WO2013158611A1

WO2013158611A1 - Methods and compositions for the treatment of glioblastomas

Info

Publication number: WO2013158611A1
Application number: PCT/US2013/036732
Authority: WO
Inventors: Daniel L. Levey; Kerry WENTWORTH; Cristina MUSSELLI; Marcel Rozencweig
Original assignee: Agenus Inc.
Priority date: 2012-04-16
Filing date: 2013-04-16
Publication date: 2013-10-24

Abstract

The invention, according to some aspects, relates to methods and compositions for the treatment of glioblastomas.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF GLIOBLASTOMAS

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 61/798,259, filed on March 15, 2013, entitled "METHODS AND COMPOSITIONS FOR THE TREATMENT OF GLIOBLASTOMAS," and U.S. Provisional Patent Application Serial No. 61/625,055, filed on April 16, 2012, entitled "METHODS AND COMPOSITIONS FOR THE TREATMENT OF GLIOBLASTOMAS," the contents of each of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to methods and compositions for treating cancer.

BACKGROUND OF THE INVENTION Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans, involving glial cells and accounting for 52% of all functional tissue brain tumor cases and 20% of all intracranial tumors. Despite being the most prevalent form of primary brain tumor, GBMs occur in only 2-3 cases per 100,000 people in Europe and North America. According to the WHO classification of the tumors of the central nervous system, the standard name for this brain tumor is "glioblastoma"; it presents two variants: giant cell glioblastoma and gliosarcoma. Treatment can involve chemotherapy, radiation, radiosurgery, corticosteroids, antiangiogenic therapy, and surgery. With the exception of the brainstem gliomas, glioblastoma has the worst prognosis of any central nervous system (CNS) malignancy.

SUMMARY OF THE INVENTION Aspects of the invention relate to methods for inhibiting recurrence of a Glioblastoma Multiforme (GBM) in a subject. In some embodiments, the GBM is a surgically resectable recurrent GBM. In some embodiments, the methods involve administering to the subject an autologous tumor-derived heat-shock protein peptide complex-96 (HSPPC-96), in which the HSPPC-96 comprises gp96 in complex with peptides derived from a GBM tumor obtained from the subject; and administering to the subject a VEGF inhibitor (e.g. , an anti-VEGF antibody (e.g. , bevacizumab)).

In other aspects, the invention relates to methods of treating a subject having a

Glioblastoma Multiforme (GBM). In some embodiments, methods provided herein are useful as a first-line therapy for GBM. However, in some embodiments, methods provided herein are useful for treating a subject who has previously received a different therapy (e.g., treatment with temozolomide, radiation, surgery, etc.) for a GBM. In some embodiments, methods provided herein are useful for treating a subject who failed to respond to a prior therapy for a GBM.

In some embodiments, the GBM is a surgically resectable GBM (e.g., a surgically resectable recurrent GBM). In some embodiments, the methods involve resecting at least a portion of a GBM tumor from the subject; preparing (e.g. , purifying) an autologous tumor- derived heat-shock protein peptide complex-96 (HSPPC-96) from resected GBM tumor tissue; administering to the subject the autologous tumor-derived HSPPC-96; and administering to the subject bevacizumab.

In some embodiments, the subject to be treated with the combination is administered HSPPC-96 concomitantly with bevacizumab. In some embodiments, the subject to be treated with the combination is administered HSPPC-96 alone followed by bevacizumab at progression (e.g. , at detection of tumor recurrence by standard tumor detection methodologies). In some embodiments, the subject to be treated with the combination is administered bevacizumab alone followed by HSPPC-96 (e.g. , HSPPC-96 administered at progression.) In other aspects, the invention relates to methods of preparing an autologous tumor- derived heat-shock protein peptide complex-96 (HSPPC-96). In some embodiments, the methods involve obtaining a sample of a GBM tumor resected from a subject, in which 90% or more of the GBM tumor was resected from the subject; and isolating HSPPC-96 from the 5 sample, wherein the HSPPC-96 comprises the heat shock protein gp96 complexed with peptides from the GBM tumor.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 provides a schematic of a protocol for evaluating efficacy of heat shock proteinic) peptide complex-96 (HSPPC-96) vaccine in combination with bevacizumab (Avastin®) in the therapy of surgically resectable recurrent Glioblastoma Multiforme (GBM).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Aspects of the invention relate to the recognition that while surgical removal of recurrent

15 glioblastoma (GBM) tumors is common practice, the resulting benefit is limited when it comes to extending survival. Further aspects of the invention relate to the recognition that existing therapeutic options are also limited post-surgery for GBM tumors. Accordingly, in some aspects of the invention, resected tumors are utilized to create a personalized, highly multivalent vaccine. In some embodiments, methods are provided for treating subjects (patients) who have

20 surgically resectable recurrent Glioblastoma Multiforme (GBM). In some embodiments,

methods are provided for treating subjects (e.g. , humans, patients) who have residual disease after resection of the GBM tumors. In some embodiments, these subjects are treated with Heat- Shock Protein Peptide Complex-96 (HSPPC-96) in combination with bevacizumab . Heat-Shock Protein Peptide Complex-96 (HSPPC-96)

In some embodiments, the multivalent vaccines comprise autologous tumor-derived heat-shock protein peptide complex-96 (HSPPC-96). HSPPC-96 is an autologous tumor derived vaccine comprising the 96-kDa heat shock protein gp96 in complex with autologous tumor derived peptides. HSPs are highly conserved, abundant, nonpolymorphic stress protein physiologically expressed in every cell. They have the function of chaperoning proteins and peptides intracellularly within different compartments; hence they bind to the intrinsic antigenic repertoire of a cell, which can be defined as the antigenic fingerprint. HSPPC-96 preparations activate T cells responses to the chaperoned peptides and to the tumors from which the complexes were derived in animal models and in human cancer patients. The specific immunogenicity and antitumor activity of this complex has been demonstrated in preclinical models, both in prophylaxis and therapy settings as well as in clinical trials. To date, results from single arm Phase 1 and 2 clinical trials in recurrent glioblastoma have shown the vaccine to be well tolerated and to be immunogenic inducing both activation of the innate as well as adaptive immune response. Relative to historical controls the vaccine also appears to provide clinical benefit as measured by overall survival.

In some embodiments, HSPPC-96 for clinical use comprises the 96-kDa heat shock protein gp96 in complex with autologous tumor-derived peptides. In some embodiments, HSPPC-96 is supplied in a single -use vial as a clear, colorless solution. In some embodiments, it is formulated in a 9% sucrose-potassium phosphate for intradermal (ID) injection. In some embodiments, each vial contains 25 μg of HSPPC-96 in a solution of 9% sucrose-potassium phosphate for intradermal (ID) injection. In some embodiments, the total volume of each vial of HSPPC-96 is 0.47 mL. In some embodiments, the total volume that is administered is 0.4 mL. In some embodiments, HSPPC-96 is administered at dose in a range of 1 μg to 25 μg.

In some embodiments, each vaccine vial is labeled with the batch number, patient number, patient initials, and patient date of birth (DOB). In some embodiments, following production of vaccine, vials are shipped to a clinical site on dry ice and are stored at -80°C ± 20°C until administration to the patient.

In some embodiments, the total volume of HSPPC-96 within a vial provided to a clinical site is 0.47 mL (this volume includes a 0.07 mL overage). In some embodiments, the total volume administered is 0.4 mL (0.07 mL overage). In some embodiments, the contents may be drawn up into a 1-mL hubless (or with small hub) tuberculin or insulin syringe without bubbles and promptly injected intradermally using an appropriate intradermal needle. In some embodiments, the injection may be given into 1 site or into 2 adjacent sites (0.2 mL each) a few centimeters apart. Syringes with slip-tip detachable needles or luer hubs that hold back greater than 0.1 mL should not be utilized.

In some embodiments, the appropriate sites for vaccination include the anterior deltoid regions, subclavicular region bilaterally, and medial inguinal regions of the upper thighs. In some embodiments, the HSPPC-96 is not administered to areas distal to lymph node basins that have been resected or in areas just distal to a surgical scar. In some embodiments, the injection sites are changed or rotated among multiple injections so injections are not repeated at the same site at 2 consecutive administrations and all potential sites are used for the patient before repeating injections at a previously used injection site.

Bevacizumab

According to some aspects of the invention, HSPPC-96 is used in combination with bevacizumab. Bevacizumab is a recombinant humanized anti-VEGF monoclonal antibody, consisting of 93% human and 7% murine amino acid sequences. The agent is composed of human IgG framework and murine antigen-binding complementarity-determining regions, bevacizumab blocks the binding of vascular endothelial growth factor (VEGF) to its receptors resulting in inhibition of angiogenesis. Bevacizumab is typically supplied as a clear to slightly opalescent, sterile liquid for parenteral administration and is supplied as a 100 mg per 4 mL single-use vial as well as a 400 mg per 16 mL single-use vial. Each glass vial contains bevacizumab with phosphate, trehalose, polysorbate 20, and Sterile Water for Injection USP. In some embodiments, bevacizumab is stored in a refrigerator (2°C to 8°C) and remains refrigerated until just prior to use. In some embodiments, solutions diluted for infusion may be stored in the refrigerator for up to 8 hours.

In some embodiments, bevacizumab is provided in vials intended for single use only. In some embodiments, the calculated dose of bevacizumab is diluted in 100 mL of 0.9% Sodium Chloride for Injection. In some embodiments, once diluted in 0.9% Sodium Chloride for Injection, the bevacizumab solution is administered within 8 hours.

In some embodiments, bevacizumab is administered as an intravenous infusion. In some embodiments, an initial dose of bevacizumab is administered over a minimum of 90 minutes. In such embodiments, if no adverse reactions occur after the initial dose, the second dose may be administered over a minimum of 60 minutes. In such embodiments, if no adverse reactions occur after the second dose, subsequent doses may be administered over a minimum of 30 minutes. However, in some embodiments, if infusion-related adverse reactions occur, subsequent infusions may be administered over the shortest period that was well tolerated.

HSPPC-96 in combination with Bevacizumab Aspects of the invention relate to methods for treating subjects (patients) who have surgically resectable recurrent Glioblastoma Multiforme (GBM). Further aspects of the invention relate to methods for treating subjects (patients) who have residual disease after resection of the GBM tumors. In some embodiments, the subjects are treated with HSPPC-96 in combination with bevacizumab .

In some embodiments, the subject to be treated with the combination is administered HSPPC-96 concomitantly with bevacizumab. In some embodiments, the subject to be treated with the combination is administered HSPPC-96 alone followed by bevacizumab at progression (e.g. , at detection of tumor recurrence by standard tumor detection methodologies). In some embodiments, the subject to be treated with the combination is administered bevacizumab alone followed by HSPPC-96 (e.g. , HSPPC-96 administered at progression.)

In some embodiments, a subject is administered HSPPC-96 starting within 26 to 30 days post-surgical resection of a GBM tumor, and is administered bevacizumab concomitantly, e.g. , starting within 26 to 30 days post-surgery. In such embodiments, the subject may be administered 6 to 12 doses of HSPPC-96. In such embodiments, the HSPPC-96 doses may be administered weekly for the first 4 doses and then bi-weekly for 2 to 8 additional doses. In such embodiments, bevacizumab administration begins within 26 to 30 days post-surgery. In such embodiments, bevacizumab is administered at a dose of 10 mg / kg by intravenous

administration. In such embodiments, bevacizumab may be administered every 2 weeks until disease progression. In some embodiments, when HSPPC-96 doses are administered bi-weekly, bevacizumab may be administered bi-weekly on alternative weeks such that a patient receives HSPPC-96 one week and bevacizumab the following week followed by a repeat of this cycle until HSPPC-96 vaccine supply is depleted. In some embodiments, a subject is administered HSPPC-96 alone starting within 26 to 30 days post-surgical resection of a GBM tumor, and if and when the subject progresses (e.g., if and when recurrence of a GBM tumor is detected), the subject is administered bevacizumab. In such embodiments, bevacizumab is administered at a dose of 10 mg / kg by intravenous administration. In such embodiments, bevacizumab may be administered every 2 weeks until further disease progression or death. In such embodiments, the subject may be administered 6 to 12 doses of HSPPC-96. In such embodiments, the HSPPC-96 doses may be administered weekly for the first 4 doses and then bi-weekly for 2 to 8 additional doses. In such

embodiments, subjects having remaining HSPPC-96 vaccines doses at the point of progression, e.g., subjects who have not received up to 6 to 12 HSPPC-96 vaccines doses, may continue to receive HSPPC-96 vaccines doses.

Cyclophosphamide

Cyclophosphamide (Elostan, Cytoxan), is a chemotherapeutic agent that has an immunomodulatory function when used at low dose, e.g., 300 mg/m given before vaccine administration. Specifically, cyclophosphamide can reduce the number and proliferative capacity of regulatory T cell (Treg) cells, e.g., CD4+CD25+FoxP3+ cells or, alternatively, CD45+CD3+CD4+CD8-FOXP3+CD25hiCD1271ow cells which otherwise can inhibit the effectiveness of vaccines. Thus, in some embodiments, cyclophosphamide is administered prior to vaccine administration according to methods herein.

B7-H1

B7 homolog 1 (B7-H1) also known as cluster of differentiation (CD274) or programmed cell death 1 ligand 1 (PD-L1) is a 40kDa type 1 transmembrane protein that in humans is encoded by the CD274 gene. B7-H1 is expressed on antigen presenting cells including CD14+ monocytes and is a negative regulator of T-cell function. B7-H1 has also been shown to be expressed on the surface of a variety of cancer cells. B7-H1 interacts with its receptor, PD1, expressed on T cells. Normally the immune system reacts to foreign antigens where there is some accumulation in the lymph nodes or spleen which triggers a proliferation of antigen- specific CD8+ T cell. The formation of PD-1 receptor / PD-L1 ligand complex transmits an inhibitory signal which reduces the proliferation of these CD8+ T cells in the lymph nodes and supplementary to that PD- 1 is also able to control the accumulation of antigen specific T cells in the lymph nodes through apoptosis which is further mediated by a down regulation of the gene Bcl-2.

In some embodiments, B7-H1 -mediated immunoresistance may be attenuated by inhibitors of the PI(3) kinase pathway or direct inhibitors of B7-H1 protein expression or function. In some embodiments, B7-H1 -mediated immunoresistance may be attenuated through the use of anti-B7-Hl antibodies that inhibit binding of B7-H1 to its receptor (e.g., program death 1 (PD-1) receptor). In some embodiments immunoresistance may be attenuated through the use of anti-PD-1 antibodies.

Surgical resection

Generally subjects undergo standard surgical resection of intracranial tumor prior to treatment. In some embodiments, a surgeon or surgical pathologist dissects the specimen in a sterile fashion. In some embodiments, a pathologist or surgeon assesses the viability of the sample and confirm histology as GBM. In such embodiments, tissue is sent for vaccine if it is histologically confirmed as GBM, necrotic, or contains cystic degeneration. Sections of viable tissues are retained until shipment for vaccine production. In some embodiments, imaging is performed after surgical resection to evaluate the percentage of tumor resected. Collection of tissue for biomarker analyses may also be performed as disclosed herein.

Subjects to be treated

The term "subject," as used herein, generally refers to a mammal. Typically the subject is a human. However, the term embraces other species, e.g., pigs, mice, rats, dogs, cats, or other primates. In certain embodiments, the subject is an experimental subject such as a mouse or rat. The subject may be a male or female. The subject may be an infant, a toddler, a child, a young adult, an adult or a geriatric. In some embodiments, the subject has or is suspected of having a GBM tumor. In some embodiments, a subject to be treated with the combination undergoes or has underwent a surgery to remove a glioblastoma.

In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has had >90 of a GBM tumor resected prior to the treatment. In some embodiments, subject to be treated with HSPPC-96 in combination with bevacizumab has had >80 of a GBM tumor resected prior to the treatment. In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has had >70 of a GBM tumor resected prior to the treatment. In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has had >60 of a GBM tumor resected prior to the treatment.

In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has a confirmed histological diagnosis of GBM.

In some embodiments, a subject to be treated has not had radiotherapy within 6 months._, within 4 months._, within 3 months._, within 2 months or within 1 month prior to administration of the combination. In some embodiments, a subject to be treated has not had a prior treatment with an anti- angiogenic agent targeting the VEGF pathway.

In some embodiments, a subject to be treated has not had a prior treatment with HSPPC- 96 or other immunotherapy.

In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab is a subject who has a histologically confirmed Glioblastoma Multiforme, and who has been treated previously with radiotherapy and/or temozolomide.

In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has not received a treatment with vincristine, nitrosureas, procarbazine, temozolomide, other chemotherapy, and/or any investigational agent within 16 weeks, 12 weeks, 8 weeks, 6 weeks, 4 weeks, or 2 weeks prior to the treatment with HSPPC-96 and bevacizumab.

In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has not received a prior adjuvant therapy.

In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has a Karnofsky Performance Status (KPS) greater than 70.

In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has a granulocyte count of >1,500/μ1. In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has a platelet count of >100,000/μ1. In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab is not lymphopenia In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has serum creatinine levels of < 1.5mg/dl. In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab has bilirubin levels that are less than or equal to 1.5 times normal upper limits of clinically normal bilirubin levels, and/or Calculated Creatinine Clearance (CCC) levels that are less than or equal to 2.5 times normal upper limits of clinically normal CCC levels.

In some embodiments, a subject to be treated with HSPPC-96 in combination with bevacizumab is less than 55 years of age. In some embodiments, a subject to be treated with the combination is 55 years of age or older.

Immune Response Evaluation

Subjects treated with the cancer vaccine may be tested for an anti-tumor immune response. In this regard, peripheral blood from patients may be obtained and assayed for markers of anti-tumor immunity. Using standard laboratory procedures, leukocytes may be obtained from the peripheral blood and assayed for frequency of different immune cell phenotypes, HLA subtype, and function of anti-tumor immune cells.

The majority of effector immune cells in the anti-tumor response are CD8+ T cells and thus are HLA class I restricted. Using immunotherapeutic strategies in other tumor types, expansion of CD8+ cells that recognize HLA class I restricted antigens is found in a majority of patients. However, other cell types are involved in the anti-tumor immune response, including, for example, CD4+ T cells, and macrophages and dendritic cells, which may act as antigen- presenting cells in the CNS. Populations of T cells (CD4+, CD8+, Treg cells), macrophages, and antigen presenting cells may be determined using flow cytometry with the HLA subtype of CD8+ T cells determined by a complement-dependent microcytotoxicity test.

To determine if there is an increase in anti-tumor T cell response, an enzyme linked immunospot assay may be performed to quantify the IFNy-producing peripheral blood mononuclear cells (PBMC). This technique provides an assay for antigen recognition and immune cell function. In some embodiments, subjects who respond clinically to the vaccine may have an increase in tumor- specific T cells and/or IFNy-producing PBMCs. In some embodiments, immune cell frequency is evaluated using flow cytometry. In some embodiments, HLA- subtype is evaluated using complement-dependent microcyto toxicity test. In some embodiments, antigen recognition and immune cell function is evaluated using enzyme linked immunospot assays.

Radiology examinations

In some embodiments, radiologic tumor evaluations are performed one or more times throughout a treatment to evaluate tumor size and status. For example, tumor evaluation scans may be performed within 30 days prior to surgery, within 48 hours after surgery (e.g., to evaluate percentage resection), 1 week (maximum 14 days) prior to the first vaccination (e.g., as a baseline evaluation), and approximately every 8 weeks thereafter for a particular duration. MRI or CT imaging may be used. Typically, the same imaging modality used for the baseline assessment is used for each tumor evaluation visit.

Assessment and Monitoring of Subjects

In some embodiments, a panel of assays may be performed to characterize the immune response generated to HSPPC-96 given in combination with bevacizumab. In some

embodiments, the panel of assays includes one or more of the following tests: whole blood cell count, absolute lymphocyte count, monocyte count, percentage of CD4⁺CD3⁺ T cells, percentage of CD8⁺CD3⁺ T cells, percentage of CD4⁺CD25⁺FoxP3⁺ regulatory T cells and other phenotyping of PBL surface markers, intracellular cytokine staining to detect proinflammatory cytokines at the protein level, qPCR to detect cytokines at the mRNA level and CFSE dilution to assay T cell proliferation. In some embodiments, the level of B7-H1 in cells of a subject, e.g., circulating monocytes (e.g., CD14⁺ monocytes), is a biomarker for the likelihood that a subject will respond to a vaccination (e.g., HSPPC-96 vaccination). Additionally, the level of B7-H1 expression expressed by tumor cells is a biomarker for the likelihood that a subject will respond to vaccination with HSPPC-96. Accordingly, in some embodiments, the level of B7-H1 in cells of a subject may be used for determining whether a subject is a candidate for a vaccination. In some embodiments, subjects having relatively high levels of B7-H1 expression compared with normal subjects (e.g., subjects who have not previously had or who have not previously been diagnosed with a glioma or a glioblastoma) are not candidates for treatments comprising a heat shock protein based cancer vaccine (e.g., HSPPC-96 vaccination). Similarly, in some embodiments, subjects having relatively low levels of B7-H1 expression compared with normal subjects are candidates for treatments comprising a heat shock protein based cancer vaccine (e.g., HSPPC-96 vaccination). In some embodiments, B7-H1 levels are assessed on isolated circulating monocytes by, e.g., FACS analysis using anti-B7-Hl antibody. In some

embodiments, subjects having circulating monocytes of which less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60% ₄ less than 70% ₄ or less than 80% are B7-H1 positive are identified as candidates for treatment according to the methods disclosed herein.

In some embodiments, B7-H1 levels are assessed on tumor tissue, by, e.g.

immunohistochemistry using anti-B7-Hl antibody. Tumor tissues or cells are considered positive for B7-H1 if there is histologic evidence of cell-surface membrane staining with B7-H1 antibody. In some embodiments, subjects having tumor tissue or cells in which less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60% ₄ less than 70% ₄ or less than 80% of cells stained by immunohistochemistry are B7-H1 positive are identified as candidates for treatment according to the methods disclosed herein.

In some embodiments, increased B7-H1 expression correlates with increased PI(3) kinase activation. Accordingly, PI(3)kinase activation may, in some embodiments, provide a surrogate or complementary biomarker for B7-H1 expression. Accordingly, in some

embodiments, subjects having relatively high levels of PI(3) kinase pathway activity (e.g., in circulating monocytes or tumor tissue) compared with normal subjects are not candidates for treatments comprising a heat shock protein based cancer vaccine (e.g., HSPPC-96 vaccination). In some embodiments, subjects having relatively low levels of PI(3) kinase pathway activity compared with normal subjects (e.g., subjects who have not previously had, or who have not previously been diagnosed with, a glioma or a glioblastoma) are candidates for treatments comprising a heat shock protein based cancer vaccine (e.g., HSPPC-96 vaccination).

In some embodiments, the immune status in peripheral blood samples is assessed to identify subjects who are candidates for treatments comprising HSPPC-96 vaccination. The immune status is evaluated by measuring, e.g., whole blood cell count, absolute lymphocyte count, monocyte count, percentage of CD4⁺CD3⁺ T cells, percentage of CD8⁺CD3⁺ T cells, percentage of CD4⁺CD25⁺FoxP3⁺ regulatory T cells and other phenotyping of PBL surface markers. The cell counts that would indicate subjects are eligible for treatments comprising HSPPC-96 vaccination are whole blood cell count (expressed as x 10⁹/L) of, e.g., 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5 or 11; absolute lymphocyte count (expressed as x 10⁹/L) of, e.g., 0.7, 1.0, 1.3, 1.9, 2.2, 2.5, 2.8, 3.1, 3.4, 3.7, 4.1, 4.4 or 4.8; or monocyte count

(expressed as x 10⁹/L) of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8.

In some embodiments, at the time that sections of tumor tissue are taken for pathological confirmation of GBM, a small section may also be allocated to perform measurements of B7-H1 expression and PI3 kinase activation levels. In some embodiments, where a post-progression biopsy is obtained, a sample of tumor tissue may be evaluated for B7-H1 expression and PI(3) kinase activation levels.

In evaluating a subject, a number of other tests may be performed to determine the overall health of the subject. For example, blood samples may be collected from subjects and analyzed for hematology, coagulation times and serum biochemistry. Hematology for CBC may include red blood cell count, platelets, hematocrit, hemoglobin, white blood cell (WBC) count, plus WBC differential to be provided with absolute counts for neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Serum biochemistry may include albumin, alkaline phosphatase, aspartate amino transferase, alanine amino transferase, total bilirubin, BUN, glucose, creatinine, potassium and sodium. Protime (PT) and partial thromboplastin time (PTT) may also be tested. One or more of the following tests may also be conducted: anti-thyroid (anti-microsomal or thyroglobulin) antibody tests, assessment for anti-nuclear antibody, and rheumatoid factor. Urinalysis may be performed to evaluated protein, RBC, and WBC levels in urine. Also, a blood draw to determine histocompatibility leukocyte antigen (HLA) status may be performed.

EXAMPLES

Example 1: Evaluating efficacy of heat shock protein-peptide complex-96 (HSPPC-96) vaccine in combination with bevacizumab (Avastin®) in the therapy of surgically resectable recurrent Glioblastoma Multiforme (GBM).

Patients with completely resected GBM (95% or greater resection of the enhancing tumor volume) are evaluated to determining efficacy of heat shock protein-peptide complex-96 (HSPPC-96) vaccine in combination with bevacizumab (Avastin®) in the therapy of surgically resectable recurrent Glioblastoma Multiforme (GBM). Clinical benefit of bevacizumab in patients who have had a complete resection of the enhancing GBM recurrence is evaluated.

Single agent activity of HSPPC-96 vaccination and the role of bevacizumab for patient population with completely resected GBM recurrences is assessed as follows. Compare a Gliadel (Carmustine) or placebo arm as the standard treatment (the control arm) to a vaccination alone arm (the experimental arm)— with provision of bevacizumab administration at progression in both arms. Alternatively, use surgical resection plus Gliadel as the standard treatment in both arms as Gliadel is approved for this indication; the control arm uses vaccine placebo and is compared to the experimental arm using vaccine in addition to surgery and Gliadel.

Alternatively, use a third treatment arm with vaccination alone as upfront treatment and administer bevacizumab at progression: this has the advantage of being able to evaluate activity of vaccination by itself by measuring PFS, and evaluates sequential administration of two treatments.

FIG. 1 provides a schematic of a protocol for evaluating efficacy of heat shock protein- peptide complex-96 (HSPPC-96) vaccine or placebo in combination with bevacizumab

(Avastin®) in the therapy of surgically resectable recurrent Glioblastoma Multiforme (GBM).

Example 2: Evaluating efficacy of heat shock protein-peptide complex-96 (HSPPC-96) vaccine in combination with bevacizumab (Avastin®) in the therapy of patients with residual disease after resection of the GBM recurrence.

Patients with measurable or evaluable disease after resection are evaluated to

determining efficacy of heat shock protein-peptide complex-96 (HSPPC-96) vaccine in combination with bevacizumab (Avastin®) in the therapy of surgically resectable recurrent Glioblastoma Multiforme (GBM). Completely resected patients can be evaluated, in which case the extent of resection can be used as a stratification factor.

Example 3: Evaluation of a GP96 + anti-VEGF Ab in mouse model This example outlines a mouse model for assessing efficacy of HSPPC-96 vaccine in combination with bevacizumab (Avastin®) for treating GBM in a first line or recurrent disease setting.

Tumor initiation

Mouse tumor cells (e.g., GL261 cells) are injected intradermally or subcutaneously into flanks of mice.

Treatment scheme 1

When tumors reach a predetermined diameter (e.g., about 0.5, 1.0 or 1.5cm), surgery is performed to resect nearly all tumor tissue (e.g., about 90%, about 95%, about 99% or more of the tumor tissue). Treatment with HSPPC-96 and anti-VEGF Ab (e.g., R&D Systems mouse VEGF 164 affinity purified polyclonal Ab, catalog no. AF-493-NA) is commenced after tumor resection.

Treatment scheme 2

Not tumor resection is performed. Treatment begins when tumor is just palpable (e.g. 2.0, 2.5 or 3mm diameter). The table below describes how human glioma derived HSPPC-96 and bevacizumab are dosed in a human clinical trial (left hand column) and how mouse glioma derived HSPPC-96 and anti- mouse VEGF antibody are dosed in a mouse preclinical model (right hand column).

M iiiiKHi cliiiionl trial protocol parameter Mouse dl.,261 model equiv alent parameter

Patient profile: recurrent or newly diagnosed GBM Scheme 1 or 2 above

in brain; tumor is essentially completely resected

Commences 26-30 days following resection Commences 2-14 days following resection for schemes 1 or when tumor is palpable for scheme 2.

Dose: 2.5, 5, 10 or 25 μg Dose: 25 μg

[may also assess lower dose in some embodiments, e.g., 5 μg, 10 μ§, or 20 μg]

sease progress on wee s

Tumors are measured bi-weekly with calipers and the longest and shortest tumor diameters are recorded. Survival of mice (in days, weeks or months) is also recorded. An alternative method for tumor initiation involves implanting a GL261 cell suspension into the frontal lobes of mice using an automated microsyringe and a stereotactic mouse frame.

Treatment with HSPPC-96 and anti-mouse VEGF antibody commences 1, 2, 3, 4, 5, 6 or 7 days after tumor cell implantation according to the doses, routes, schedule and regimens specified in the table above.

The methods may optionally involve administering cyclophosphamide prior to the first vaccination. Cyclophosphamide (Elostan, Cytoxan), is a chemotherapeutic agent that has an immunomodulatory function when used at low dose. Specifically, the modulatory effect acts upon Treg cell population and immune-suppressive network when used as a single low dose

(e.g., in a range of 50 mg/m 2 to 500 mg/m 2 , e.g., 300 mg/m 2 ) given before vaccine

administration. The cyclophosphamide may be administered as a dose of, for example, about 50 mg/m 2 , about 100 mg/m 2 , about 200 mg/m 2 , about 300 mg/m 2 , about 400 mg/m 2 , or about 500 mg/m².

Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention.

Accordingly, the foregoing description and drawings are by way of example only and the invention is described in detail by the claims that follow.

As used herein, the terms "approximately" or "about" in reference to a number are generally taken to include numbers that fall within a range of 1%, 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

The entire contents of all references, publications, abstracts, and database entries cited in this specification are incorporated by reference herein.

Claims

CLAIMS What is claimed is:

1. A method for inhibiting recurrence of a Glioblastoma Multiforme (GBM) in a subject, the method comprising: administering to the subject an autologous tumor-derived heat-shock protein peptide complex-96 (HSPPC-96), wherein the HSPPC-96 comprises gp96 in complex with peptides derived from a GBM tumor obtained from the subject; and administering to the subject bevacizumab.

2. The method of claim 1, wherein the GBM is a surgically resectable recurrent

GBM.

3. A method of treating a subject having a Glioblastoma Multiforme (GBM), the method comprising: resecting at least a portion of a GBM tumor from the subject; preparing an autologous tumor-derived heat-shock protein peptide complex-96 (HSPPC 96) from the resected GBM tumor tissue; administering to the subject the autologous tumor-derived HSPPC-96; and administering to the subject bevacizumab.

4. The method of any one of claims 1 to 3, wherein the autologous tumor-derived HSPPC-96 is administered concomitantly with the bevacizumab .

5. The method of any one of claims 1 to 4, wherein a dose of the autologous tumor derived HSPPC-96 is administered within 26 to 30 days following resection of a GBM tumor from the subject.

6. The method of any one of claims 1 to 5, wherein a dose of the bevacizumab is administered within 26 to 30 days following resection of a GBM tumor from the subject.

7. The method of any one of claims 1 to 3, wherein the autologous tumor-derived HSPPC-96 is administered prior to bevacizumab.

8. The method of claim 7, wherein bevacizumab is administered following detection of GBM tumor recurrence in the subject.

9. The method of any one of claims 1 to 8, wherein 6 to 12 doses of the autologous tumor-derived HSPPC-96 are administered to the subject.

10. The method of claim 9, wherein the subject is administered the autologous tumor- derived HSPPC-96 doses weekly for the first 4 doses.

11. The method of claim 10, wherein the subject is administered the autologous tumor-derived HSPPC-96 doses bi-weekly for an additional 2 to 8 doses.

12. The method of any one of claims 1 to 11, wherein the HSPPC-96 is administered at a dose of 25 μg.

13. The method of any one of claims 1 to 11, wherein the bevacizumab is

administered at a dose of 10 mg / kg.

14. The method of any one of claims 1 to 12, wherein the bevacizumab is

administered every 2 weeks until disease progression.

15. The method of any preceding claim further comprising evaluating activity of PI(3) Kinase in cells of the subject.

16. The method of any preceding claim further comprising evaluating expression of B7-H1 in cells of the subject.

17. The method of claim 15 or 16, wherein the cells of the subject are monocytes or tumor cells.

18. The method of claim 16 or 17, wherein the cells of the subject are monocytes and wherein expression of B7-H1 is evaluated by isolating circulating monocytes from the subject and detecting expression of B7-H1 in the isolated monocytes.

19. The method of claim 18, wherein the subject is administered the autologous tumor-derived HSPPC-96 after determining that less than 50% of the isolated monocytes are B7- Hl positive.

20. The method any one of claims 1 to 19 further comprising administering cyclophosphamide to the subject.

21. The method of 20, wherein the cyclophosphamide is administered in a dose in a range of 50 mg/m 2 to 500 mg/m 2.

22. The method of claim 20 or 21, wherein the cyclophosphamide is administered in a dose in a range of about 300 mg/m .

23. The method of any one of claims 20 to 22, wherein the cyclophosphamide is administered once during a treatment course of HSPPC-96, wherein the treatment course of HSPPC-96 comprises one or more administrations of HSPPC-96.

24. The method of any one of claims 20 to 23, wherein the cyclophosphamide is administered in a single dose 2 to 5 days prior to a first vaccine with HSPPC-96.

25. A method of preparing an autologous tumor-derived heat-shock protein peptide complex-96 (HSPPC-96), the method comprising: obtaining a sample of a GBM tumor resected from a subject, wherein 90% or more of the GBM tumor was resected from the subject; and isolating GP96 from the sample, wherein the GP96 is complexed with peptides from the GBM tumor.