Method for treating malignant glioma

Abstract

A method of treating cancer in a subject is disclosed, the method comprising (i) administering a pharmaceutical composition comprising a therapeutically effective amount of mutagenic IL13 moiety (mIL13), and then (ii) performing radiotherapy.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 368,283, filed Jul. 13, 2022, which is incorporated herein by reference.

[0002] Technical field The present disclosure relates to cancer treatment, and more specifically to the treatment of malignant glioma.

Background Art

[0003] The prognosis for malignant glioma remains poor due to the lack of effective therapeutic treatments. Given the limited effectiveness of current treatment modalities for certain cancers, there is a strong need to develop new effective treatments for patients with these diseases.

Summary of the Invention

Means for Solving the Problems

[0004] Disclosed herein is a method of treating cancer in a subject, the method comprising (i) administering a pharmaceutical composition comprising a therapeutically effective amount of mutagenized IL13 subunit (mIL13), and (ii) performing radiation therapy.

Brief Description of the Drawings

[0005]

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DETAILED DESCRIPTION OF THE INVENTION

[0006] Reference to electronic sequence listings The content of the electronic sequence listing (Targepeutics Sequence Listing.xml; size: 27,000 bytes; and creation date: July 10, 2022) is hereby incorporated by reference in its entirety.

[0007] Detailed description Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For testing the present disclosure, any methods and materials similar or equivalent to those described herein may be used in practice, but the preferred materials and methods are described herein.

[0008] For the purposes of the following description, it should be understood that the present disclosure may assume various alternative variations and step sequences, unless the contrary is explicitly specified. Further, all numerical values, such as those representing values, amounts, percentages, ranges, sub-ranges, and ratios, other than when an operation example or otherwise indicated, may be read as being preceded by the term "about" even if the term does not explicitly appear. Thus, unless the contrary is indicated, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending upon the desired results to be obtained by the present disclosure. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and in reference to the ordinary rounding techniques. When a closed or open-ended numerical range is recited herein, all numbers, values, amounts, percentages, sub-ranges, and ratios within or encompassed by that numerical range are to be specifically included in the original disclosure of this application and considered to belong thereto as if the entirety of those numbers, values, amounts, percentages, sub-ranges, and ratios were explicitly recited.

[0009] Numerical ranges and parameters setting forth broad ranges of the present disclosure are approximations, although they are accurately reported in specific examples. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective test measurements.

[0010] Unless otherwise indicated, plural terms used herein can include their singular counterparts and vice versa. For example, reference to "a" catheter or "an" IL13 herein can include any combination (i.e., plural) of these components. Additionally, unless otherwise specifically described, use of "or" in this application means "and / or," although "and / or" may be explicitly used in certain situations.

[0011] As used herein, "including," "containing," and similar terms are synonymous with "comprising" in the context of this application and are thus open-ended, understood not to exclude the presence of additional unrecited and / or undescribed elements, materials, components, and / or method steps.

[0012] As used herein, "consisting of" is understood to exclude the presence of any unspecified element, component, and / or method step in the context of this application.

[0013] As used herein, "consisting essentially of" is understood to include the specified elements, materials, components, and / or method steps in the context of this application, and those that do not "substantially affect the basic and novel features (singular or plural) of what is described."

[0014] As used herein, terms such as "patient", "subject", and "individual" are used interchangeably herein and mean an animal that can accept the treatment methods described herein, which includes mammals including humans, dogs, cats, cows, horses, pigs, primates, and / or rodents.

[0015] As used herein, "administering" or "administration" of a composition in an amount (e.g., dosage) may be performed by the subject himself / herself or another subject (e.g., a medical professional, caregiver, or family member). The composition may be provided by the subject or an administrator to the subject along with instructions for administration of the composition (e.g., instructions written on the label of the container containing the composition).

[0016] As used herein, "disorder", "disease", and "illness" are used interchangeably and refer to a condition of a subject that adversely affects the health of the subject.

[0017] As used herein, "treat", "treatment", or "treating" means treating a disease or disorder as defined herein in a subject, which includes (1) suppressing the disease or disorder, (2) halting the development of the disease or disorder, (3) slowing the progression of the disease or disorder, and / or (4) suppressing, alleviating, or delaying the progression of one or more symptoms of the disease or disorder. A disease, disorder, and / or illness, or a reduction in the severity and / or frequency of symptoms associated therewith, is "treated" when experienced by the subject.

[0018] As used herein, "cancer" refers to a disease characterized by abnormal proliferation of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, brain cancers in which histiocytes express IL13Rα2, such as glioma.

[0019] As used herein, "glioma" refers to a tumor that arises in the glial cells of the brain or spinal cord.

[0020] As used herein, a "therapeutically effective amount" is defined as the amount required to improve the symptoms of a disease in a treated patient compared to an untreated patient. The effective amount of an active compound(s) used to therapeutically treat a disease varies depending on the mode of administration, as well as the age, weight, and general health of the subject.

[0021] As used herein, "composition" refers to a solution or a dispersion.

[0022] As used herein, "pharmaceutical composition" refers to any chemical or biological composition, material, or agent, etc., that is capable of inducing a therapeutic effect when properly administered to a subject, and includes the inactive form of the composition, material, or agent, etc., as well as its active metabolites, which may be formed in vivo.

[0023] As used herein, "radiation therapy" refers to a method of treating a patient, including internal or external radiation treatment.

[0024] As used herein, "radiation treatment" refers to the delivery of radioactive energy, eg, electrons, protons, or heavy ions, to a target volume, eg, a tumor or lesion, within a patient.

[0025] As used herein, "measuring" or "measurement" or "detecting" or "detection" means evaluating the presence, absence, quantity, or amount (which may be an effective amount) of a given substance in a clinical sample or a subject-driven sample, including deriving qualitative or quantitative levels of such substances, or otherwise evaluating the value or categorization of a clinical parameter of a subject.

[0026] As used herein, "sample" or "biological sample" refers to biological material isolated from an individual, such as a liquid or solid biological sample collected via a biopsy (i.e., "liquid biopsy" or "solid biopsy"). Liquid biopsies may include, for example, blood, plasma, saliva, urine, cerebrospinal fluid, and / or other body fluids. Liquid biopsies may contain extracellular vesicles and / or cell-free genetic material. Solid biopsies may include, for example, organs and / or tissues such as tumors. A biological sample may contain any biological material suitable for detecting a desired biomarker and may include cellular and / or acellular material obtained from an individual.

[0027] As used herein, "control" refers to any experimental condition that is either not treated at all or is treated by a method not disclosed herein. For example, a sample or subject treated only with radiation therapy is a control. In another example, a sample or subject treated only with a pharmaceutical composition containing a therapeutically effective amount of mutagenic IL13 is a control.

[0028] As used herein, "interleukin 13" or "IL13", unless the contrary is indicated, refers to any wild-type or native IL13 derived from any vertebrate source, including mammals such as primates and rodents, which includes untreated IL13, any form of IL13 resulting from intracellular processing, and any naturally occurring variants of IL13, such as splice or allelic variants. An exemplary amino acid sequence of human IL13 is shown in SEQ ID NO:1. An amino acid sequence of a second exemplary human IL13 is shown in SEQ ID NO:2.

[0029] "Mutation" in a polypeptide means any amino acid substitution, deletion (e.g., a truncated version of a protein such as a polypeptide), insertion, and / or modification of a protein, such as by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, and amidation. In one example, "mutant IL13" or "mutagenized IL13" refers to an IL13 in which one or more of its amino acids differ from the corresponding amino acids in the wild-type form of IL13. Mutant IL13 and / or mutagenized IL13 may be derived from the wild-type form of IL13 found in humans, non-human primates, rats, mice, pigs, cows, and dogs, etc. Mutant IL13 and / or mutagenized IL13 may be referred to herein as "mIL13". As further discussed herein, mIL13 may be connected to a cytotoxin ("cmIL13"). As used herein, "mIL13" includes "cmIL13".

[0030] As used herein, the term "IL13 receptor" or "IL13R" refers to a receptor that binds to IL13.

[0031] As used herein, the terms "IL13 receptor α2" or "IL13Rα2" refer to the monomeric IL13 receptor, which is expressed on the surface of certain cell subsets and binds to IL13 or any homolog of IL13 such as, for example, mIL13, or is contained in extracellular vesicles and binds to IL13 independently of IL4.

[0032] As used herein, "affinity" refers to the strength of the overall non-covalent interaction between a single binding site of a receptor and a ligand. The affinity of a receptor for a ligand can be expressed as the dissociation constant (K D ), which is the ratio of the dissociation rate constant K off to the association rate constant K on . Affinity can be measured by methods known to those of skill in the art.

[0033] "Increase in binding" means that the binding level of mIL13 is at least 10% or more, for example 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% higher or higher than that of wild-type IL13, or 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 100-fold, 1000-fold higher or higher than that of wild-type IL13, and any whole or partial increase therebetween.

[0034] "Decrease in binding" means that the binding level of mIL13 is at least 10% or less, for example 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% lower or lower than that of wild-type IL13, or 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 100-fold, 1000-fold lower or lower than that of wild-type IL13, and any whole or partial increase therebetween.

[0035] As used herein, "fusion" or "fused" is defined as linking one gene to a second gene prior to translation.

[0036] As used herein, "determining the level of a marker (or biomarker)" means assessing the degree of expression or presence of a marker in a sample at the nucleic acid or protein level using techniques available to those of skill in the art to detect a sufficient portion of any marker expression product, such as "determining the level of IL13Rα2".

[0037] As used herein, "level" of one or more markers (or biomarkers) means the absolute or relative amount or concentration of the marker (or biomarker) in a sample.

[0038] As used herein, "ng / ml" means the mass of protein in a volume of 1 ml.

[0039] The present disclosure is directed to a method of treating cancer in a subject, the method comprising, consisting essentially of, or consisting of administering a pharmaceutical composition comprising a therapeutically effective amount of mutagenized IL13 moiety (mIL13) and then performing radiation therapy.

[0040] The pharmaceutical composition may comprise, consist essentially of, or consist of mIL13. mIL13 may be engineered to have an increased affinity for IL13Rα2 compared to wild-type human IL13. The affinity of mIL13 for IL13Rα2 may be increased compared to wild-type IL13, which may be, for example, a 2-fold increase, 3-fold increase, 5-fold increase, 10-fold increase, 100-fold increase, 1000-fold increase, or more in kinetic K D of up to 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, 1000-fold, or more.

[0041] In addition, mIL13 may be engineered to have a decreased affinity for interleukin 13 receptor α1 (IL13Rα1) compared to wild-type human IL13. For example, mIL13 may have a reduced affinity for IL13Rα1 compared to wild-type IL13, which may be, for example, a decrease in kinetic K DA decrease by a factor of 2, 3, 5, 10, 100, or more, while the binding or activation of IL13Rα2 may be at least retained.

[0042] In an example, mIL13 may be produced by cDNA mutagenesis, DNA synthesis, peptide / protein synthesis, or any method known to those skilled in the art.

[0043] mIL13 may be a full-length IL13 molecule, such as a human full-length IL13 molecule. In an example, mIL13 may contain amino acid changes to wild-type IL13 at positions corresponding to residue 13 of human IL13 (SEQ ID NO:1), residue 66 of SEQ ID NO:1, residue 69 of SEQ ID NO:1, and / or residue 105 of SEQ ID NO:1. mIL13 may contain a substitution for glutamic acid at position 13 of human IL13 (SEQ ID NO:1). For example, lysine may be substituted for glutamic acid at position 13. mIL13 may contain a substitution for arginine at position 66 of human IL13 (SEQ ID NO:1). For example, aspartic acid may be substituted for arginine at position 66. mIL13 may contain a substitution for serine at position 69 of human IL13 (SEQ ID NO:1). Aspartic acid may be substituted for serine at position 69. mIL13 may contain a substitution of lysine at position 105 of human IL13 (SEQ ID NO:1). Arginine may be substituted for lysine at position 105. mIL13 may contain changes at positions E13, R66, S69, and / or K105. mIL13 may be named IL13.E13K.R66D.S69D.K105R. In one example, mIL13 may contain the amino acid sequence shown in SEQ ID NO:3. In other examples, mIL13 may contain any of SEQ ID NOs: 4-22, or a sequence that shares at least 50, 60, 70, 80, 85, 90, 95, or 99% homology with any of SEQ ID NOs: 1-24, or a sequence that differs from any of SEQ ID NOs: 1-24 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or fewer residues.

[0044] mIL13 and its analogs provide selective binding to the IL13 receptor. mIL13 may be a highly selective antagonist and / or a selective agonist of wild-type IL13 activity. mIL13 is engineered to have one or more of the following properties: (a) an altered affinity for IL13Rα2 relative to wild-type human IL13 (decreased affinity or increased affinity), (b) an altered affinity for IL13Rα1 relative to wild-type human IL13 (decreased affinity or increased affinity), and / or (c) disruption of the binding site for IL4Rα1. mIL13 may include amino acid modifications at one or more amino acids within a set of contacting residues that interact with IL13Rα1, IL13Rα2, or IL4Rα1, and these residues include, for example, L10, R11, I14, E12, V18, R65, R86, D87, T88, K89, L101, K104, K105, F107, and R108 (for reference purposes, the sequence of wild-type human IL13 is provided as SEQ.ID.NO:1 and the amino acid numbers refer thereto). mIL13 may include modifications at two or more, three or more, four or more, five or more, and up to 14 amino acids within a combined set of contacting residues as defined herein.

[0045] mIL-13 may include one or more of the following amino acid substitutions: (1) L10F; L10I; L10V; L10A; L10D; L10T; L10H; (2) R11S; R11N; R11H; R11L; R11I; (3) I14L; I14F; I14V; I14M; (4) V18L; V18F; V18I; (5) E12A; (6) R65D; (7) R86K; R86T; R86M; (8) D87E; D87K; D87R; D87G; (9) T88I; T88K; T88R; (10) K89R; K89T; K89M; (11) L101F; L101I; L101Y; L101H; L101N; (12) K104R; K104T; K104M; (13) K105T; K105A; K105R; K105E; (14) F107L; F107I; F107V; F107M; and (15) R108K; R108T; R108M. These substitutions cause a change in the affinity for one or both of IL-13Rα1 and IL-13Rα2. mIL-13 may include modified residues in two or more, three or more, four or more, five or more, and 14 or fewer amino acids of a combined set of contacting residues as defined herein.

[0046] The terms "homology" and "homologous" refer to the identity of the subunit sequences between two molecules, such as two protein or peptide molecules. When the subunit positions of both molecules are occupied by the same subunit, those molecules are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions. For example, when half of the positions of two sequences are homologous, those two sequences are 50% homologous, and when 70% (i.e., 7 out of 10) of the positions are matching or homologous, those two sequences are 70% homologous. The homologs of the present disclosure can be the result of variations of natural alleles, including natural mutations. The homologs of the present disclosure can be the result of variations of natural alleles, including natural mutations. The homologs of the present disclosure can also be generated using techniques known in the art, including direct modification of proteins using recombinant DNA techniques such as those that result in random or targeted mutagenesis.

[0047] mIL13 may be complexed with extracellular vesicles (EVs) to form an extracellular vesicle complex (EV complex). The extracellular vesicles may be obtained from a specific type of biological sample and / or may be derived from a specific type of cell, such as, for example, glioma stem cells. In an example, the glioma stem cells may be mesenchymal glioma stem cells or proneural glioma stem cells. The extracellular vesicles may include exosomes, such as, for example, tumor-associated exosomes. The extracellular vesicles may be purified or concentrated from a biological sample using, for example, fractionation centrifugation, ultracentrifugation, and / or other methods known to those skilled in the art.

[0048] A subpopulation of extracellular vesicles may be isolated using a biological marker. The biological marker may be a receptor, such as, for example, a tumor-associated receptor. The tumor-associated receptor may be IL13Rα2. Each exosome may express, for example, 1, 2, 5, 10, 15, 20, 25, 50, 100, 250, 500, or 1000 or more biological markers.

[0049] A cytotoxin may be fused to one of the mIL13s disclosed herein (“cmIL13”). For example, the fusion may occur by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (“EDC”) chemistry or purified gel filtration, among others.

[0050] The cytotoxin may comprise, consist essentially of, or consist of a bacterially-derived toxin. The bacterially-derived toxin may include, for example, Pseudomonas exotoxin A.

[0051] The pharmaceutical composition may be administered by a catheter. For example, the pharmaceutical composition may be administered by convection enhanced delivery (CED). As used herein, "convection enhanced delivery" or "CED" refers to a method of drug administration that creates a pressure gradient at the tip of an infusion catheter for directly delivering a therapeutic agent to a structure to be treated, such as an ablation cavity or an intact tumor, through the interstitial spaces of the central nervous system. The pressure gradient may be a positive pressure gradient, which means that the pharmaceutical composition may be administered under positive pressure. In addition, the pharmaceutical composition may be administered by CED at a constant pressure. Any suitable catheter known in the art may be used. The pharmaceutical composition may be administered by convective infusion at a specified flow rate controlled by an external syringe pump. The pharmaceutical composition may be administered at a flow rate of up to 2 mL / hour. This method includes positioning the tip of the catheter within the area to be treated. An external pump may be connected to the catheter, which supplies a composition containing a therapeutically effective dose of a therapeutic agent, such as mIL13, while maintaining a positive pressure gradient during delivery. Administration of mIL13 may or may not be accompanied by co-infusion of a contrast component that acts as an alternative marker for drug delivery, such as gadolinium or gadolinium-DTPA.

[0052] The pharmaceutical composition may be administered acutely. As used herein, "acute administration" and the like refer to a procedure for inpatients in which a medical professional administers the pharmaceutical composition to a patient. For example, administration of the pharmaceutical composition may be carried out over a period not exceeding one week. For example, administration of the pharmaceutical composition may be carried out over a period of at least 4 hours. In other examples, administration of the pharmaceutical composition may be carried out over a period of 96 hours or less. Administration of the pharmaceutical composition may be carried out over a period of 4 to 96 hours.

[0053] A pharmaceutical composition containing mIL13 may be administered, and the dosage of mIL13 is at least 0.03 μg / mL to 1 μg / mL or less.

[0054] The method of treating cancer in the subject disclosed herein further comprises, consists essentially of, or consists of performing radiation therapy. The radiation therapy may be any suitable radiation therapy known to those skilled in the art. By way of example, the radiation therapy may be fractionated radiation therapy.

[0055] The radiation therapy may be administered subsequent to the administration of the pharmaceutical composition containing mIL13. In addition to being administered before the radiation therapy, the pharmaceutical composition may also be administered concurrently with the radiation therapy.

[0056] Any type of tumor containing cells that express IL13Rα2 may be treated by the method disclosed herein. For example, the method disclosed herein may be used to treat cancers of the brain and / or central nervous system (CNS), such as, for example, malignant glioma. Examples of malignant gliomas that may be treated by the method disclosed herein include, but are not limited to, adult glioblastoma, pediatric glioblastoma, anaplastic astrocytoma, such as diffuse midline glioma, anaplastic oligodendroglioma, anaplastic oligoastrocytoma, anaplastic ependymoma, and / or anaplastic ganglioglioma. In other examples, the method disclosed herein may be used to treat soft tissue sarcoma.

[0057] Prior to administration of mIL13, expression of IL13Rα2 in tumor samples may be detected. In an example, prior to detection of expression of IL13Rα2, tumor samples may be purified. Detection of expression of IL13Rα2 may be detected by mass spectrometry, analytical assays, immunohistochemistry, and / or sequencing. In an example, the analytical assay may include an enzyme-linked immunosorbent assay (ELISA). In another example, immunohistochemistry may include immunohistochemistry (IHC) and / or fluorescence cytochemistry. In another example, sequencing may include whole genome sequencing, exome sequencing, proteomic sequencing, and / or RNA sequencing. Expression of IL13Rα2 by a tumor indicates that the tumor is responsive to treatment with mIL13.

[0058] Surprisingly, it has been discovered that treating tumors expressing IL13Rα2 with a pharmaceutical composition containing mIL13 prior to administration of radiotherapy enhances the anti-tumor effects of those treatments alone.

[0059] In addition, surprisingly, it has been discovered that treating tumors expressing IL13Rα2 with a pharmaceutical composition containing mIL13 prior to administration of radiotherapy reduces the intensity of radiation required to achieve the same level of tumor cell death when compared to treatment with radiotherapy alone.

[0060] Those skilled in the art will recognize that numerous modifications and variations are possible in light of the above disclosure without departing from the broad inventive concepts described and illustrated herein. Accordingly, the foregoing disclosure is merely illustrative of various exemplary aspects of the present application, and it is understood that numerous modifications and variations can be readily made by those skilled in the art within the spirit and scope of the present application and the appended claims.

Examples

[0061] Example 1: The cytotoxicity of cmIL13 correlates with IL13Rα2 expression The selected DIPG cell lines were collected and the lysates were processed for immunoblotting with IL13Rα2, IL13Rα1, and GAPDH antibodies. Figure 1 shows the immunoblotting assay.

[0062] Cell lines with various IL13Rα2 expression levels were tested in a cell potency assay. Various concentrations of mutant IL13 fused to a cytotoxin (cmIL13) were added to cells plated on plates for 72 hours, and then cell viability was determined by the MTS assay. As shown in Figure 2, cell lines with high IL13Rα2 were sensitive (SF6828 IC50 = 0.05 ng / ml; PED17 IC50 = 9.8 ng / ml), whereas cells lacking IL13Rα2 were insensitive to the highest treatment (SU-DIPG-IV IC50 > 2600 ng / ml).

[0063] Example 2: Administration of cmIL13 before radiation enhances the antitumor effect on DIPG and GBM cells in vitro On Day 1, DIPG and GBM cells were seeded in well plates and allowed to attach to the plates. 25,000 SF8628 cells (DIPG cell line), 50,000 U87-MG cells (GBM cell line), and 50,000 DIPG IV cells (DIPG cell line) were seeded in 6-well plates, respectively. The SF8628 cells were treated in triplicate with 1 ng / mL of cmIL13, the U87-MG cells were treated in triplicate with 50 ng / ml of cmIL13, and the DIPG IV cells were treated in triplicate with 0.25 ng / mL. Then all plates were placed in an incubator. 16 hours after cmIL13 administration, the cells were irradiated with a single dose of X-ray radiation (2 Gy for SF8628 cells, 10 Gy for U87-MG cells, and 2 Gy for DIPG IV cells). The plates were returned to the incubator and the cell numbers were counted with a hemocytometer 72 hours, 96 hours, and 120 hours after radiation.

[0064] Figure 3 shows the results of the SF6828 DIPG cell assay, Figure 4 shows the results of the U87-MG GBM assay, and Figure 5 shows the results of the DIPG IV assay. All test cell types had a decrease in total cell number when treated with radiotherapy following cmIL13, compared to untreated cells or cells treated with either cmIL13 or radiotherapy alone. An additive effect was seen when radiotherapy followed cmIL13 treatment, with the lowest total cell numbers in all treatments investigated. These results also demonstrate that cmIL13 does not negatively impact the effects of radiation.

[0065] Example 3: The combination of cmIL13 and radiation induces enhanced caspase - mediated cell death 100,000 SF8628 cells were seeded into five separate 60 mm plates. The cells in each plate were treated under one of the following five conditions: (1) vehicle, (2) vehicle followed by treatment with a single dose of 4 Gy X-ray radiation 24 hours after vehicle administration, (3) treatment with 1 ng / mL cmIL13, (4) treatment with 2.5 ng / mL cmIL13, or (5) treatment with 1 ng / mL cmIL13 followed by treatment with a single dose of 4 Gy X-ray radiation 24 hours after cmIL13 administration. SF8628 cells were treated in triplicate with 1 ng / mL cmIL13. Then, after radiation treatment, all plates were placed in an incubator for 72 hours.

[0066] Tumor cells were lysed and sonicated in Triton X-100 lysis buffer containing protease inhibitor. The collected protein lysates were stored at -20°C. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Cat#23227; Thermo Fisher Scientific). 30 μg of total protein was size fractionated by 12.5% SDS-PAGE. Proteins separated by electrophoresis were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane, washed in PBST buffer, blocked with 2% non-fat milk for 1 h at room temperature, and then incubated overnight at 4°C with the primary antibody. Following primary antibody blotting, specific signals were detected with a peroxidase-conjugated secondary antibody (Thermo Fisher Scientific) appropriate for the species using the SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat#34580; Thermo Fisher Scientific), and imaged using a BioRad GelDoc XR imaging system (BioRad, USA). Details regarding the antibodies used for Western blot are shown in Table 1.

[0067]

Table 1

[0068] Figure 6 provides data collected from Western blot. As shown in Figure 6, tumor cells treated with combination therapy of 4 Gy radiation following 1 ng / mL of cmIL13 showed strong staining for cleaved caspase 3 and cleaved caspase 7, apoptosis markers. These data indicate that treating cells with cmIL13 prior to radiotherapy resulted in a substantial increase in cell death compared to other treatment conditions.

[0069] Example 4: cmIL13 plus radiation inhibits colony formation of DIPG cells The SF8628 cells were seeded in 6-well plates at a density empirically determined based on the plating efficiency for each radiation dose that would result in non-overlapping cell colonies after 14 days. The densities were as follows: 0 Gy - 500 cells per well; 2 Gy - 1000 cells per well; 4 Gy - 3000 cells per well; and 6 Gy - 8000 cells per well. The SF8628 cells were treated with either solvent or 0.5 ng / mL of cmIL13. Twenty-four hours after cmIL13 or solvent treatment, the SF8628 cells were treated with either 0 Gy, 2 Gy, 4 Gy, or 6 Gy of radiation and incubated for 14 days. At the end of the incubation, the cells were fixed with ice-cold methanol and stained with crystal violet. For all conditions, colonies with more than 50 cells were counted. As shown in Figure 7, pre-radiation co-treatment with cmIL13 substantially decreased the survival rate of SF8628 cells compared to treatment with radiation alone. Thus, pre-radiation treatment with cmIL13 resulted in enhanced anti-tumor effects.

[0070] Example 5: Treatment of SU - DIPG - XVII malignant glioma in mice with cmIL13 and radiation Preparation of tumors : SU-DIPG-XVII cells were directly injected into the pons of mice. The presence of tumors was determined by MRI. Animals with confirmed tumors were divided into the following four treatment groups. (1) Solvent pump + sham radiation, (2) cmIL13 pump + sham radiation, (3) solvent pump + radiation, or (4) cmIL13 pump + radiation.

[0071] c Administration of mIL13 : ALZET® osmotic pumps (model 2001; infusion rate: 1 μL / hr) were prepared according to the manufacturer's instructions. Each pump received a solution of 0.005 mg / mL of cmIL13 (total dose 1 μg) or solvent. The pumps were connected to catheters that were inserted into a 30-gauge cannula (protruding 5 mm under the pedestal) filled with the drug solution.

[0072] The animals underwent Alzet pump implantation surgery approximately 85 days after tumor transplantation. The animals were anesthetized and a midline incision was made to expose the skull. Using a Foredom MH-130 portable drill (Foredom), a burr hole was drilled at coordinates 1 mm posterior to the lamboid suture, 1 mm lateral to the mid-sagittal plane, and a depth of 4.2 mm. Next, a subcutaneous pocket was created between the scapulae using a hemostatic agent at the posterior end of the incision. An ALZET® pump connected to an infusion cannula via a vinyl catheter was inserted into the subcutaneous pocket. A dental glue primer (Kulzer) was applied to the skull surrounding the burr hole using a 0.3 mL insulin syringe (BD), and the primer was activated by UV light. Each animal was placed in a stereotaxic frame and an ALZET® cannula (Plastics One, 7 mm below the pedestal) was inserted using a stereotaxic insertion arm. A dental adhesive (Ivoclair Vivadent) was used to attach the guide cannula to the skull. The skin incision was closed with wound clips. Then, cmIL13 or vehicle was continuously administered over a 7-day period at an infusion rate of 1 μL / h such that the total dose for the treatment group receiving cmIL13 was 1 μg. The ALZET® pump was removed 8 days after pump implantation. The animals were anesthetized and a small incision was made between the scapulae near the pump. The catheter connection was removed, sealed with bone wax, and the pump was removed. The incision was closed with 4.0 Vicryl sutures (Ethicon).

[0073] Radiation treatment: Two days after the start of cmIL13 or solvent administration, animals in treatment groups (1) and (2) were exposed to sham radiation, and treatment groups (3) and (4) were exposed to 2 Gy of ionizing radiation for five consecutive days using image-guided focused radiation. Radiation was delivered by using an X-RAD SmART irradiator that enables image-guided focused implementation with shielding. To deliver the radiation, mice were anesthetized with isoflurane and fixed head-side up on the positioning stage using a bite block without pins.

[0074] After treatment, the animals were monitored for animal survival rate. The results can be found in Figure 8. As shown, treatment with cmIL13 before radiation substantially increased the survival rate of the mice.

[0075] Although specific embodiments of the present invention have been described in detail, it will be recognized by those skilled in the art that various modifications and alternative forms can be developed in light of the overall teachings of the disclosure. Accordingly, the specific arrangements disclosed are merely illustrative and are not meant to be limitations on the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalents thereof.

Claims

1. A pharmaceutical composition for treating cancer, which is administered to the site of cancer in a patient undergoing radiation therapy, comprising a pharmaceutical composition containing a therapeutically effective amount of mutagenic IL13 moiety (mIL13).

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered to the site of cancer in the subject during the radiotherapy.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered to the site of cancer in the subject before the radiotherapy.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered via a catheter and / or by convection-enhanced delivery.

5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is administered via a catheter and / or by convection-enhanced delivery using stereotactic image-guided catheter insertion.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered acutely.

7. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered by convection-enhanced delivery at a flow rate of up to 2 mL / hour.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein the therapeutically effective dose of mIL13 is 0.03 μg / mL to 1 μg / mL.

9. The pharmaceutical composition according to any one of claims 1 to 3, wherein the expression of interleukin 13 receptor α2 (IL13Rα2) in the cancer is detected.

10. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered into a tumor and / or a resection cavity.

11. The pharmaceutical composition according to any one of claims 1 to 3, wherein the mIL13 is fused with a cytotoxin.

12. The pharmaceutical composition according to claim 11, wherein the cytotoxin includes a toxin derived from bacteria.

13. The aforementioned mIL13, Compared to wild-type human IL-13, it is engineered to have increased affinity for IL-13Rα2 and / or decreased affinity for interleukin 13 receptor α1 (IL-13Rα1) compared to wild-type human IL-13; It contains at least one amino acid substitution when compared to SEQ ID NO: 1 or SEQ ID NO: 2; This includes amino acid modifications at positions E13, R66, S69, and / or K105 relative to wild-type IL13; It comprises one or more amino acid substitutions E13K, R66D, S69D, and K105R; The amino acid sequence represented by one of SEQ ID NO: 3 to 24 or its homologue, or comprising either SEQ ID NO: 1 or SEQ ID NO: 2 homologue; and / or SEQ ID NO: 1 or SEQ ID NO: 2 differs from residues 20 or less. The pharmaceutical composition according to claim 11.

14. The pharmaceutical composition according to any one of claims 1 to 3, wherein the cancer includes a malignant glioma.

15. The pharmaceutical composition according to any one of claims 1 to 3, wherein administration of the pharmaceutical composition to the site of cancer in a subject undergoing radiotherapy results in an increase in the survival rate of the subject compared to a control subject having a malignant glioma that has not received radiotherapy and a pharmaceutical composition containing a therapeutically effective amount of mIL13.

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