Treatment of mgmtp unmethylated glioblastoma

Abstract

Provided herein are compositions and methods for treating MGMT promoter unmethylated glioblastoma using mature dendritic cells that are autologous to the patient and that have been loaded with mRNA and soluble lysate derived from the patient's glioblastoma. The methods further comprise administered to the patient a Type I interferon (IFN).

Description

TREATMENT OF MGMTp UNMETHYLATED GLIOBLASTOMAREFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of United States provisional application number 63 / 574,441, filed April 4, 2024, the entire contents of which are incorporated herein by reference.BACKGROUND1. Field

[0002] The present invention relates generally to the fields of cancer biology and immunology. More particularly, it concerns the use of dendritic cancer cell vaccines in the treatment of MGMTp unmethylated GBM.2. Description of Related Art

[0003] Glioblastoma (GBM) management remains a great challenge to cancer medicine. GBM is the most common primary malignant brain tumor with an annual incidence of 3.19 per 100,000 persons in the United States. Despite aggressive management, median survival remains between 14-18 months, with less than 10% five-year survival (Sener et al., 2022). Standard of care for GBM patients follows the Stupp protocol, established in 2005. This protocol includes post-operative radiation and temozolomide chemotherapy, which increases median survival by 2.3 months to 14.6 months. The addition of tumor treating field therapy achieved a 4-month improvement in median survival, but is not yet universally accepted. Additional improvements to care standards have not occurred despite continued GBM biomedical research. Therefore, effective therapeutic interventions remain a critical need in glioblastoma (GBM).SUMMARY

[0004] Provided herein are methods of treating MGMT promoter unmethylated glioblastoma, the methods comprising: (a) administering a population of mature dendritic cells to a patient that has MGMT promoter unmethylated glioblastoma, wherein the cells are autologous to the patient, wherein the cells have been loaded with mRNA and soluble lysate derived from the patient’s glioblastoma; and (b) administered to the patient a Type I interferon (IFN). The Type I IFN may be IFN-a.

[0005] The mature dendritic cells may be administered proximal to a deep cervical lymph node. The mature dendritic cells may be administered within 0.5-5 cm of a deep cervical lymph node, for example, within 0.5 cm, 1 cm, 1.5 cm, 2 cm, 2.5 cm, 3 cm, 3.5 cm, 4 cm, 4,5 cm, or 5 cm of a deep cervical lymph node. The mature dendritic cells may be administered within 0.5-5 cm of one right-sided and within 0.5-5 cm of one left-sided deep cervical lymph node, wherein each administration is independently with 0.5 cm, 1 cm, 1.5 cm, 2 cm, 2.5 cm, 3 cm, 3.5 cm, 4 cm, 4,5 cm, or 5 cm of the deep cervical lymph node. The mature dendritic cells may be administered under the guidance of imaging. The mature dendritic cells may be administered by ultrasound-guided injection.

[0006] The Type I IFN may be administered simultaneously with the mature dendritic cells or 0.5-48 hours after the administration of the mature dendritic cells. For example, the Type I IFN may be administered about 0, 0.01, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after the mature dendritic cells. The Type I IFN may be administered subcutaneously.

[0007] The mature dendritic cells may be administered at least a second time. Each administration may occur about 2 weeks apart. The mature dendritic cells may be administered biweekly for a total of at least three administrations.

[0008] The Type I IFN may be administered with or after each administration of the mature dendritic cells. The Type I IFN may be administered weekly. The Type I IFN may be administered weekly for a total of at least six administrations. As such, the Type I IFN may be administered with or after each administration of the mature dendritic cells as well as between each biweekly dose of mature dendritic cells.

[0009] The mature dendritic cells may have been loaded with amplified mRNA. The mature dendritic cells may have been loaded with non-amplified mRNA. The mature dendritic cells may have been loaded with mRNA followed by soluble lysate.

[0010] The patient may have completed standard-of-care treatment. The standard-of- care treatment may comprise surgery, craniospatial radiation, and / or temozolomide chemotherapy. The patient may have relapsed. The patient may have undergone reoperation. However, reoperation was identified as an independent negative prognostic co-variable. As such, preferably, the patient may have not undergone reoperation. In addition, the patient may not undergo reoperation during the treatment method.

[0011] Provide herein are vaccine compositions for use in the treatment of MGMT promoter unmethylated glioblastoma in a patient, the vaccine compositions comprising a population of mature dendritic cells, wherein the cells are autologous to the patient, wherein the cells have been loaded with mRNA and soluble lysate derived from the patient’s glioblastoma; wherein said vaccine composition is to be used in conjunction with a Type I interferon (IFN).

[0012] Provided herein is the use of a population of mature dendritic cells in the manufacture of a medicament for treating MGMT promoter unmethylated glioblastoma in a patient, wherein the cells are autologous to the patient, wherein the cells have been loaded with mRNA and soluble lysate derived from the patient’s glioblastoma, and wherein the cells are to be used in conjunction with a Type I interferon (IFN).

[0013] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF DRAWINGS

[0014] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0015] FIG. 1. Study Calendar of Events Timeline. Beginning adjuvant day #1 , patients will receive TMZ cycles consisting of 5 consecutive days of TMZ every 28 day cycle, for 6- 12 cycles. Patients will obtain a brain MRI every 2 TMZ cycles to assess response, progression and / or recurrence.

[0016] FIG. 2. Overall survival (OS) of newly diagnosed unmethylated GBM patients (n = 13) (green) compared to standard of care (SOC) historical data of unmethylated GBM patients (red) with a median OS of 12.7 months. With an average of 13 months follow-up,median survival of the unmethylated GBM patients has not been reached. Historical data derived from Fisher et al. (2021) Biomedicines.

[0017] FIG. 3. Kaplan Meier survival analysis of ten patients treated with DOC 1021 whom would generally have been excluded from a GBM study, in comparison to the expected OS of such patients (as determined by Skaga et al., Neurooncol Adv, 2021), indicates a highly significant improvement (p=0.006 by log-rank [Mantle-Cox]) from 8.9 months to not yet reached with an average follow-up time of 13.2 months.

[0018] FIG. 4. Kaplan Meier survival analysis of patients stratified by reoperation vs. no reoperation. Also shown is a comparison to a matched control historical cohort (Skaga et al., 2024). Two patients in the non-reoperative cohort were censored: one patient who died of unknown causes is censored at time of last contact with the healthcare system (16.8 months) and another who committed suicide is censored at the time of her death (17.2 months).

[0019] FIG. 5. Longitudinal radiologic study of three long-lived patients. Contrast axial MRI imaging from two of the longest-lived newly diagnosed patients (006, DL1, 26,4 mos and 0014, DL2, 19.6 mos) and one recurrent patient (0027R, DL4, 11.6 mos) treated with DC vaccine injections (DOC1021) indicate Tl-weighted signal largely resolved by ~18 months post-treatment among the two newly-diagnosed patients and the recurrent patient exhibited substantial signal diminution between 7-12 months post-treatment.DETAILED DESCRIPTION

[0020] Glioblastoma (GBM) is a devastating tumor of the central nervous system that exhibits a median survival of 14.5 months. Diagnoses in GBM are stratified by the methylation status of the O6-methylguanine-DNA methyltransferase promoter (MGMTp) as determined by bisulfite sequencing.

[0021] In MGMTp methylated GBM (> 20% promoter methylation), MGMT expression levels are lower, temozolomide chemotherapy is metabolized less efficiently, and survival is enhanced (median OS = 21.7 months).

[0022] In MGMTp unmethylated GBM, temozolomide is metabolized more rapidly and median OS is concomitantly decreased to only 12.7 months. The MGMTp unmethylated subtype comprises -60% of all GBM cases and remains highly refractory to therapeuticintervention or advances in treatment (Pinson et al., 2020). In the last 5 years, there were only 6 completed clinical trials that specifically focused on unmethylated GBM (NCT00813943, NCT03367715, NCT02336165, NCT02573324, NCT03150862, NCT02617589). The most significant improvement in overall survival was an additional 4.2 months.

[0023] Provided herein is a cell therapy vaccination protocol that has demonstrated surprising efficacy against MGMTp unmethylated GBM in early stage clinical trials when administered in conjunction with standard-of-care Stupp protocol (surgery, craniospatial radiation, temozolomide chemotherapy). Trial eligibility stipulated only minimal exclusion criteria. Among patients eligible for analysis, 1-year median OS of a cohort that included 7 MGMTp unmethylated patients and 1 MGMTp methylated patient was 88% (p=0.07 by log rank), comparing favorably to 1 year OS in MGMTp methylated (71 %) and MGMTp unmethylated (53%) disease. 1 year OS among MGMTp unmethylated patients in this cohort was 86% (6 / 7). The data therefore disclose a promising therapeutic approach against GBM in general and MGMTp unmethylated GBM specifically. The results provided herein demonstrate enhanced survival, particularly among participants that did not receive reoperation.I. Definitions

[0024] As used herein, a “complete response” requires all of the following: complete disappearance of all enhancing measurable and non-measurable disease sustained for at least 4 weeks; no new lesions; stable or improved non-enhancing (T2 / FLAIR) lesions; subjects must be off corticosteroid (or on physiologic replacement doses only); and stable or improved clinically.

[0025] As used herein, a “partial response” requires all of the following: > 50% decrease compared with baseline in the sum of products of perpendicular diameters of all measurable enhancing lesions sustained for at least 4 weeks; no progression of non-measurable disease; no new lesions; stable or improved non-enhancing (T2 / FLAIR) lesions on same or lower dose of corticosteroids compared with baseline scan; the corticosteroid dose at the time of the scan evaluation should be no greater than the dose at time of baseline scan; and stable or improved clinically.

[0026] As used herein, “stable disease” requires all of the following: does not qualify for complete response, partial response, or progression; stable non-enhancing (T2 / FLAIR) lesions on same or lower dose of corticosteroids compared with baseline scan. In the event thatthe corticosteroid dose was increased for new symptoms and signs without confirmation of disease progression on neuroimaging, and subsequent follow-up imaging shows that this increase in corticosteroids was required because of disease progression, the last scan considered to show stable disease will be the scan obtained when the corticosteroid dose was equivalent to the baseline dose.

[0027] As used herein, “progression” is defined by any of the following: > 25% increase in sum of the products of perpendicular diameters of enhancing lesions compared with the smallest tumor measurement obtained either at baseline (if no decrease) or best response, on stable or increasing doses of corticosteroids; significant increase in T2 / FLAIR nonenhancing lesions on stable or increasing doses of corticosteroids compared with baseline scan or best response after initiation of therapy not cause by comorbid events (e.g., radiation therapy, demyelination, ischemic injury, infection, seizures, postoperative changes, or other treatment effects); any new lesion; clear clinical deterioration not attributable to other causes apart from the tumor (e.g., seizures, medication adverse effects, complications of therapy, cerebrovascular events, infection, and so on) or changes in corticosteroid dose; failure to return for evaluation as a result of death or deteriorating condition; or clear progression of non-measurable disease.

[0028] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01 %. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0029] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0030] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0031] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method beingemployed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.II. DC Vaccine Preparation

[0032] Homologous antigenic loading is an ex vivo technique that leverages p38MAPK and mTORC 1 signaling cascades to initiate powerful eDC 1 -like skewing in monocyte-derived DC, leading to downstream induction of CD161mtCD8+cytolytic memory effectors. The resulting DC vaccine generates downstream immune responses with a marked THI bias.

[0033] The DC vaccines are prepared from mobilized patient peripheral blood and autologous patient tumor.

[0034] Prior to mobilizing the patient’s peripheral blood following completion of standard-of-care treatment, the patient’s peripheral CD4+ T-cell counts may be allowed to recover to >200 / pL. In order to mobilize the patient’s peripheral blood, the patient may be mobilized with G-CSF, which is a human recombinant growth factor that acts on hematopoietic cells to stimulate mobilization. G-CSF may be administered to the patient by subcutaneous injection at a dose of 5 mcg / kg / dose every 24 hours for five days (8 doses total) with a final (9th) dose of 10 mcg / kg given 12 hours before collection. Following mobilization, the patient’s peripheral blood monocytes may be obtained by leukapheresis. A minimum of IxlO10total nucleated cells should be collected for DC preparations. The apheresis product may be cryopreserved or the fresh apheresis product may be immediately used for preparing the DC vaccine.

[0035] Methods for isolating cell populations enriched for dendritic cell precursors and immature dendritic cells from various sources, including blood and bone marrow, are known in the art. For example, dendritic cell precursors and immature dendritic cells can be isolated by collecting heparinized blood, by apheresis or leukapheresis, by preparation of buffy coats, resetting, centrifugation, density gradient centrifugation (e.g., using Ficoll (such as FICOLL- PAQUE®), PERCOLL® (colloidal silica particles (15-30 mm diameter) coated with non- dialyzable polyvinylpyrrolidone (PVP)), sucrose, and the like), differential lysis of cells, filtration, and the like. In certain embodiments, a leukocyte population can be prepared, such as, for example, by collecting blood from a subject, defribrinating to remove the platelets and lysing the red blood cells. Dendritic cell precursors and immature dendritic cells can optionally be enriched for monocytic dendritic cell precursors by, for example, centrifugation through aPERCOLL® gradient. In other aspects, dendritic cell precursors can be selected using CD 14 selection of G-CSF mobilized peripheral blood.

[0036] Dendritic cell precursors and immature dendritic cells optionally can be prepared in a closed, aseptic system. As used herein, the terms “closed, aseptic system” or “closed system” refer to a system in which exposure to non-sterilize, ambient, or circulating air or other non-sterile conditions is minimized or eliminated. Closed systems for isolating dendritic cell precursors and immature dendritic cells generally exclude density gradient centrifugation in open top tubes, open air transfer of cells, culture of cells in tissue culture plates or unsealed flasks, and the like. In a typical embodiment, the closed system allows aseptic transfer of the dendritic cell precursors and immature dendritic cells from an initial collection vessel to a sealable tissue culture vessel without exposure to non-sterile air.

[0037] In certain embodiments, monocytic dendritic cell precursors are isolated by adherence to a monocyte-binding substrate. For example, a population of leukocytes (e.g., isolated by leukapheresis) can be contacted with a monocytic dendritic cell precursor adhering substrate. When the population of leukocytes is contacted with the substrate, the monocytic dendritic cell precursors in the leukocyte population preferentially adhere to the substrate. Other leukocytes (including other potential dendritic cell precursors) exhibit reduced binding affinity to the substrate, thereby allowing the monocytic dendritic cell precursors to be preferentially enriched on the surface of the substrate.

[0038] Suitable substrates include, for example, those having a large surface area to volume ratio. Such substrates can be, for example, a particulate or fibrous substrate. Suitable particulate substrates include, for example, glass particles, plastic particles, glass-coated plastic particles, glass-coated polystyrene particles, and other beads suitable for protein absorption. Suitable fibrous substrates include microcapillary tubes and microvillous membrane. The particulate or fibrous substrate usually allows the adhered monocytic dendritic cell precursors to be eluted without substantially reducing the viability of the adhered cells. A particulate or fibrous substrate can be substantially non-porous to facilitate elution of monocytic dendritic cell precursors or dendritic cells from the substrate. A “substantially non-porous” substrate is a substrate in which at least a majority of pores present in the substrate are smaller than the cells to minimize entrapping cells in the substrate.

[0039] Adherence of the monocytic dendritic cell precursors to the substrate can optionally be enhanced by addition of binding media. Suitable binding media include monocytic dendritic cell precursor culture media (e.g., AIM-V®, RPMI 1640, DMEM, X- VIVO 15®, and the like) supplemented, individually or in any combination, with for example, cytokines (e.g., Granulocyte / Macrophage Colony Stimulating Factor (GM-CSF), Interleukin 4 (IE-4), or Interleukin 13 (IE- 13)), blood plasma, serum (e.g., human serum, such as autologous or allogenic sera), purified proteins, such as serum albumin, divalent cations (e.g., calcium and / or magnesium ions) and other molecules that aid in the specific adherence of monocytic dendritic cell precursors to the substrate, or that prevent adherence of non-monocytic dendritic cell precursors to the substrate. In certain embodiments, the blood plasma or serum can be heated-inactivated. The heat-inactivated plasma can be autologous or heterologous to the leukocytes.

[0040] Following adherence of monocytic dendritic cell precursors to the substrate, the non-adhering leukocytes are separated from the monocytic dendritic cell precursor / substrate complexes. Any suitable means can be used to separate the non-adhering cells from the complexes. For example, the mixture of the non- adhering leukocytes and the complexes can be allowed to settle, and the non-adhering leukocytes and media decanted or drained. Alternatively, the mixture can be centrifuged, and the supernatant containing the non- adhering leukocytes decanted or drained from the pelleted complexes.

[0041] Isolated dendritic cell precursors can be cultured ex vivo for differentiation, maturation and / or expansion. (As used herein, isolated immature dendritic cells, dendritic cell precursors, T cells, and other cells, refers to cells that, by human hand, exists apart from their native environment, and are therefore not a product of nature. Isolated cells can exist in purified form, in semi-purified form, or in a non-native environment.) Briefly, ex vivo differentiation typically involves culturing dendritic cell precursors, or populations of cells having dendritic cell precursors, in the presence of one or more differentiation agents. Suitable differentiating agents can be, for example, cellular growth factors (e.g., cytokines such as (GM-CSF), Interleukin 4 (IL-4), Interleukin 13 (IL- 13), and / or combinations thereof). In certain embodiments, the monocytic dendritic cells precursors are differentiated to form monocyte- derived immature dendritic cells.

[0042] The dendritic cell precursors can be cultured and / or differentiated in suitable culture conditions. Suitable tissue culture media include AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, and the like. The tissue culture media can be supplemented with serum, amino acids, vitamins, cytokines, such as GM-CSF and / or IL-4, divalent cations, and the like, to promote differentiation of the cells. In certain embodiments, the dendritic cell precursors can be cultured in the serum-free media. Such culture conditions can optionally exclude any animal-derived products. A typical cytokine combination in a typical dendritic cell culture medium is about 500 units / ml each of GM-CSF (50 ng / ml) and IL-4 (10 ng / ml). Dendritic cell precursors, when differentiated to form immature dendritic cells, are phenotypically similar to skin Langerhans cells. Immature dendritic cells typically are CD 14- and CD1 lc+, express low levels of CD86 and CD83, and are able to capture soluble antigens via specialized endocytosis. The immature DC expressed very high levels of CD86. Also, the population was mixed in terms of CD14 and CD11C. Though the majority were CDl lc+, there were distinct subpopulations that were CD1 1c- and CD 14+.

[0043] The immature dendritic cells are matured to form mature dendritic cells. Mature DC lose the ability to take up antigen and display up-regulated expression of costimulatory cell surface molecules and various cytokines. Specifically, mature DC express higher levels of MHC class I and II antigens than immature dendritic cells, and mature dendritic cells are generally identified as being CD80+, CD83+, CD86+, and CD14-. Greater MHC expression leads to an increase in antigen density on the DC surface, while up regulation of costimulatory molecules CD80 and CD86 strengthens the T cell activation signal through the counterparts of the costimulatory molecules, such as CD28 on the T cells. Mature dendritic cells of the present invention can be prepared (i.e.. matured) by contacting the immature dendritic cells with effective amounts or concentrations of a tumor nucleic acid composition and a tumor lysate composition.

[0044] The autologous patient tumor tissue may be collected at the time of debulking surgery during the standard-of-care treatment phase. At the time of collection, the sample should be flash frozen, for example, by immersion in liquid nitrogen for five minutes, after which the sample should be stored at a minimum of -80° C.

[0045] The RNA and tumor lysate may be prepared from the surgically resected tumor tissue. For example, the isolated tumor tissue can be minced and placed into a container with a buffer solution containing a proteinase (e.g., collagenase) to dissociate the tissue, producing liberated tumor cells. Following filtering of the tissue digest, liberated tumor cells may be centrifuged into a pellet. The cell pellet may then be suspended in a small volume of culturemedium and subjected to cell disruption methods such as sonication or freeze-thaw cycles. After disruption, the tumor RNA and lysate can be isolated.

[0046] Total RNA is isolated from the autologous patient tumor tissue for use in dendritic cell loading. If sufficient RNA is obtained from the tissue, then the RNA may be used directly in loading the dendritic cells. For example, if about 200 pg of mRNA is obtained from the tissue, then the mRNA may be used without amplification. If insufficient RNA is obtained from the tissue, then an mRNA amplification may be performed by first performing RT-PCR to obtain cDNA that includes a T7 promoter, and then in vitro transcribing the cDNA. For example, the RNA may be amplified according to the protocol described in WO 2019 / 118978, which is incorporated herein by reference in its entirety. This amplified mRNA is intended to fall within the definition of mRNA derived from the patient’s tumor tissue.

[0047] Soluble tumor lysate is also isolated from the autologous patient tumor tissue for use in dendritic cell loading. For example, tumor lysate may be prepared by the suspension of tumor cells in medium, followed by three successive freeze / thaw cycles at -80°C / 37°C. Insoluble material may be pelleted and the soluble fraction was placed at -80°C until needed for use. Protein concentration may be determined by the bicinchoninic acid (BCA) assay.

[0048] The cells obtained from the patient’s apheresis product may be loaded with RNA and lysate as follows. Preferably, cells are loaded with RNA first, followed by loading with lysate. For loading with mRNA, immature dendritic cells may be mixed with tumor mRNA (amplified or not) to a concentration of 1 pg mRNA / 106cells and incubated for 10 min on ice in an electroporation cuvette. Cells may then be electroporated. Following electroporation, cells are loaded with soluble lysate. For loading with soluble lysate, the mRNA-loaded dendritic cells may be cultured for three hours at 37 °C in a four- fold dilution of blast lysate at 2 mg / mL final concentration. Following three hours of antigenic loading, cells may be pelleted, washed once to remove residual lysate, and matured for 24 h in the presence of ITIP [10 ng / mL IL-1, 10 ng / mL TNF-a, 15 ng / mL IL-6, and 1 g / mL PGE2I. Additional details regarding homologous loading of dendritic cells can be found in U.S. Patent 8,728,806, which is incorporated herein by reference in its entirety.

[0049] The patient-specific DC vaccine product should be stored in cryopreservation tubes in the vapor phase of liquid nitrogen at a temperature of at least - 140°C . Just prior to use, the DC vaccine should be rapidly thawed, for example, in a 37°C water bath.III. Methods of Administration

[0050] The DC vaccine may be administered to the patient using ultrasound sonography (US)-guided injection targeting deep cervical lymph nodes. This perinodal injection may be within 0.5 cm of each deep cervical lymph node, one on the left side and one on the right side. A total of three pairs of sequential DC vaccines may be administered perinodally under sonographic guidance. Each pair of injections may consist of a bilateral (left and right) neck injection. Two weeks may separate each set of injections.

[0051] The injections may be performed as follows: 1.5 mL sterile clinical grade saline may be drawn into a 3 mL syringe through a 1.5 inch, 20 gauge needle. After complete thawing, the DC vaccine product should be resuspended by agitation and the complete volume (approximately 0.5 mL) drawn into the same syringe with the saline. The syringe may be agitated and the suspension administered in the vicinity of the left deep cervical node as determined by US. This process may then be repeated for the vaccine dose to be delivered to the vicinity of the right deep cervical node. The sonographic-guided delivery system may consist of a standard ultrasound unit (e.g. Ultrasonix SonixTouch or similar) using a high frequency linear transducer.

[0052] While on vaccine therapy, patients may also receive weekly doses of interferon alpha (e.g., pegylated interferon alpha), starting on the same day as the first vaccination, and continuing until one week after the last DC vaccination, for a total of six treatments. Each dose of pegylated interferon alpha may be 180 mcg, administered subcutaneously.IV. Examples

[0053] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, 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 invention.Example 1

[0054] The inventors report the results of a completed phase I clinical trial for a personalized dendritic cell vaccine in adult patients with resectable GBM. Adult patients diagnosed with resectable GBM and deemed good candidates for postoperative adjuvant chemoradiation were enrolled in this phase I clinical study. Inclusion criteria included: adequate kidney, liver, bone marrow, and immune function with an ECOG performance status < 2. Exclusion criteria were minimal. After completion of an initial standard-of-care chemoradiation regime, as defined by the Stupp protocol, dendritic cell vaccines, which were prepared through homologous antigenic loading, were administered to newly diagnosed glioblastoma (GMB) patients bilaterally in the vicinity of the deep cervical lymph nodes, assisted by ultrasound sonography. The DC vaccine was every 2 weeks for a total of three doses. Immediately after the first set of DC vaccine injections, the patients began interferon treatments with pegylated interferon alpha. Weekly interferon treatments continued until one week after the last DC vaccine, for a total of six treatments (FIG. 1).

[0055] Four dose levels from 3.5 x 106to 3.6 x 107total vaccine cells were tested, none of which resulted in AEs > grade 2 attributable to the investigational regimen. Immunohistochemistry of tumors derived from early post-vaccination second resections displayed enhanced CD8+T-cell infiltration and pathologic findings consistent with residual rather than relapsed GBM in 2 / 3 patients. Radiologic pseudoprogression was also routinely observed among patients of all cohorts. Analysis of post- vaccination circulating PBMC indicated expansion of both CD4+and CD8+central memory T-cell compartments (p<0.00006 and p<0.003 respectively for each by student’s two-tailed t-test) as well as expansion of CD8+CD127+T cells (p<0.002 by student’s two tailed t-test) among 13 patients analyzed. Median survival of this largely MGMT promoter unmethylated cohort was 19.7 months, statistically greater (p<0.05 by log-rank[Mantle-Cox]) than that of matched historical controls.

[0056] FIG. 2 shows the overall survival (OS) of newly diagnosed unmethylated GBM patients (n = 13) (green) compared to standard of care (SOC) historical data of unmethylated GBM patients (red) with a median OS of 12.7 months. With an average of 13 months followup, median survival of the unmethylated GBM patients has not been reached. Historical data derived from Fisher et al. (2021) Biomedicines.

[0057] FIG. 3 shows a Kaplan Meier survival analysis of ten patients treated with DC vaccine injections (DOC1021) whom would generally have been excluded from a GBM study, in comparison to the expected OS of such patients (as determined by Skaga et al., Neurooncol Adv, 2021), indicates a highly significant improvement (p=0.006 by log-rank [Mantle-Cox]) from 8.9 months to not yet reached with an average follow-up time of 13.2 months.

[0058] FIG. 4 shows a Kaplan Meier survival analysis of patients stratified by reoperation vs. no reoperation. Reoperation was determined to be detrimental to patient outcomes. Also shown is a comparison to a matched control historical cohort (Skaga et al., 2021). Two patients in the non-reoperative cohort were censored: one patient who died of unknown causes is censored at time of last contact with the healthcare system (16.8 months) and another who committed suicide is censored at the time of her death (17.2 months).

[0059] FIG. 5 shows a longitudinal radiologic study of three long-lived patients. Contrast axial MRI imaging from two of the longest- lived newly diagnosed patients (006, DL1, 26.4 mos and 0014, DL2, 19.6 mos) and one recurrent patient (0027R, DL4, 11.6 mos) treated with DC vaccine injections (DOC1021) indicate Tl-weighted signal largely resolved by ~18 months post-treatment among the two newly-diagnosed patients and the recurrent patient exhibited substantial signal diminution between 7-12 months post-treatment.Table 1. Patient Characteristics* * *

[0060] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.Decker et al., “Th-1 polarization is regulated by dendritic-cell comparison of MHC class I and class II antigens,” Blood (2009).Pinson et al., “Weak MGMT gene promoter methylation confers a clinically significant survival benefit in patients with newly diagnosed glioblastoma: a retrospective cohort study,” Journal of Neurooncology (2020).Sener et al., “Immunotherapy in Glioblastoma: Current Approaches and Future Perspectives,” International Journal of Molecular Sciences (2022).Skaga et al., “Real-world validity of randomized controlled phase III trials in newly diagnosed glioblastoma: to whom do the results of the trials apply?” Neurooncol Adv 3:vdab008, 2021.

Claims

CLAIMS1. A method of treating a patient having a MGMT promoter unmethylated glioblastoma, the method comprising:(a) administering to the patient a population of mature dendritic cells, wherein the cells are autologous to the patient, wherein the cells have been loaded with mRNA and soluble lysate derived from the patient’s glioblastoma; and(b) administered to the patient a Type I interferon (IFN).

2. The method of claim 1, wherein the Type I IFN is IFN-a.

3. The method of claim 1 or 2, wherein the mature dendritic cells are administered proximal to a deep cervical lymph node.

4. The method of any one of claims 1-3, wherein the mature dendritic cells are administered within 0.5-5 cm of a deep cervical lymph node.

5. The method of any one of claims 1-4, wherein the mature dendritic cells are administered within 0.5-5 cm of one right-sided and one left-sided deep cervical lymph node,6. The method of any one of claims 3-5, wherein the mature dendritic cells are administered by ultrasound-guided injection.

7. The method of any one of claims 1-6, wherein the mature dendritic cells are administered at least a second time,8. The method of claim 7, wherein each administration occurs about 2 weeks apart.

9. The method of any one of claims 1-8, wherein the mature dendritic cells are administered biweekly for a total of at least three administrations.

10. The method of any one of claims 1-9, wherein the Type I IFN is administered 0.5-48 hours after the administration of the mature dendritic cells.

11. The method of any one of claims 1-10, wherein the Type I IFN is administered subcutaneously.

12. The method of any one of claims 7-11, wherein the Type I IFN is administered after each administration of the mature dendritic cells.

13. The method of any one of claims 1-12, wherein the Type I IFN is administered weekly.

14. The method of any one of claims 1-13, wherein the Type I IFN is administered weekly for a total of at least six administrations.

15. The method of any one of claims 1-14, wherein the mature dendritic cells have been loaded with amplified mRNA.

16. The method of any one of claims 1-15, wherein the mature dendritic cells have been loaded with mRNA followed by soluble lysate.

17. The method of any one of claims 1-16, wherein the patient has completed standard-of- care treatment.

18. The method of claim 17, wherein the standard-of-care treatment comprises surgery, craniospatial radiation, and / or temozolomide chemotherapy.

19. A vaccine composition for use in the treatment of MGMT promoter unmethylated glioblastoma in a patient, the vaccine composition comprising a population of mature dendritic cells, wherein the cells are autologous to the patient, wherein the cells have been loaded with mRNA and soluble lysate derived from the patient’s glioblastoma; wherein said vaccine composition is to be used in conjunction with a Type I interferon (IFN).

20. Use of a population of mature dendritic cells in the manufacture of a medicament for treating MGMT promoter unmethylated glioblastoma in a patient, wherein the cells are autologous to the patient, wherein the cells have been loaded with mRNA and soluble lysate derived from the patient’ s glioblastoma, and wherein the cells are to be used in conjunction with a Type I interferon (IFN).

Images