Methods for Treating Malignant Gliomas

Abstract

A method of treating a subject having a malignant glioma is disclosed, the method comprising administering a therapeutically effective amount of cytotoxin-linked mutagenized IL13 (cmIL13) by convection-enhanced drug delivery (CED) for up to 96 hours, the malignant glioma expressing interleukin-13 receptor alpha 2 (IL13Rα2). Also disclosed is mutagenized IL13 for use in a method of treating a subject having a malignant glioma expressing IL13Rα2, the method comprising administering a therapeutically effective amount of cytotoxin-linked mutagenized IL13 (cmIL13) by convection-enhanced drug delivery (CED) for up to 96 hours.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 277,866, filed November 10, 2021, which is incorporated herein by reference.

[0002] Technical Field The present disclosure relates to cancer treatment. [Background technology]

[0003] The prognosis of malignant gliomas remains poor due to the lack of effective therapeutic treatments. As current treatment modalities for certain cancers have limited effectiveness, there is a significant need to develop effective and novel treatments for patients suffering from these diseases. Summary of the Invention [Means for solving the problem]

[0004] Disclosed herein is a method of treating a subject having malignant glioma, the method comprising administering a therapeutically effective amount of cytotoxin-linked mutagenized IL13 (mIL13) (cmIL13) by convection-enhanced delivery (CED) for up to 96 hours, wherein the malignant glioma expresses interleukin-13 receptor alpha 2 (IL13Rα2).

[0005] Also disclosed herein is a cytotoxin-linked mutated IL13 (cmIL13) for use in a method of treating a subject having a malignant glioma that expresses IL13Rα2, the method comprising administering a therapeutically effective amount of cmIL13 by convection-enhanced drug delivery (CED) for up to 96 hours. [Brief description of the drawings]

[0006] [Figure 1A]RNA sequencing of diffuse midline glioma (DMG) and adult glioblastoma (GBM) cell models is shown. [Figure 1B] Immunoblots of DMG and GBM cell models are shown. [Figure 2A] Cell proliferation and dose response curves of the DMG cell line are shown as percentage response as a function of cmIL13 dose. [Figure 2B] Cell viability and dose response curves for DMG cell lines are shown as percentage response as a function of cmIL13 dose. [Figure 2C] 1 shows a graph of the inverse relationship between IL13Rα2 expression in DMG cells and sensitivity to cmIL13. [Figure 2D] Immunoblots of SU-DIPG XIII-P, SF8628, and PED17 cell lines after 8, 24, 48, and 72 hours of exposure to wild-type IL13 (10 ng / mL) are shown. [Figure 2E] Immunoblots of SU-DIPG XIII-P, SF8628, and PED17 cell lines are shown after 8, 24, 48, and 72 hours of cmIL13 (cell line specific IC50) exposure. [Figure 2F] Immunofluorescence staining of SF8628 cells 72 hours after treatment with IC50 of cmIL13 is shown. [Diagram 3] Cell viability and dose response curves of adult GBM cell lines are shown as percent response as a function of cmIL13 dose. [Figure 4] The IC50 values ​​of cmIL13 on DMG and GBM cell lines are shown as a function of IL-13Rα2 expression. [Figure 5A] FIG. 1 is a schematic diagram of the workflow of tumor cell injection and CED. [Figure 5B] Bioluminescence (BLI) signal is shown as a function of days after injection of 1 μg cmIL13 into GBM6-bearing animals. [Figure 5C]The increased survival of GBM6-bearing animals is shown by percent survival as a function of days after injection of 1 μg of cmIL13. [Figure 5D] BLI signal as a function of days after injection of 1 μg cmIL13 into PED17 xenografts. [Figure 5E] The increased survival of PED17 xenografts is shown by percent survival as a function of days after injection of 1 μg of cmIL13. [Figure 5F] BLI signal as a function of days after injection of 1 μg cmIL13 in SU-DIPG-XIII-P animals. [Figure 5G] The increased survival of SU-DIPG-XIII-P animals is shown by percent survival as a function of days after injection of 1 μg of cmIL13. [Figure 6A] Immunohistochemistry of HGG-bearing mouse brains harvested on days 55 (control) and 86 (experimental) following CED of vehicle solution or 1 μg cmIL13, respectively. [Figure 6B] IHC analysis of IL-13Rα2 levels, apoptosis induction, and cell proliferation in mice treated with cmIL13 compared to controls. [Figure 6C] The density of cells containing Ki-67, cleaved caspase 3, and NeuN for control and 1 μg cmIL13 treated mice is shown. [Figure 7A] Immunofluorescence (IF) of SF8628 cells 8, 24, 48, and 72 hours after exposure to cmIL13 at IC50 is shown. cmIL13 is shown in red, IL-13Rα2 in green, and DAPI in blue. [Figure 7B] IF of SF8628 cells exposed to cmIL13 at IC50 at 8, 24, 48, and 72 hours is shown. Cleaved caspase 3 is shown in green, Ki-67 in red, and DAPI in blue. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] 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. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the testing of this disclosure, the preferred materials and methods are described herein.

[0008] For the purposes of the following detailed description, unless expressly indicated otherwise, it should be understood that the present disclosure can assume various alternative variations and sequence of steps. Moreover, except in the operational examples or unless otherwise indicated, all numbers, such as numbers expressing values, amounts, percentages, ranges, subranges, and fractions, can be read as if they were prefixed with the word "about" even if not expressly indicated. Thus, unless otherwise indicated, the numerical parameters set forth in the following specification and appended claims are approximations and may vary depending on the desired results sought to be obtained by the present disclosure. At the very least, and without any attempt to further limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of significant digits and by applying ordinary rounding techniques. When closed-ended or open-ended numerical ranges are set forth herein, all numbers, values, amounts, percentages, subranges, and fractions that are within or encompassed by that numerical range are to be considered as specifically included and included in the original disclosure of this application, as if those numbers, values, amounts, percentages, subranges, and fractions were expressly set forth in their entirety.

[0009] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0010] As used herein, unless otherwise indicated, a plural term can include its corresponding singular term and vice versa. For example, although the present specification refers to "a" mutagenized IL13, or "a" cytotoxin, combinations (i.e., a plurality) of those components can be used. Furthermore, in this application, even if "and / or" can be explicitly used in certain cases, the use of "or" means "and / or" unless specifically stated otherwise.

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

[0012] As used herein, "consisting of" is understood to exclude the presence of any not specified elements, components, and / or method steps within the context of this application.

[0013] As used herein, "consisting essentially of" is understood in the context of this application to include the elements, materials, components, and / or method steps specified, as well as those that "do not materially affect the basic and novel characteristics" of what is being described.

[0014] As used herein, the terms "patient," "subject," "individual," and the like are used interchangeably and refer to any animal or cells thereof, whether in vitro or in situ, including mammals, including humans, dogs, cats, cats, cows, horses, pigs, primates, and / or rodents.

[0015] As used herein, the term "interleukin-13" or "IL13" refers to any native or wild-type IL13 from any vertebrate source, including mammals such as primates and rodents, unless otherwise indicated, and includes any naturally occurring variant of IL13, such as unprocessed IL13 and any form of IL13 resulting from processing in cells, as well as splice or allelic variants. An exemplary amino acid sequence of human IL13 is set forth in SEQ ID NO:1. A second exemplary amino acid sequence of human IL13 is set forth in SEQ ID NO:2.

[0016] A "mutation" in a polypeptide is intended to encompass proteins having any amino acid substitution, deletion (e.g., truncated versions of proteins, such as peptides), insertion, and / or modification by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation, etc. In one example, "mutated IL13" or "mutagenized IL13" refers to IL13 in which one or more of the amino acids differ from the corresponding amino acids in native IL13. Mutant and / or mutagenized IL13 may be derived from native IL13 found in humans, non-human primates, rats, mice, pigs, cows, dogs, etc. Mutant and / or mutagenized IL13 may be referred to herein as "mIL13."

[0017] As used herein, the term "IL-13 receptor" or "IL13R" refers to a receptor that binds IL-13.

[0018] As used herein, the term "IL-13 receptor alpha 2" or "IL13Rα2" refers to the monomeric IL13 receptor that is expressed on the surface of specific cell subsets and binds IL13.

[0019] As used herein, the term "cmIL13" or "cmIL-13" refers to cytotoxin-linked mutagenized IL13.

[0020] As used herein, "treat," "treatment," or "treating" refers to a therapeutic measure provided to a patient or subject with the intent of preventing the onset of or altering a pathology or symptoms experienced by the patient or subject, such as those caused by a disease. A "treatment" administered to a patient or subject can achieve any clinically or quantitatively measurable decrease in the condition for which the patient or subject is being treated, including complete elimination.

[0021] As used herein, a "therapeutically effective amount" is defined as an amount that, when administered to a patient for treating a disease (e.g., cancer), is sufficient to treat the disease. For example, a therapeutically effective amount of a compound for treating cancer can be, for example, an amount sufficient to reduce the volume of a malignant tumor or to increase the survival time of a patient.

[0022] As used herein, a "cytotoxin" is defined as a substance, such as a toxin or antibody, that interferes with the function of cells, causes destruction of cells, or both.

[0023] As used herein, the term "affinity" is defined as the strength of the total non-covalent interactions between a single binding site of a receptor and a ligand. The affinity of a receptor for a ligand is determined by the dissociation rate constant, K off and the association rate constant K on The dissociation constant (K D Affinity can be measured by methods known to those skilled in the art.

[0024] "Increased binding" refers to a binding level of mIL13 that is at least 10% or more, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more higher than wild-type IL13, or 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 100-fold, 1000-fold or more higher, and any whole or partial increment therebetween.

[0025] "Decreased binding" refers to binding levels of mIL13 that are at least 10% or more, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more lower than wild-type IL13, or 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 100-fold, 1000-fold or more lower, and any whole or partial increment therebetween.

[0026] As used herein, the term "link" is defined as the chemical attachment (covalent or non-covalent) of one gene to a second gene, for example, by splicing or fusion. As used herein, the term "linking" is defined as the chemical attachment (covalent or non-covalent) of one gene to a second gene. As used herein, the term "conjugation" is defined as the post-translational linking of one gene to a second gene. As used herein, the term "fusion" is defined as the pre-translational linking of one gene to a second gene.

[0027] As used herein, the term "control" or "reference" or "comparator control" is defined as a subject to which cmIL13 is not administered via convection-enhanced drug delivery, such that a control or reference standard can serve as a comparator to which experimental samples can be compared.

[0028] As used herein, the term "determining the expression level of a marker (or biomarker)" is intended to assess the degree of expression or presence of a marker in a sample at the nucleic acid or protein level using techniques available to one of skill in the art to detect a sufficient portion of any marker expression product, such as "determining the level of IL13Rα2."

[0029] As used herein, the "level" of one or more markers (or biomarkers) refers to the absolute or relative amount or concentration of the marker (or biomarker) in a sample.

[0030] "Measuring" or "measurement," or alternatively, "detecting" or "detection," means evaluating the presence, absence, quality, or amount (which may be an effective amount) of any given substance in a clinical sample or a sample from a subject, including deriving a qualitative or quantitative concentration level of such substance, or otherwise assessing the value or categorization of a clinical parameter of the subject.

[0031] "Sample" or "biological sample," as used herein, refers to biological material separated from an individual, such as a liquid or solid biological sample obtained by biopsy (i.e., "liquid biopsy" or "solid biopsy"). Liquid biopsies can include, for example, blood, plasma, saliva, urine, cerebrospinal fluid, and / or other bodily fluids. Liquid biopsies can contain extracellular vesicles and / or acellular genetic material. Solid biopsies can include, for example, tissues, such as organs and / or tumors. Biological samples can contain any biological material suitable for detecting the desired biomarkers and can include cellular and / or non-cellular material obtained from an individual.

[0032] The term "cancer" as used herein is defined as a disease characterized by abnormal proliferation of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include brain tumors, including but not limited to gliomas, in which tissue cells express IL13Rα2.

[0033] The term "glioma" is defined as a tumor that arises in the glial cells of the brain or spinal cord.

[0034] The present disclosure is directed to a method of treating a subject having malignant glioma, comprising, consisting essentially of, or consisting of administering a therapeutically effective amount of cytotoxin-linked mutagenized IL13 (cmIL13) by convection-enhanced drug delivery (CED) for up to 96 hours, wherein the malignant glioma expresses interleukin-13 receptor alpha 2 (IL13Rα2).

[0035] The present disclosure is also directed to a cytotoxin-linked mutagenized IL13 (cmIL13) for use in a method of treating a subject having a malignant glioma that expresses IL13Rα2, the method comprising administering a therapeutically effective amount of cmIL13 by convection-enhanced drug delivery (CED) for up to 96 hours.

[0036] The method comprises, consists essentially of, or consists of administration of mutated IL13 (mIL13). The mIL13 may be linked to a cytotoxin (cmIL13).

[0037] mIL13 can be modified to have increased affinity for IL13Rα2 compared to native human IL13 and / or decreased affinity for interleukin-13 receptor alpha 1 (IL13Rα1) compared to native human IL13. For example, mIL13 can have a reduced affinity, e.g., kinetic K, for IL13Rα1 compared to the wild-type polypeptide, while at least maintaining binding or activity for IL13Rα2. D In some examples, the affinity of mIL13 for IL13Rα1 can be reduced by 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or more than 2-fold, while maintaining binding or activity of IL13Rα2. D In some cases, the affinity of mIL13 for IL13Rα2 is at least maintained, or in some cases is reduced, e.g., by a kinetic K D It can be increased by 2 times, 3 times, 5 times, 10 times, 100 times, or even 1000 times or more.

[0038] In some examples, mIL13 can be produced by cDNA mutagenesis, DNA synthesis, peptide / protein synthesis, or any method known to one of skill in the art.

[0039] mIL13 can be a full-length IL13 molecule, such as a human full-length IL13 molecule. In some examples, mIL13 can include an amino acid change relative to wild-type IL13 at a position corresponding to residue 13 of human IL13 (SEQ ID NO:1), at a position corresponding to residue 66 of SEQ ID NO:1, at a position corresponding to residue 69 of SEQ ID NO:1, and / or at a position corresponding to residue 105 of SEQ ID NO:1. mIL13 can include a substitution for glutamic acid at position 13 of human IL13 (SEQ ID NO:1). For example, glutamic acid at position 13 can be substituted with lysine. mIL13 can include a substitution for arginine at position 66 of human IL13 (SEQ ID NO:1). For example, arginine at position 66 can be substituted with aspartic acid. mIL13 can include a substitution for serine at position 69 of human IL13 (SEQ ID NO:1). Serine at position 69 can be substituted with aspartic acid. mIL13 can include a substitution for lysine at position 105 of human IL13 (SEQ ID NO:1). The lysine at position 105 may be substituted with arginine. mIL13 may include changes at positions E13, R66, S69, and / or K105. mIL13 may be referred to as IL13.E13K.R66D.S69D.K105R. In one example, mIL13 may include the amino acid sequence set forth in SEQ ID NO:3. In other examples, mIL13 may include any of SEQ ID NOs:4-22, or may include 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 may include a sequence that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more residues from any of SEQ ID NOs:1-24. In some examples, U.S. Patent Application No. 15 / 597,823, filed May 17, 2017, entitled "Therapeutic IL13 Polypeptides," cols. 2, 1. 16-47 and cols. 2, 1. 56-cols. 3, 1. 3, which are incorporated herein by reference, disclose wild-type IL13 (disclosed herein as SEQ ID NO: 2), and specific mutations in IL13Rα2, provided herein as SEQ ID NOs: 3-24.The terms "homology" and "homologous" refer to the identity of subunit sequences between two molecules, such as two protein or peptide molecules. When a position of a subunit in both molecules is occupied by the same subunit, the molecules are homologous at that position. The homology between two sequences is a linear function of the number of matching or homologous positions. For example, if half of the positions of two sequences are homologous, the two sequences are 50% homologous, and if 70% (i.e., 7 out of 10) of the positions are matching or homologous, the two sequences are 70% homologous. Homologs of the present disclosure can be the result of natural allelic variation, including natural mutations. Homologs of the present disclosure can also be generated using techniques known in the art, including direct modifications to proteins, for example, using recombinant DNA techniques to perform random or targeted mutagenesis.

[0040] mIL13 can be complexed with extracellular vesicles (EVs) to form extracellular vesicle complexes (EV complexes). Extracellular vesicles can be obtained from a certain type of biological sample (urine, serum, plasma, cerebrospinal fluid, organs, tissues, etc.) and / or can be collected from a certain type of cell, such as glioma stem cells. In some examples, the glioma stem cells can be mesenchymal glioma stem cells or proneural glioma stem cells. The extracellular vesicles can include exosomes, such as tumor-associated exosomes. The extracellular vesicles can be purified or enriched from biological samples using differential centrifugation, ultracentrifugation, and / or other methods known to those skilled in the art.

[0041] Subpopulations of extracellular vesicles can be separated by using biological markers. The biological marker can be a receptor, such as a tumor-associated receptor. The tumor-associated receptor can be IL13Rα2. Each exosome can express, for example, 1, 2, 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000 or more biological markers.

[0042] A cytotoxin can be linked to one of the mIL13s disclosed herein to form cmIL13. For example, linking can occur by EDC chemistry or purification gel filtration.

[0043] 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.

[0044] A therapeutically effective amount of cmIL13 can be administered by convection-enhanced drug delivery (CED). CED involves inserting at least one catheter, for example, into the interstitial space of the brain, into a resection cavity of the brain, or directly into an intact brain tumor. A person skilled in the art can select an appropriate catheter for delivery of cmIL13 by CED. As used herein, "convection-enhanced drug delivery" or "CED" is defined as a low-flow positive pressure injection method that directly administers a therapeutic agent to a structure to be treated, for example, a malignant glioma tumor or a resection cavity. cmIL13 can be administered by convection injection at a defined flow rate controlled by an external syringe pump. The method includes positioning the tip of the catheter in the area to be treated. An external pump can be connected to the catheter, thereby delivering a therapeutically effective dose of a therapeutic agent, for example, a composition containing cmIL13, while maintaining a positive pressure gradient. Administration of cmIL13 may or may not be accompanied by co-injection of an imaging moiety that serves as a surrogate marker for drug delivery, for example, gadolinium or gadolinium-DTPA.

[0045] Administration of cmIL13 can occur over a period of at least 4 hours. In some examples, administration of cmIL13 can occur over a period of 4 to 96 hours.

[0046] cmIL13 may be administered at a dose of at least 0.03 μg / mL up to and including 1 μg / mL. Administration of cmIL13 may be administered at a flow rate of up to 1 mL / hour.

[0047] The malignant gliomas treated by the methods disclosed herein may comprise, consist essentially of, or consist of high-grade gliomas. Examples of malignant gliomas that can be treated using the methods disclosed herein include, but are not limited to, anaplastic astrocytomas, such as adult glioblastomas, pediatric glioblastomas, diffuse midline gliomas, anaplastic oligodendroglioma, anaplastic oligoastrocytoma, anaplastic ependymoma, and / or anaplastic ganglioneuroma.

[0048] The malignant glioma treated by the disclosed methods may express an IL13-specific receptor. The IL13-specific receptor may include interleukin-13 receptor alpha 2 (IL13Rα2). Expression of IL13Rα2 may be detected in a sample of the malignant glioma prior to administration of cmIL13. In some examples, the sample of the malignant glioma may be purified prior to detection of expression of IL13Rα2. Detection of expression of IL13Rα2 may be detected by mass spectrometry, analytical assays, immunostaining, and / or sequencing. In some examples, the analytical assay may include enzyme-linked immunosorbent assay (ELISA). In other examples, the immunostaining may include immunohistochemistry (IHC) and / or fluorescent cytochemistry. In other examples, the sequencing includes whole genome sequencing, exome sequencing, proteomic sequencing, and / or RNA sequencing. Expression of IL13Rα2 by malignant gliomas indicates that they will respond to treatment with cmIL13.

[0049] Surprisingly, it has been found that the methods of treating malignant glioma described herein improve the survival rate of subjects with malignant glioma relative to control subjects with malignant glioma who are not treated with the methods described herein. The improved survival rate can be determined, for example, by Kaplan-Meier survival curve analysis. Survival curve analysis includes, but is not limited to, a comparison of survival probability between subjects receiving cmIL13 treatment and a control, where survival probability (S t) is defined as [number of subjects alive at start-number of subjects dead] / number of subjects alive at start. The data obtained are plotted on a graph where the survival probability is on the y-axis and the time since entering the study is on the x-axis. Statistical differences between at least two different graphed data sets can be determined by either a log-rank test, where a chi-square is calculated for each event time in each group and the results are summed, or by a hazard ratio, where a chi-square is calculated for each event time and the results are summed to give the final observed and predicted results for the entire curve. Statistical significance is defined as p ≤ 0.05.

[0050] Surprisingly, it has also been found that the methods of treating malignant glioma described herein do not result in detectable changes in clinically relevant biomarkers in healthy cells and / or healthy tissues (e.g., non-glioma cells and tissues). Examples of clinically relevant biomarkers include, but are not limited to, NeuN and CD68+ protein.

[0051] Finally, it is surprisingly found that the method of treating malignant glioma disclosed herein can reduce the volume of malignant glioma.The reduction of glioma volume can be determined by statistical analysis known to those skilled in the art, for example, by one-way or two-way analysis of variance (ANOVA) test or Student's two-tailed t-test, and statistical significance can be determined by p<0.05.Therefore, the method of treating malignant glioma described herein can effectively treat malignant glioma without harming healthy cells and tissues.

[0052] Aspects Certain non-limiting aspects of the present disclosure are summarized below.

[0053] Aspect 1 1. A method of treating a subject having a malignant glioma, comprising: Therapeutically effective doses of cytotoxin-linked mutated IL13 (mIL13) (cmIL13) administered by convection-enhanced drug delivery (CED) for up to 96 hours Including, The method comprises the steps of: (a) expressing interleukin-13 receptor alpha 2 (IL13Rα2);

[0054] Aspect 2 The method of embodiment 1, wherein cmIL13 is administered over a period of 4 to 96 hours.

[0055] Aspect 3 The method of embodiment 1 or embodiment 2, wherein cmIL13 is administered at a dose of 0.03 μg / mL to 1 μg / mL.

[0056] Aspect 4 The method of any of the preceding embodiments, wherein the administration of cmIL13 is administered at a flow rate of up to 1 mL / hr.

[0057] Aspect 5 The method of any of the preceding embodiments, wherein the cmIL13 is modified to have increased affinity for IL13Rα2 relative to native human IL13 and / or decreased affinity for interleukin-13 receptor alpha 1 (IL13Rα1) relative to native human IL13.

[0058] Aspect 6 The method of any of the preceding embodiments, wherein the cmIL13 comprises amino acid changes relative to wild-type IL13 at positions E13, R66, S69, and / or K105.

[0059] Aspect 7 The method of any of the preceding embodiments, wherein the cmIL13 comprises one or more amino acid substitutions E13K.R66D.S69D.K105R.

[0060] Aspect 8 The method of any of the preceding embodiments, wherein cmIL13 comprises the amino acid sequence set forth in one of SEQ ID NOs: 3 to 24 or a homolog thereof, or a homolog of one of SEQ ID NO: 1 or SEQ ID NO:2.

[0061] Aspect 9

[0023] The method of any of the preceding embodiments, wherein cmIL13 differs from SEQ ID NO:1 or SEQ ID NO:2 by no more than 20 residues.

[0062] Aspect 10 8. The method of any preceding embodiment, wherein the cytotoxin comprises a bacterially derived toxin.

[0063] Aspect 11 11. The method of embodiment 10, wherein the bacterial-derived toxin comprises Pseudomonas exotoxin A.

[0064] Aspect 12 The method of any of the preceding aspects, wherein the malignant glioma comprises high grade glioma.

[0065] Aspect 13 The method of any of the preceding aspects, wherein the malignant glioma comprises adult glioblastoma, pediatric glioblastoma, anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic oligoastrocytoma, anaplastic ependymoma, and / or anaplastic ganglioneuroma.

[0066] Aspect 14 The method of embodiment 13, wherein the anaplastic astrocytoma comprises diffuse midline glioma.

[0067] Aspect 15 The method of any preceding aspect, further comprising co-injection of an imaging moiety.

[0068] Aspect 16 16. The method of embodiment 15, wherein the imaging moiety comprises gadolinium, gadolinium-DTPA, or a combination thereof.

[0069] Aspect 17

[0023] The method of any of the preceding aspects, further comprising detecting expression of IL13Rα2 in the sample, wherein expression of IL13Rα2 indicates that the malignant glioma will respond to treatment with cmIL13.

[0070] Aspect 18 The sample is Liquid biopsies of blood, plasma, saliva, urine, and / or cerebrospinal fluid, and / or Solid biopsy of organs and / or tissues 20. The method of embodiment 17, comprising:

[0071] Aspect 19 The method of embodiment 18, wherein the tissue comprises a tumor.

[0072] Aspect 20 20. The method of embodiment 19, wherein the tumor comprises a malignant glioma.

[0073] Aspect 21 21. The method of any of embodiments 18 to 20, wherein the liquid biopsy comprises extracellular vesicles and / or cell-free genetic material.

[0074] Aspect 22 22. The method of any of embodiments 18 to 21, further comprising purifying the sample.

[0075] Aspect 23 The method according to any of embodiments 18 to 22, wherein the detection of the expression of IL13Rα2 is carried out by mass spectrometry, analytical assay, immunostaining, and / or sequencing.

[0076] Aspect 24 (a) the analytical assay includes an enzyme-linked immunosorbent assay (ELISA); (b) the immunostaining method includes immunohistochemistry (IHC) and / or fluorescent cytochemistry; and / or (c) the sequencing comprises whole genome sequencing, whole exome sequencing, RNA sequencing, and / or proteomics.

[0077] Aspect 25 The method of any of the preceding embodiments, wherein the administration improves survival of the subject relative to a control subject having a malignant glioma to which the therapeutically effective amount of cmIL13 is not administered.

[0078] Aspect 26 The method of embodiment 25, wherein the improved survival is determined by Kaplan-Meier survival curve analysis.

[0079] Aspect 27 13. The method of any of the previous aspects, wherein the method does not result in a detectable change in a clinically relevant biomarker in non-glioma cells and / or tissue.

[0080] Aspect 28 28. The method of embodiment 27, wherein the biomarkers comprise NeuN and CD68+ protein.

[0081] Aspect 29 Administration of cmIL13 to cells expressing IL13Rα2 resulted in (i) the volume of malignant gliomas is reduced; (ii) there is an increase in detectable cleaved caspase-3 as measured by IHC or fluorescent cytochemistry; and / or (iii) The method of any of the preceding embodiments, wherein the level of Ki-67 positive cells is reduced by at least 10%, as measured by IHC or fluorescent cytochemistry.

[0082] Aspect 30 The method of embodiment 29, wherein the reduction in the volume of the malignant glioma is determined by an analysis of variance (ANOVA) test, and statistical significance is determined by a p-value of <0.05.

[0083] Aspect 31 30. The method of embodiment 29, wherein the reduction in malignant glioma volume is determined by a two-tailed Student's t-test, with statistical significance being determined by a p-value of <0.05.

[0084] Aspect 32 The method of any of the preceding aspects, wherein the mutated IL-13 is characterized by at least one amino acid substitution when compared to SEQ ID NO:1 or SEQ ID NO:2.

[0085] Aspect 33 A cytotoxin-linked mutated IL13 (cmIL13) for use in a method for treating a subject having a malignant glioma expressing interleukin-13 receptor alpha 2 (IL13Rα2), the method comprising administering a therapeutically effective amount of cmIL13 by convection-enhanced drug delivery (CED) for up to 96 hours, and / or the method of treatment is further optionally characterized by any of the properties described in the preceding aspects.

[0086] The following examples illustrate the disclosure, but are not intended to limit the disclosure to the details of those examples. Unless otherwise specified, all parts and percentages in the following examples and throughout the specification are by weight. EXAMPLES

[0087] Research 1 material and method material cmIL13 (IL13.E13K‐PE4E) was obtained from Targepeutics, Inc. (Hershey, PA). cmIL13 was dissolved in phosphate-buffered saline (PBS) and stored at −80°C as a 2.6 mg / mL stock. Human IL13 recombinant protein (catalog number: A2525) was obtained from Invitrogen (Thermo Fisher Scientific). IL13 was dissolved in double-distilled water (ddH2O) according to the manufacturer's protocol and stored at −80°C as a 5 μg / mL stock.

[0088] Cell lines and culture Informed consent and Institutional Review Board approval was obtained for all patient-derived cell lines. Details regarding the cell lines are shown in Table 1.

[0089] [Table 1]

[0090] Early passage HGG lines were used, and all cell lines were verified annually by short tandem repeat DNA fingerprinting and tested for mycoplasma contamination every 3 months. Cell lines harboring H3K27M mutations were verified for histone expression of the K27M mutation every 3 months by Western blot and Sanger sequencing. All patient-derived tumor cell lines were maintained in cell line-appropriate media, the details of which are shown in Table 2.

[0091] [Table 2]

[0092] Cells cultured as neurospheres were passaged every 1-2 weeks. Cells cultured as adherent monolayers were passaged 1-2 times per week.

[0093] RNA sequencing and data analysis Total RNA was extracted from whole cell lysates using RNeasy Plus micro kit (Cat#74034; QIAGEN, Germantown, MD, USA) according to the manufacturer's instructions. RNA-seq studies were performed as single replicates to screen large libraries of cell lines. RNA library preparation and sequencing were performed by Novogene (Beijing, China). NEBNext UltraTM RNA Library Prep Kit for Illumina sequencers (New England Biolabs, Ipswich, MA, USA) was used for library preparation, and then cDNA libraries were size-selected using AMPure XP magnetic beads (Beckman Coulter, Pasadena, CA, USA). Samples were sequenced on a NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA) using single-end or paired-end sequencing depending on the time frame in which samples were available and the available sequencing technology. Paired-end sequencing data of adult GBM cell lines was obtained from cBioPortal. cBioPortal is a web-based free tool that contains RNA-seq data for brain tumor patient-derived xenografts from Mayo Clinic. FASTQC was used to quality assess the generated FASTQ files. Trimmed reads were mapped to hg38 using STARv2.7.3a and annotated gene counts were obtained using the -quantMode geneCounts function. Transcript counts (TPM) or reads per kilo base per million (RPKM) values ​​were calculated using RSEM for single-end or paired-end library status.

[0094] Immunoblotting Patient-derived tumor cells for immunoblotting were lysed in Triton X-100 lysis buffer containing protease inhibitors and sonicated. Collected protein lysates were stored at -20°C. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Cat#23227; Thermo Fisher Scientific). 15 μg of total protein was size-fractionated by 12.5% ​​SDS-PAGE. Electrophoretically separated proteins were electrically transferred to polyvinylidene difluoride (PVDF) membranes, washed with PBST buffer, blocked with 2% nonfat milk for 1 h at room temperature, and then incubated with primary antibodies overnight at 4°C. After primary antibody blotting, specific signals were detected with species-appropriate peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific) using SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat#34580; Thermo Fisher Scientific) and the signals were imaged using an Azure 600 Western Blot Imaging System (Azure Biosystems, Dublin, CA, USA). Details regarding the antibodies used for Western blots can be found in Table 3.

[0095] [Table 3]

[0096] Cell proliferation and viability assays Cells were seeded in single cell suspension at a concentration of 2,500 cells per well for adult GBM cell lines (GBM6, GBM10, GBM14, GBM39, GBM43, and GBM108) or 5,000 cells per well for DMG cell lines (SU‐DIPG XIII‐P, SU‐DIPG XVII, SF8628, SF8628‐B23, and PED17) in culture medium in 96‐well clear‐bottom black microplates (Cat #3917; Corning Costar, Corning, NY, USA) and cultured overnight at 37°C in 5% CO2. The next day, cells were treated in triplicate with vehicle (ddH20 or PBS) or serial dilutions of IL13 (to final concentrations of 100ng / mL, 50ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 1ng / mL, and 0.5ng / mL) or cmIL13 (to final concentrations of 320ng / mL, 100ng / mL, 32ng / mL, 10ng / mL, 3.2ng / mL, 1ng / mL, 0.32ng / mL, 0.1ng / mL, 0.032ng / mL, 0.01ng / mL, 0.0032ng / mL, and 0.001ng / mL). Cells were incubated for 72 hours and then assayed with the CellTiter-Glo Luminescent Cell Viability Assay (Cat#G7570; Promega, Madison, WI, USA) according to the manufacturer's recommendations. Luminescence was measured using an Infinite M200 PRO multimode microplate reader (Tecan Group, Maennedorf, Switzerland) and normalized to control wells (ddH20 or PBS only), and the relative luminescence treatment was plotted as a function of drug concentration. The efficacy of each treatment (50% inhibitory concentration, IC 50 ) was calculated by nonlinear least-squares curve fitting using Prism 9 (GraphPad, San Diego, CA, USA).

[0097] Immunofluorescence Cells were seeded in single cell suspension at a density of 10,000 cells per well on 4 Chamber Cell Culture Slides (Cat#50-114-9053; CELLTREAT Scientific Products, Pepperell, MA, USA) and cultured overnight at 37°C with 5% CO2. After 24 h, cells were incubated with vehicle (PBS) or IC40 as determined by CellTiter-Glo Luminescent Cell Viability Assay (Promega). 50 The cells were then washed with PBS at specific time points (8 h, 24 h, 48 h, and 72 h) and fixed with 4% paraformaldehyde for 20 min. The cells were washed three times for 5 min each with PBS and incubated with 0.5% Triton X-100 in PBS for 5 min. To wash the coverslips of the permeabilization buffer, the cells were incubated three times with PBS for 5 min each and then blocked with 3% BSA in PBS-T for 1 h at room temperature. Up to two different primary antibodies were then added in 1% BSA in PBST and left overnight at 4°C. Dilution buffer was used instead of the primary antibody for cell-specific negative controls. The next day, the cells were washed three times with PBS-T for 5 min each. The cells were then incubated with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) in 1% BSA in PBS-T for 1 h at room temperature in the dark. One control per primary antibody condition was included by applying other secondary antibodies to the primary antibodies to test for cross-reactivity. After three further 5 min washes with PBS, the chambers were removed and the slides were rinsed three times with dH20. Slides were mounted using ProLong Gold Antifade reagent with DAPI (Cat#P36935; Thermo Fisher Scientific) and stored at 37°C until imaging by microscope. All slides were examined and images were captured using an LSM780 confocal laser scanning microscope (Carl Zeiss Microscopy, White Plains, NY, USA). Detailed information on the antibodies used for immunofluorescence can be found in Table 3 above.

[0098] Patient-derived xenograft All animal studies were performed in accordance with NIH and IACUC guidelines for the use of laboratory animals and were approved by the Mayo Clinic Institutional Committee for Animal Research. HGG cell lines (GBM6, PED17, and SU-DIPG XIII-P) were transduced with a luciferase reporter system (eGFP / fLuc2) that allows a bioluminescent readout of tumor volume. Welby JP, Kaptzan T, Wohl A, Peterson TE, Raghunathan A, Brown DA, Gupta SK, Zhang L, Daniels DJ(2019)Current Murine Models and New Developments in H3K27M Diffuse Midline Gliomas, Front Oncol9:92 and Carlson BL, Pokorny JL, Schroeder MA, Sarkaria JN(2011)Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme(GBM) xenograft models for translational biology studies and drug discovery.Orthotopic tumor inoculation was performed with cultured cells as previously described in Curr Protoc Pharmacol Chapter 14:Unit 14.16, which are incorporated herein by reference. Briefly, cells were placed in single cell suspension to provide 300,000 cells in 3 μl of sterile PBS for engraftment in each mouse. Burr holes of 0.5 mm were made at the following coordinates: 1 mm posterior and 2 mm right of the bregma (GBM6) or 1 mm posterior and 1 mm lateral to the midsagittal plane of the lambda suture (PED17 and SU-DIPGXIII). Six- to seven-week-old female Hsd:Athymic Nude-Foxn1 mice were obtained from Envigo (Madison, WI, USA).nu Mutant mice were stereotaxically injected with tumor cells at a constant flow rate of 0.5 μl / min using a 26-gauge (51 mm, point style AS) syringe (Cat. #203185; Hamilton Company, Bonaduz, Switzerland) into the cerebral hemispheres (GBM6) or pons (PED17 and SU-DIPGXIII). The injection depth was 4 mm in all groups. In vivo tumor engraftment and progression were monitored by bioluminescence imaging (BLI). Animals were dosed with Cycluc 10 mg / kg by intraperitoneal injection. Ten minutes later, mice were imaged under soflurane anesthesia using an IVIS-200 imaging system (Xenogen Corporation, Berkeley, CA). Image analysis was performed using LivingImage 4.3 (PerkinElmer, Waltham, MA, USA) to quantify the total flux (photons per second) within the region of interest.

[0099] For brain-targeted drug delivery, animals were randomized into control (PBS) and treatment (cmIL13) groups based on the BLI signal to ensure equal distribution of tumor size at the beginning of the study (when BLI reached approximately 1,000,000 total log flux). Mice were placed under anesthesia with ketamine 100 mg / kg and xylazine 10 mg / kg. A 2 cm midline skin incision was made, extending from behind the eye to ear level. The previously made burr hole was reopened and the mouse was secured on a stereotaxic table with an automated heat assist using a Rodent Warmer X1 (Cat#53800M; Stoelting, Wood Dale, IL, USA). A 33-gauge inner cannula (Cat#8IC315IS5SPC; P1 Technologies, Roanoke, VA) protruding 4 mm below the base was inserted into a 26-gauge guide cannula (Cat#8IC315GS5SPC; P1 Technologies) protruding 3.5 mm below the base, both connected to PE tubing and secured with a single connector assembly (#C313C / SPC; P1 Technologies). The entire unit was secured vertically using a cannula holder (Cat#505254; World Precision Instruments, Sarasota, FL, USA) and connected to a 22-gauge (51 mm, point style AS) syringe (Cat#80400; Hamilton Company) placed in a Legato130 syringe pump (Cat#788130; KD Scientific, Holliston, MA, USA). Vehicle (PBS) and drug (cmIL13 at concentrations of 50 μg / ml (1 μg dose), 15 μg / ml (0.3 μg dose), or 5 μg / ml (0.1 μg dose)) solutions were then primed through the inner cannula and associated tubing. The cannula holder with the inner cannula attached was lowered until it was flush with the mouse skull to reach the desired injection depth of 4 mm (GBM6) or 4.2 mm (PED17 and SU-DIPGXIII).All study groups received the same incremental CED infusion protocol with a total volume infused of 20 μl and infusion rates as follows: 3 μl at 0.2 μl / min, 5 μl at 0.5 μl / min, and 12 μl at 0.8 μl / min. The cannula was removed 10 min after completion of the infusion to avoid backflow into the injection tubing. Animals were monitored daily and euthanized if they showed progressive neurological deficits or were found to be moribund.

[0100] Immunohistochemistry Brains were harvested after euthanasia of the animals by carbon dioxide inhalation and fixed in 4% paraformaldehyde overnight at room temperature. Brains were then embedded in paraffin and sectioned (5 μm / section) in the coronal plane using a microtome (CM1860UV; Leica Biosystems, Buffalo Grove, IL, USA). Hematoxylin and eosin (H&E) staining was performed according to standard procedures. For immunohistochemistry, paraffin-embedded tissue sections were dewaxed in xylene and rehydrated in ethanol. Antigen retrieval was performed by baking slides in preheated sodium citrate buffer (10 mM trisodium citrate, 0.05% Tween 20, pH 6.0) for 30 min. Sections were cooled to room temperature and rinsed in dH20 for 1 min. Sections were then immersed in 0.6% hydrogen peroxide in MeOH for 20 min. Sections were then blocked with 10% normal goat serum (NGS) in Tris-buffered saline (TBS) for 30 min at room temperature. Primary antibodies were diluted in 2% NGS and 0.5% Triton X-100 in TBS and added to sections overnight at 4°C. Dilution buffer was used instead of primary antibodies for tissue-specific negative controls. The next day, sections were washed three times for 5 min in 2% NGS and 0.5% Triton X-100 in TBS. Biotinylated secondary antibodies were added to sections using the VECTASTAIN Elite ABC kit (Cat# PK-6100; VECTOR Laboratory, Burlingame, CA) diluted in TBS with 1.5% NGS according to the manufacturer's recommendations. After three further 5-min washes in TBS, sections were incubated with avidin / biotinylated enzyme complex (ABC) solution (Cat#PK-6100; VECTOR Laboratory) for 30 min at room temperature.For visualization, sections were then developed using the SignalStain DAB Substrate Kit (Cat#8059P; Cell Signaling, Danvers, MA, USA) according to the manufacturer's protocol, counterstained with hematoxylin, and mounted with Permount (Cat#SP15‐100, Thermo Fisher Scientific). Images were acquired with a digital slide scanner (Axio Scan.Z1; Carl Zeiss Microscopy) and displayed at 40x magnification. Detailed information on the antibodies used for immunohistochemistry (IHC) is provided in Table 3 above. Low magnification images were included to show the consistency of staining in tissue sections.

[0101] statistical analysis Data were obtained and presented as the mean ± standard deviation or standard error where appropriate. Direct statistical comparisons between two groups were performed using a two-tailed Student's t-test. Nonlinear least-squares curve fitting analysis was used to estimate the efficacy of in vitro cmIL13 treatment (IC 50 ) was determined. Survival analysis was performed using Kaplan-Meier estimates with the log-rank test. Statistical tests and analyses were performed using Prism 9 (GraphPad), statistical significance was set at an alpha threshold of 0.05, and figures are marked with an asterisk at p<0.05.

[0102] result IL-13R.ALPHA.2 is expressed at different levels in HGG tumor cell models To identify baseline transcript and protein levels of IL-13Rα2 in HGG cells, RNA sequencing and immunoblot were performed on 10 patient-derived HGG cell lines (four DMG and six adult GBM) (Figure 1A, B). In accordance with previous studies, the sequenced cohort of HGG transcriptomes confirmed that IL-13Rα2 RNA expression patterns differed among HGG cell lines (Figure 1A). In both DMG and adult GBM models, IL-13Rα2 transcript levels ranged from low expression (SU-DIPG XIII-P, GBM39, GBM108) to intermediate expression (SU-DIPG XVII, SF8628, GBM43, GBM6) and high expression (PED17, GBM10, GBM14). We next assessed IL-13Rα2 protein levels in available HGG cell lines. Correspondingly, IL-13Rα2 protein levels were consistent with gene expression in both DMG and adult GBM cell lines (Figure 1B). Several cell models, including PED17, GBM10, GBM14, GBM59, and GBM118, showed high IL-13Rα2 expression, whereas other cell models, such as SU-DIPG XVII, SF8628, SF8628-B23, GBM6, GBM12, and GBM43, showed significantly (although not none) lower IL-13Rα2 levels. A third category of HGG cell lines, including SU-DIPG XIII-P, GBM39, GBM108, and GBM123, showed IL-13Rα2 protein levels below the detection threshold of the assay.

[0103] Functional impact of IL13Rα2 on proliferation and survival of HGGs Given the cell line-dependent overexpression of IL-13Rα2 in tumor cell models of DMG and adult GBM, we investigated the role of IL-13Rα2 signaling in HGG (Figures 2A-F). To determine whether cytokine stimulation affects cell proliferation in vitro, we treated HGG cells with various concentrations of IL13, the canonical ligand for IL-13Rα2. Although the lack of response of SU-DIPG XIII-P was consistent with the low expression of IL-13Rα2 in the cell models assayed, neither cell lines with intermediate nor high expression of IL-13Rα2 stimulated with IL13 showed a significant increase in cell proliferation versus vehicle as a control (Figure 2A). Based on previous reports showing that IL-13Rα2 is more related to cell survival than cell proliferation and invasion, we hypothesized that cytokine stimulation is associated with increased IL-13Rα2 expression, which enhances its antiapoptotic effect. To test this, HGG cells were stimulated with IL-13 (10 ng / mL) and protein levels were examined at various time points (Figure 2C). Stimulation with IL13 led to robust upregulation of IL-13Rα2 after 8, 24, 48, and 72 hours in cell lines with intermediate and high IL-13Rα2 expression. Conversely, levels of IL-13Rα1 were not affected by IL13 stimulation in any of the HGG cell models assayed.

[0104] cmIL13 exerts potent antitumor effects in HGG cell models To assess whether IL-13Rα2 expression confers sensitivity to IL-13Rα2-targeted therapy in vitro, we tested the pharmacological response of HGG cells to cmIL13. Eleven HGG cell lines (five DMG and six adult GBM) were selected and exposed to various concentrations of cmIL13 ranging from 0.001 ng / mL to 320 ng / mL. The results showed a direct relationship between IL-13Rα2 expression and cmIL13 sensitivity (Figure 2B, Figure 3). cmIL13 exhibited strong cytotoxicity in cell lines with high IL-13Rα2, which was characterized by a significant left shift on the dose-response curve relative to the relatively insensitive cell models with low IL-13Rα2. The IC of cmIL13 in cells with high IL-13Rα2 was 0.001 ng / mL. 50 The values ​​were 0.02 ng / mL for PED17 cells, 0.06 ng / mL for GBM14, and 0.58 ng / mL for GBM10. Cells with intermediate IL-13Rα2 had the following IC 50 The IC values ​​for cmIL13 in IL-13Rα2-low cells were: 0.10 ng / mL for SF8628, 0.75 ng / mL for SU-DIPG XVII, 0.81 ng / mL for SF8628-B23, 0.12 ng / mL for GBM6, and 9.08 ng / mL for GBM43. 50 Values ​​were 10.63 ng / mL in SU‐DIPG XIII‐P, 15.74 ng / mL in GBM 108, and 53.82 ng / mL in GBM 39. Both DMG and adult GBM cell models showed similar sensitivity to cmIL13 depending on IL‐13Rα2 status.

[0105] Next, to clarify the effect of cmIL13 on IL-13Rα2, HGG cells were treated with IC 50Concentrations of the drug were treated and protein levels were investigated at 8, 24, 48, and 72 h (Fig. 2D). Similar to IL13 stimulation, cmIL13 did not induce downregulation of IL-13Rα2, but rather protein levels were stable or increased over time. Interestingly, IL-13Rα1 was upregulated in several IL-13Rα2-moderate and IL-13Rα2-high cell models exposed to cmIL13. Furthermore, apoptosis induction was characterized by elevated levels of cleaved caspase 3 and / or cleaved PARP. The results were confirmed by confocal microscopy (Fig. 2E, Fig. 4), where significant IL-13Rα2 levels were found at baseline, which were maintained in cells treated with cmIL13 for up to 72 h. Staining of the PE domain of cmIL13 confirmed colocalization of the drug with the receptor as well as internalization into the cytoplasm and cell nucleus. In addition to increased levels of apoptosis, cell proliferation was reduced in the presence of cmIL13.

[0106] Intratumoral administration of cmIL13 reduced tumor burden and extended survival in vivo To validate the in vivo antitumor efficacy of cmIL13, we utilized orthotopic patient-derived murine xenograft models of HGG, including models with low IL-13Rα2 expression (SU-DIPG XIII-P), intermediate IL-13Rα2 expression (GBM6), and high IL-13Rα2 expression (PED17). Tumor-bearing animals were randomized into four cohorts, with 4–5 animals per treatment group treated by CED of a single brain-targeted dose of cmIL13 (Figure 5A). Drug delivery systems were first established in adult GBM animals by injecting vehicle solution (PBS) or various doses of cmIL13 into the GBM6 tumor region of the cerebral hemisphere. All CED systems were placed and tolerated without complications. No treatment-related deaths occurred, and all clinical evaluations of the animals after completion of the infusion were unremarkable, with no signs of acute or delayed toxicity or neurological deficits. Tumor volumes measured by BLI were significantly lower in animals treated with 1 μg cmIL13 (p=0.01) compared to the 0.3 μg (p=0.14), 0.1 μg (p=0.08), and vehicle treatment groups (Figure 5B). A single dose of 1 μg cmIL13 significantly extended survival with a median survival of 84 days (p=0.01) compared to 64 days for 0.3 μg cmIL13 (p=0.35), 68 days for 0.1 μg cmIL13 (p=0.17), and 57 days for the vehicle group (Figure 5C).

[0107] Histological assessment of the brains of mice euthanized in moribund condition showed that tissue architecture was maintained and tumor size was reduced after cmIL13 treatment (Figure 6A). The on-target effect of the drug in the tumor was verified by IHC analysis of IL-13Rα2 levels, apoptosis induction, and cell proliferation in drug-treated mice compared to controls. Consistent with the in vitro data, elevated IL-13Rα2 was maintained in GBM6 cells (Figure 6B). Cell proliferation, as determined by Ki-67 staining, was reduced after exposure to cmIL13 (Figures 6B and 6C). Interestingly, strong staining for the apoptotic marker cleaved caspase 3 was evident throughout the tumor area and in all cmIL13 groups, but was absent in vehicle-treated animals for several weeks after cmIL13 administration (Figure 6B). To account for toxicity that may accompany delivery of immunotoxins to the brain, additional IHC analysis was performed for NeuN, a marker of mature neurons, and CD68, which is highly expressed by microglia and monocytes. CED of cmIL13 did not reduce NeuN-positive cells in the injected ipsilateral hemisphere compared to vehicle (Figure 6B and Figure 6C). No immune cell infiltration was evident in either study group (Figure 6B and Figure 6C).

[0108] These findings were validated in a DMG xenograft model in which IL-13Rα2 was upregulated. PED17 cells were implanted orthotopically in the pons, and tumor-bearing animals were treated again with vehicle solution, 0.1 μg, 0.3 μg, or 1 μg of cmIL13. Consistent with previous observations, all animals tolerated the CED treatment, but at the highest dose (CED injection of 1 μg cmIL13), 5 / 5 animals showed signs of developing toxicity (neurological deficits such as hemiparesis or ataxia, hunched posture, dermatitis) within 24 hours after injection, and 4 / 5 animals had to be euthanized within 72 hours after drug administration. Postoperative clinical evaluation was normal for animals treated with 0.1 μg or 0.3 μg cmIL13. Comparison of BLI signals showed that a single injection of 0.1 μg or 0.3 μg cmIL13 significantly reduced tumor volume (p=0.0001 and 0.0004, FIG. 5D) and significantly extended median survival (147 days for 0.1 μg cmIL3 (p=0.003) and 155 days for 0.3 μg cmIL13 (p=0.003)) compared to the vehicle group (128 days) (FIG. 5E). Similar to the findings in hemispheric GBM6 tumors, neither the 0.1 nor 0.3 μg doses affected NeuN+ cell density and CD68+ cell infiltration. Consistent with the observed differences in clinical toxicity, the 1 μg dose of cmIL13 led to a significant reduction in NeuN-positive cells in the brainstem compared to the lower dose of cmIL13 or the vehicle control. No infiltration of monocytic cells was observed after exposure to 1 μg cmIL13. The levels of IL-13Rα2 detected remained constant between treatment groups. A decrease in H3 K27M and an increase in H3 K27me3, characteristic of DMG, were evident in drug-treated tumors. Additional IHC findings were comparable to those of the first study (Figures 7A and 7B).

[0109] Finally, we used the DMG cell line SU-DIPG XIII-P to establish an HGG xenograft model with low IL-13Rα2 protein levels. Based on the in vitro data, we did not expect cmIL13 to affect tumor volume or survival time using the previously established dosing regimen. Indeed, CED of 0.1 μg, 0.3 μg, or 1 μg cmIL13 failed to show a significant reduction in tumor growth (p=0.16, 0.18, and 0.27, respectively, Figure 5F) and did not provide a significant survival benefit compared to control animals (vehicle for 24.5 days, 0.1 μg cmIL13 for 23 days (p=0.92), 0.3 μg cmIL13 for 24 days (p=0.68), and 1 μg cmIL13 for 24 days (p=0.57)) (Figure 5G). CED treatment proved to be feasible and safe among all treatment groups, and no immune cell infiltration or reduction of NeuN-positive cells was observed in mice treated with 0.1 μg or 0.3 μg cmIL13, but there was also no reduction in NeuN+ cell density in the brainstem of animals treated with 1 μg cmIL13. IHC did not show increased staining of cleaved caspase 3 in cmIL13-treated xenografts with low IL-13Rα2, and high cell proliferation was maintained in those tumors after cmIL13 injection. In accordance with in vitro protein level analysis, IHC staining of IL-13Rα2 was absent in SU-DIPG XIII-P xenografts. Furthermore, H3 K27M and H3 K27me3 remained largely unchanged between vehicle versus cmIL13-treated tumors. These results indicate that upregulation of IL-13Rα2 is required for targeted therapy such as cmIL13 to confer therapeutic benefit in HGG orthotopic xenograft models.

[0110] Although certain aspects of the present disclosure have been described in detail, it will be appreciated by those skilled in the art that various modifications and alterations to those details may be devised in light of the overall teachings of the present disclosure. Accordingly, the particular configurations disclosed are for illustrative purposes only and are not intended to limit the scope of the present disclosure, which is to be accorded the full scope of the appended claims and aspects and any equivalents thereof.

Claims

1. 1. A method of treating a subject having malignant glioma, comprising: administering a therapeutically effective amount of cytotoxin-linked mutagenized IL13 (mIL13) (cmIL13) via convection-enhanced drug delivery (CED) for up to 96 hours; Including, The method, wherein the malignant glioma expresses interleukin-13 receptor alpha 2 (IL13Rα2).

2. 10. The method of claim 1, wherein the cmIL13 is administered at a dose of 0.03 μg / mL to 1 μg / mL and / or at a flow rate of up to 1 mL / hour for 4 to 96 hours.

3. The method of claim 1 or 2, wherein the cytotoxin comprises a bacterial toxin.

4. The method of claim 1 or 2, further comprising co-injecting an imaging moiety.

5. The method of claim 1 or 2, further comprising detecting expression of IL13Rα2 in the sample, wherein expression of said IL13Rα2 indicates that said malignant glioma will respond to treatment with cmIL13.

6. The sample is Liquid biopsies of blood, plasma, saliva, urine, and / or cerebrospinal fluid, and / or Solid biopsy of organs and / or tissues The method of claim 5 , comprising:

7. The method of claim 5, wherein the detection of IL13Rα2 expression is performed by mass spectrometry, analytical assay, immunostaining, and / or sequencing.

8. 3. The method of claim 1 or 2, wherein said administration improves survival of said subject relative to a control subject having malignant glioma who is not administered a therapeutically effective amount of cmIL13.

9. The administration of the cmIL13 to cells expressing IL13Rα2 results in (i) the volume of the malignant glioma is reduced; (ii) an increase in detectable cleaved caspase 3 as measured by IHC or fluorescent cytochemistry, and / or (iii) the level of Ki-67 positive cells is reduced by at least 10% as measured by IHC or fluorescent cytochemistry.

10. The method of claim 1 or 2, wherein the mIL13 is modified to have increased affinity for IL13Rα2 compared to native human IL13 and / or decreased affinity for interleukin-13 receptor α1 (IL13Rα1) compared to native human IL13.

11. 3. The method of claim 1 or 2, wherein said m13 is characterized by at least one amino acid substitution when compared to SEQ ID NO: 1 or SEQ ID NO:

2.

12. 3. The method of claim 1 or 2, wherein the mIL13 comprises amino acid changes relative to wild-type IL13 at positions E13, R66, S69, and / or K105.

13. 3. The method of claim 1 or 2, wherein the mIL13 comprises one or more amino acid substitutions E13K, R66D, S69D, K105R.

14. 3. The method of claim 1 or 2, wherein the mIL13 comprises an amino acid sequence set forth in one of SEQ ID NOs: 3 to 24 or a homolog thereof, or a homolog of one of SEQ ID NO: 1 or SEQ ID NO:

2.

15. 3. The method of claim 1 or 2, wherein the mIL13 differs from SEQ ID NO: 1 or SEQ ID NO: 2 by no more than 20 residues.

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