be important first to shut down protein synthesis with an active DNA crosslinking agent like mitomycin C. An active inhibitor of glycosylation may also be necessary to globally eliminate the additional of any more glycan sequences. The famous AIDS drug AZT is an excellent inhibitor of glycosylation at a final concentration of about 20 μM. These two drugs can be given to the tumor cells, which may be incubated with a variety of different glycosidases (neuraminidase, β-galactosidase, (β- and α-N-acetylgalactosaminidase, α-fucosidase, α-mannosidase, and endo-β-galactosidase). These enzymes may be able to extensively digest the surface glycans on the tumor cells, enabling immune cell access to tumor associated epitopes that are newly exposed and for which tolerance has not been induced. The digested tumor cells may be collected and screened for the loss of specific glycan epitopes with antibodies and lectins to confirm their removal. Once deglycosylated on is achieved, cells may be injected intramuscularly or into the peritoneal cavity of cancer patients along with alum and monophoryl lipid A adjuvants.

[0147] The inventor has initiated a prospective, single arm efficacy trial of an adjuvanted deglycosylated tumor vaccine in canines. Tumor specimens (about 3 cm.sup.3) were collected surgically during treatment of naive dogs that had developed either melanoma or apocrine gland anal sac adenocarcinoma (AGASACA). In some cases, draining lymph nodes were not removed to determine the efficacy of immune activation. Tumor cells were isolated and treated with exoglycosidases and chemical oxidation procedures to remove CFGs. Successful elimination of CFGs was confirmed by lectin binding analysis of the treated cells. The tumor cells were subjected to 5× freeze-thaw cycles to kill them and generate tumor cell lysates. This lysate was mixed with AS04 adjuvant (alum, monophosphoryl lipid A) in physiological saline (0.14 M). This combination is routinely employed in vaccinations against viruses and bacteria in humans. Dogs were inoculated subcutaneously at different sites on their limbs with lysates from 7-10×10.sup.6 cells at 0, 2, 4 and 6 weeks.

[0148] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Example 1. Vaccine Immunization Therapy on a Dog with Cancer

[0149] The mass in the patient dog, a 14 year old neutered male cocker spaniel, was identified in the mouth in addition to multiple metastatic lymph nodes. A 2.5 cm.sup.3 metastatic lymph node was surgically removed and confirmed as a melanoma. Initial thoracic radiographs were unremarkable. The mass was surgically removed again and tumor cells were isolated and expanded in culture using standard methods. Tumor cells were deglycoslated as described herein. Lysates of deglycoslated tumor cells were prepared and mixed with an AS04 adjuvant system for preparation of autologous vaccine. The patient dog was vaccinated with the resulting autologous vaccine four times at 2 week intervals (0, 2, 4, and 6 weeks). Each inoculation was performed intradermally over the right popliteal region. Each dose contained lysates from 7 million deglycoslated tumor cells and 550 mg of AS04 suspended in 0.5 ml of physiological saline.

[0150] Refer to FIG. 1. Radiographs of the thorax on before first surgical intervention revealed the development of metastatic disease in the lung fields.

[0151] Refer to FIG. 2. Radiographs of the thorax on before surgical harvest of the metastatic lymph node revealed progression of metastatic disease.

[0152] Refer to FIG. 3. Radiographs of the thorax after the fourth inoculation revealed resolution of metastatic disease. No other therapy was administered to this dog.

Example 2. Preparation of an Adjuvanted, Deglycosylated Autologous Tumor Vaccine from Canine Tumor Cells

[0153] Melanoma or AGASACA tumors or metastatic nodes (about 3-4 cm.sup.3) were surgically excised from the canine patient and placed in a sterile tube containing chilled RPMI 1640 media (Gibco) on ice and immediately transferred to a tissue culture suite. Tumor cells were separated from non-tumor cell types (e.g., blood cells, fibroblasts, supportive cell types) and connective tissues using the Miltenyi Biotec tumor dissociation kit (for human tumor cells) in conjunction with a gentleMACS Dissociator. The resulting dissociated tumor cells were plated out in RPMI 1640 media (Gibco) containing 10% fetal calf serum (Sigma-Aldrich), 100 units of penicillin-streptomycin mixture/ml (Gibco), Glutamax (L-alanyl-L-glutamine; 1 ml/100 ml of media; Gibco) and Fungizone (amphotericin B (1 ml/500 ml)). Tumor cells were allowed to adhere to the plate, while other cells stayed in suspension. After three days, nonadherent contaminating cells were removed, leaving about 500,000-1 million tumor cells/cm.sup.3 tumor tissue. These tumor cells were seeded into two T-75 flasks. The tumor cells were expanded in culture until about 35-40 million tumor cells were obtained.

[0154] To deglycosylate the tumor cells, cells were suspended in new media (2 million cells/nil) supplemented with 5 μg/mltunicamycin (Sigma-Aldrich), 2 mM benzyl 2-acetamido-2-deoxy-α-D-galactopyrano side (Sigma-Aldrich) and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (Matreya LLC) to inhibit N-glycosylation, O-glycosylation and glycosphingolipid synthesis, respectively. The media was also supplemented with the following glycosidases: neuraminidase from Clostridium perfringens (New England BioLabs, 100 units/ml), β1-4 galactosidase from Streptococcus pneumoniae (Prozyme, 10 mU/ml) and endo-β-galactosidase (QA-Bio, 30 mU/ml). The tumor cells were incubated for 3 hours, centrifuged (500×g, 10 minutes), and washed with physiological saline (0.85% NaCl). They were then resuspended in 50 mM sodium phosphate buffer (pH 6.0) containing 0.15 M NaCl and freshly prepared 50 mM sodium-m-periodate. The cells were incubated for 1 hour in the dark at 23° C. Periodate oxidation was terminated by the addition of 50 (0.1.1 of ethylene glycol/ml of buffer.

[0155] Deglycosylated tumor cells were next pelleted by centrifugation (500×g, 10 minutes) resuspended in physiologic saline and transferred to a cryovial. The cells were pelleted again by centrifugation. The cells were subjected to five freeze-thaw cycles (−80° C., 37° C.) to break the cellular membrane and generate lysates.

[0156] Tumor cell lysates were mixed with 2.2 mg of Adjuvant System 04 (AS04). AS04 is a mixture of Alhydrogel® adjuvant 2% and 220 μg of synthetic monophosphoryl lipid A that induces a transient localized innate immune response that enhances adaptive immunity in humans. The lysate and AS04 were suspended in sterile physiological saline to a final volume of 2.2 ml. This vaccine was immediately refrigerated and stored at 4° C.

Example 3. Assessment of an Adjuvanted, Deglycosylated Autologous Tumor Vaccine on Metastatic Progression in Canines

[0157] Four dogs with naturally-occurring oral melanoma were enrolled into a single-arm trial to assess the efficacy of an adjuvanted, deglycosylated autologous tumor vaccine. To be included in the study, dogs could be of any sex but a minimum weight of 10 kg was required. Dogs in the study had an oral melanoma lesion of at least 2 cm in longest diameter or metastatic lymph nodes that could be surgically resected for vaccine preparation. Dogs could be of any stage of disease progression, but a performance score of 0 or 1 with a minimum estimated life expectancy of 12 weeks was required for inclusion. Dogs could not be receiving immunosuppressive therapy. Metastatic lesions could not have been treated with radiation therapy. Dogs with melanoma could not have received the ONCEPT® vaccine prior to enrollment or during the trial. Prior chemotherapy was allowed with a three week washout prior to trial enrollment. Comorbidities, including kidney disease with azotemia, liver disease or hepatic enzymopathy greater than twice the upper limit of basal state, or chronic infection were not allowed.

[0158] Prior to the start of therapy, dogs were evaluated by thoracic radiographs and draining lymph node aspirate for accurate staging. Abdominal ultrasound was performed at clinician discretion to evaluate for abdominal metastasis as indicated. Dogs underwent surgery at the start of the trial to resect the tumor, with clear margins where possible. Tumor cells were isolated from a portion of the tumor and treated with exoglycosidases and chemical oxidation procedures to remove carbohydrate functional groups (CFGs). Successful elimination of CFGs was confirmed by lectin binding analysis of the treated cells. The tumor cells were subjected to five freeze-thaw cycles to kill the cells and generate tumor cell lysates. The resulting lysate was mixed with AS04 adjuvant (alum, monophosphoryl lipid A) in physiological saline. Dogs were inoculated subcutaneously at different sites on their limbs with lysates from 7-10×10.sup.6 cells at 0, 2, 4 and 6 weeks. Prior to the first vaccine administration, and at each subsequent administration, then monthly thereafter, blood was collected for humoral and cellular assays to evaluate tumor-specific immune response. Thoracic radiographs were repeated every 2 months until 6 months, then every 3 months thereafter. Draining lymph nodes was monitored by palpation and aspiration on the same schedule as thoracic radiographs are made.

[0159] None of the treated dogs developed adverse events following vaccination. As shown in Table 1, the metastasis was resolved in three of the four dogs. Of those three dogs, the average age of survival was 447±108 days following diagnosis.

TABLE-US-00001 Date of Node Distant Stage of Outcome of Date of Dog Diagnosis Diagnosis Metastasis Metastasis Diagnosis Metastases Death Survival 1 Caudal Aug. 5, 2014 + + IV Progressive Oct. 15, 2014 71 Mandible Aug. 5, 2014 Sep. 11, 2014 Melanoma 2 Left Apr. 27, 2015 + + II Resolved Feb. 4, 2017 646 Mandible May 1, 2015 Aug. 12, 2015 progressed Amelanotic to IV Melanoma 3 Left Dec. 15, 2015 + − III Resolved Alive 423 Cervical Dec. 15, 2015 Lymph Node Metastatic Melanoma 4 Right May 23, 2016 − − II None Alive 273 Maxillary Melanoma

[0160] The description herein is merely exemplary in nature and, thus, variations that do not depart from the gist of that which is described are intended to be within the scope of the teachings. Such variations are not to be regarded as a departure from the spirit and scope of the teachings.