Noninvasive tracking of a targeted toxin

Abstract

A labeled cell targeting agent for in vivo imaging of a subject is disclosed. The labeled cell targeting includes, for example, a radiolabel or a paramagnetic ion or compound or a labeling agent-binding moiety; and a polypeptide comprising an IL13 or mIL13 amino acid sequence that binds IL13Rα2 and optionally a PE cytotoxin amino acid sequence. Methods of preparing and using the agent also are provided. The labeled cell targeting agent may be used in a method of in vivo imaging, wherein the method comprises administering the labeled therapeutic agent to a subject such that a cell of the subject becomes labeled for in vivo imaging, wherein the cell may be an IL13Rα2-expressing cell, such as a diffuse midline glioma cell or a glioblastoma cell.

Description

Attorney Docket No. TPI-7GCA1WO NONINVASIVE TRACKING OF A TARGETED TOXIN CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 689,538, filed August 30, 2024, and U.S. Provisional Patent Application No. 63 / 726,591, filed December 1, 2024, the disclosures of each of which are incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This disclosure was made with Government support under HT94252310681 awarded by USAMRAA. The government has certain rights in the subject matter claimed herein. SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (Targepeutics Sequence Listing.xml; Size: 27,000 bytes; and Date of Creation: July 10, 2022) are incorporated herein by reference in its entirety.

[0004] Provided herein are methods of labeling a targeting agent for medical imaging, and methods for medical imaging of a subject using a targeting agent labeled with a labeling moiety.

[0005] Delivery of drugs to a subject, for example targeted delivery to the brain by convection enhanced delivery (CED), can affect the response to a treatment. Tissue distribution of drugs delivered to the brain, for example by CED can be assessed by surrogate methods, such as mathematical modeling using MRI-based assessment of catheter placement, MRI of tissue distribution of gadolinium-conjugated agents like Gd- DTPA co-infused with a drug, and single-photon emission computerized tomography. A disadvantage of these surrogate methods is that they need to be validated with real- time assessment of the tissue distribution of the administered drug. A quantitative and sensitive imaging method is needed to assess the real-time tissue distribution of administered drugs, for example, those targeting the brain.Attorney Docket No. TPI-7GCA1WO SUMMARY

[0006] The overall poor survival of patients with Diffuse midline gliomas (DMGs) and Glioblastoma (GBM) has led to a worldwide effort to improve treatment options. Quantitative and sensitive imaging methods like positron emission tomography (PET) can be beneficial for assessing the real-time tissue distribution of an administered drug, such as a targeted therapeutic agent, following delivery, for example by CED. To achieve this assessment, a labeled targeting agent described herein. A targeting agent comprising an IL13 amino acid sequence can be coupled to a chelating moiety. The chelating moiety is capable of bonding to a radioisotope for use in positron emission tomography (PET) imaging and for tracking of the targeting agent using other suitable detection systems and methods. The targeting agent may comprise a fusion protein comprising an IL13 amino acid sequence and a cytotoxin amino acid sequence.

[0007] A cell targeting agent is provided, comprising a binding moiety that binds to a radiolabel or a paramagnetic ion or compound; a polypeptide comprising a naturally occurring IL13 (IL13) amino acid sequence or a mutagenized IL13 (mIL13) amino acid sequence that binds IL13Rα2; and optionally a radiolabel or a paramagnetic ion or compound bound to the binding moiety. The cell targeting agent may further comprise a cytotoxin, such as a Pseudomonas exotoxin (PE) amino acid sequence included in the polypeptide.

[0008] A method of in vivo imaging also is provided, comprising administering a labeled cell targeting agent to a subject such that an IL13Rα2-expressing cell, such as a diffuse midline glioma (DMG) cell or a glioblastoma (GBM) cell, of the subject becomes labeled for in vivo imaging. The cell targeting agent may comprise a binding moiety that binds to a radiolabel or a paramagnetic ion or compound; a polypeptide comprising a naturally occurring IL13 (IL13) amino acid sequence or a mutagenized IL13 (mIL13) amino acid sequence that binds IL13Rα2; and a radiolabel or a paramagnetic ion or compound bound to the binding moiety.

[0009] A method of preparing the cell targeting agent is provided. The method comprises conjugating a compound comprising a binding moiety that binds to a radiolabel or a paramagnetic ion or compound with a polypeptide comprising the IL13 or mIL13 amino acid sequence, and optionally a PE cytotoxin amino acid sequence, toAttorney Docket No. TPI-7GCA1WO produce a pre-radiolabeled cell targeting agent. The method may further comprise mixing the pre-radiolabeled cell targeting agent with a radiolabel or a paramagnetic ion or compound to produce a labeled cell targeting agent.

[0010] A kit also is provided. The kit comprises a first container comprising a cell targeting agent. The cell targeting agent comprises a binding moiety that binds to a radiolabel or a paramagnetic ion or compound; and a polypeptide comprising a naturally occurring IL13 (IL13) amino acid sequence or a mutagenized IL13 (mIL13) amino acid sequence that binds IL13Rα2 and optionally a cytotoxin, such as a PE cytotoxin amino acid sequence. The kit also includes a second container, comprising a radiolabel or a paramagnetic ion or compound capable of binding to the binding moiety. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawings provided herein are intended to be illustrative of the subject matter described herein.

[0012] FIG.1 is a schematic of a positron emission tomography (PET) system.

[0013] FIG.2 shows a scheme of a conjugation of p-SCN-Bn-DFO to TC1 to form DFO-TC1.

[0014] FIG.3 shows MALDI mass spectrometric characterization of TC1 having a mass of 79,211 Da.

[0015] FIG. 4 shows the mass characterization of example conjugation reactions where the reaction conditions include a reaction time of 30 minutes and a ratio of TC1:DFO-NCS that is 1:3 (FIG.4, Panel A) or 1:6 (FIG.4, Panel B).

[0016] FIG. 5 shows the mass characterization of example conjugation reactions where the reaction conditions include a reaction time of 60 minutes and a ratio of TC1:DFO-NCS that is 1:3 (FIG.5, Panel A) or 1:6 (FIG.5, Panel B).

[0017] FIG.6 shows a table of the mass characterization results using MALDI mass spectrometry of example conjugation reactions.

[0018] FIG. 7 shows a scheme of an example of a radiolabeling procedure for radiolabeling DFO-TC1 to form [89Zr]Zr-DFO-TC1.

[0019] FIG. 8 shows a scheme of an example of the steps of the synthesis of [89Zr]Zr-DFO-TC1, according to an aspect of the disclosure.Attorney Docket No. TPI-7GCA1WO

[0020] FIG.9 shows example data for radiolabeling yields of [89Zr]Zr-DFO-TC1 as compared to the protein concentration.

[0021] FIG. 10 shows a general scheme for r-TLC method development for the characterization of [89Zr]Zr-DFO-TC1.

[0022] FIG. 11 shows an example of r-TLC method development data and conditions for the characterization of unlabeled89Zr.

[0023] FIG. 12 shows an example of r-TLC method development data and conditions for the characterization of [89Zr]Zr-DFO-TC1.

[0024] FIG.13 shows example data for the purification of [89Zr]Zr-DFO-TC1.

[0025] FIG.14 shows example data for a stability analysis of [89Zr]Zr-DFO-TC1 in formulation and in mouse serum.

[0026] FIG.15 shows example data for cell lines used for uptake study with [89Zr]Zr- DFO-TC1.

[0027] FIG.16 shows example data for the uptake of [89Zr]Zr-DFO-TC1 in DMG cell lines SU-DIPG-XVII and PED 17 and the GBM cell lines GBM6 and GBM59 where the conjugation ratio of TC1:DFO-NCS is 1:3.

[0028] FIG.17 shows example data for the uptake of [89Zr]Zr-DFO-TC1 in DMG cell lines SU-DIPG-XVII and PED 17 and in the GBM cell lines GBM6 and GBM59, where the conjugation ratio of TC1:DFO-NCS is 1:6.

[0029] FIG.18 shows example data for the uptake of [89Zr]Zr-DFO-TC1 in DMG cell lines SU-DIPG-XVII and PED 17 and in the GBM cell lines GBM6 and GBM59, where the conjugation ratio of TC1:DFO-NCS is 1:1.

[0030] FIG. 19 shows example of an autoradiograph of [89Zr]Zr-DFO-TC1 in an acrylamide gel.

[0031] FIG. 20 shows an example scheme for a process of bioluminescence imaging of IL-13Rα2 expression in a diffuse midline glioma tumor model in an animal followed by PET / X-ray imaging.

[0032] FIG. 21 shows the bioluminescence imaging results of animals with a luciferase expressing (Luc-PED17) tumor.

[0033] FIG.22 shows an example scheme for a process of PET imaging IL-13Rα2 in diffuse midline glioma tumor model using [89Zr]Zr-DFO-TC1 in animals.Attorney Docket No. TPI-7GCA1WO

[0034] FIG. 23 shows a comparison of biodistribution of [89Zr]Zr-DFO-TC1 in PED17 tumor with and without co-infusion of Galbumin at different time points post- infusion.

[0035] FIG. 24 shows bioluminescence imaging of luciferase expressing Luc- DIPGXVII tumor prior to [89Zr]Zr-DFO-TC1 imaging with and without Galbumin.

[0036] FIG. 25 shows a comparison (PET-CT) of biodistribution of [89Zr]Zr-DFO- TC1 in DIPGXVII tumor with and without co-infusion of Galbumin at different time points post-infusion.

[0037] FIG.26 shows a comparison of uptake of [89Zr]Zr-DFO-TC1 in PED17 and DIPGXVII tumors. IVIS images confirm the presence of the tumor prior to PET / CT or PET / X-ray imaging.

[0038] FIG.27 shows a comparison of biodistribution of [89Zr]Zr-DFO-TC1 when co- infused with Galbumin in PED17 and DIPGXVII tumor models.

[0039] FIG. 28 shows MR images with Gd-Albumin contrast agent in mice with DIPGXVII tumors as a control experiment, showing clearance of the contrast agent.

[0040] FIG. 29 shows MR images with Gd-Albumin contrast agent in mice with PED17 tumors as a control experiment, showing clearance of the contrast agent.

[0041] FIG. 30 shows surface plasma resonance (SPR) data demonstrating significantly higher binding affinity of TC1-DFO for IL13Rα2 over IL13Rα1 human and mouse proteins. This shows that after conjugation of the DFO chelator, the TC1 retains its function and selectivity towards the target IL13Rα2 receptor.

[0042] FIG. 31 shows the determination of Vd for [89Zr]Zr-DFO-TC1 alone, or in combination with magnetic resonance imaging (MRI) contrast agents.

[0043] FIG.32 provides amino acid sequences of IL13 and mutated IL13 (SEQ. ID NOS: 1-24). DETAILED DESCRIPTION

[0044] For purposes of this detailed description, it is to be understood that the disclosure may assume alternative variations and step sequences, except where expressly specified to the contrary.

[0045] The numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errorsAttorney Docket No. TPI-7GCA1WO necessarily resulting from the standard variation found in their respective testing measurements.

[0046] Also, any numerical range recited herein is intended to include all sub- ranges subsumed therein. For example, a range of “1 to 10” is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.

[0047] As used herein, “including,” “containing,” and like terms are understood in the context of this application to be synonymous with “comprising” and are therefore open-ended and do not exclude the presence of additional undescribed or unrecited elements, materials, ingredients, or method steps. As used herein, “consisting of” is understood in the context of this application to exclude the presence of any unspecified element, ingredient, or method step. As used herein, “consisting essentially of” is understood in the context of this application to include the specified elements, materials, ingredients, or method steps “and those that do not materially affect the basic and novel characteristic(s)” of what is being described.

[0048] In addition, in this application, the use of “or” means “and / or” unless specifically stated otherwise, even though “and / or” may be explicitly used in certain instances.

[0049] As used herein, the term “patient,” “subject,” “individual,” and the like are used interchangeably herein and mean animals amenable to the methods described herein, including mammals, including a human, a canine, a feline, a bovine, an equine, a porcine, a primate, and / or a rodent.

[0050] As used herein, “administering” or “administration” of an amount (e.g., a dose) of a composition may be done by the subject himself / herself or another subject (e.g., a medical professional, a caretaker, or a family member). The composition may be provided by the subject or the administrator for the subject along with instructions for the administration of the composition (e.g., written instructions on the label of a container containing the composition).

[0051] As used herein, “disorder,” “disease,” and “illness” are used interchangeably and refer to a condition in a subject that negatively impacts the health of the subject.Attorney Docket No. TPI-7GCA1WO

[0052] As used herein, “treat,” “treatment,” or “treating” means treatment of a disease or disorder, as defined herein, in a subject, including: (1) inhibiting a disease or disorder; (2) arresting the development of a disease or disorder; (3) slowing progression of the disease or disorder; and / or (4) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. A disease or disorder is “treated” if the subject experiences a reduction in the severity and / or frequency of the disease, disorder, and / or illness or a symptom associated therewith.

[0053] As used herein, “cancer” refers to a disease characterized by the abnormal growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include those brain cancers in which tissue cells express IL13Rα2, including but not limited to gliomas and the like.

[0054] As used herein, “glioma” refers to a tumor that originates in glial cells of the brain or spinal cord.

[0055] As used herein, a “therapeutically effective dose” is defined as an amount required to ameliorate the symptoms of a disease in a treated patient relative to an untreated patient. The effective amount of active compound(s) used to therapeutically treat a disease varies depending upon the manner of administration, as well as the age, body weight, and general health of the subject.

[0056] As used herein, “composition” refers to a solution or dispersion.

[0057] As used herein, “pharmaceutical composition” refers to any chemical or biological composition, material, agent, or the like that is capable of inducing a therapeutic effect when properly administered to a subject, including the composition, material, agent, or the like in an inactive form and active metabolites thereof, where such active metabolites may be formed in vivo.

[0058] As used herein, “measuring” or “measurement” or “detecting” or “detection” means assessing the presence, absence, quantity, or amount (which can be an effective amount) of either a given substance within a clinical or subject-driven sample, including the derivation of qualitative or quantitative levels of such substance, or otherwise evaluating the values or categorization of a subject’s clinical parameters.Attorney Docket No. TPI-7GCA1WO

[0059] As used herein, “sample” or “biological sample” refers to a biological material isolated from an individual, such as a liquid or solid biological sample collected via a biopsy (e.g., a “liquid biopsy” or a “solid biopsy”). A liquid biopsy may comprise, for example, blood, plasma, saliva, urine, cerebral spinal fluid, and / or other body fluid. The liquid biopsy may contain extracellular vesicles and / or cell-free genetic material. A solid biopsy may comprise, for example, an organ and / or a tissue, such as a tumor. The biological sample may contain any biological material suitable for detecting the desired biomarkers and may comprise cellular and / or non-cellular material obtained from the individual.

[0060] As used herein, “control” refers to any experimental condition that is either not treated at all or is treated by a method that is not disclosed herein.

[0061] A “moiety” refers to a portion of a larger molecule often having a function, such as a reactive group, a chelating group, a spacer, or a portion of a fusion protein amino acid sequence, such as the targeting and cytotoxic moieties described herein in the case of a fusion protein.

[0062] As used herein, a “label” is a detectable marker typically conjugated with (e.g., covalently linked to or complexed with) a moiety, such as a targeting polypeptide in the context of the present disclosure. A label may be a radioactive label, e.g. a “radiolabel”, or a paramagnetic ion or compound complexed with a chelating moiety.

[0063] A “mutation” in a polypeptide is meant to encompass proteins having any amino acid substitution(s), deletion(s) (e.g., a truncated version of the protein, such as a polypeptide), insertion(s), and / or modification(s), such as by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation, and the like. In an example, a “mutated IL13” or a “mutagenized IL13” refers to an IL13 in which one or more of the amino acids differs from the corresponding amino acids in a wild-type form of IL13. Mutated IL13 and / or mutagenized IL13 may be derived from the wild-type form of IL13 found in humans, non-human primates, rats, murine, porcine, bovine, canine, and the like. A mutated IL13 and / or mutagenized IL13 may be referred to herein as “mIL13.” mIL13 may be connected to a cytotoxin, e.g., in a fusion protein. An mIL13 amino acid sequence may have at least 80%, at least 90%, at least 95%, or at least 99% sequence identity with a naturally-occurring human IL13, for exampleAttorney Docket No. TPI-7GCA1WO SEQ. ID NOS: 1 or 2. An mIL13 amino acid sequence may have 1, 2, 3, 4, 5, 6, or more amino acid substitutions, insertions, or deletions as compared to a naturally- occurring human IL13, for example SEQ. ID NOS: 1 or 2. An mIL13 may be a truncated naturally-occurring IL13, for example SEQ. ID NOS: 1 or 2. For uses herein, an mIL13 binds specifically to an IL13 receptor, such as IL13Rα2, GBM cells, and / or DMG cells.

[0064] As used herein, “interleukin-13” or “IL13” refers to any wild-type or native, IL13 of any vertebrate source, including mammals, such as primates and rodents, unless indicated to the contrary, and includes unprocessed IL13 and any form of IL13 that results from processing in a cell and any naturally occurring variants of IL13, such as splice or allelic variants. The amino acid sequence of an exemplary human IL13 is shown in FIG. 32 (SEQ. ID NO:1). An amino acid sequence of a second exemplary human IL13 is shown in FIG. 32 (SEQ. ID NO:2). IL13 and mIL13 may be used as targeting moieties in targeting agents described herein, such as [89Zr]Zr-DFO-TC1 (e.g., [89Zr]Zr-DFO-IL13.E13K-PE4E).

[0065] As used herein, the term “IL13 receptor” or “IL13R” refers to a receptor that binds IL13.

[0066] As used herein, the term “IL13 receptor α 2” or “IL13Rα2” refers to the monomeric IL13 receptor that is expressed on the surface of specific cell subsets and that binds IL13 or an analog thereof, such as mIL13 or is contained in extracellular vesicles and that binds IL13 independent of IL4.

[0067] As used herein, “affinity” refers to the strength of the total of non-covalent interactions between a single binding site of a receptor and a ligand. The affinity of a receptor for a ligand can be represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants, Koff and Kon, respectively. Affinity can be measured by methods known to those of skill in the art.

[0068] “Increased binding” refers to binding levels of an mIL13, e.g., to an IL13 receptor such as IL13Rα2, which are at least 10% or more, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% higher, or more, or 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 100-fold, 1000-fold higher, or more, and any and whole or partial increments therebetween, than binding levels of a wild-type IL13 to, e.g., an IL13 receptor such as IL13Rα2.Attorney Docket No. TPI-7GCA1WO

[0069] “Decreased binding” refers to binding levels of an mIL13, e.g., to an IL13 receptor such as IL13Rα2, which are at least 10% or less, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% lower or less, or 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 100- fold, 1000-fold lower or less, and any and whole or partial increments therebetween, than a wild-type IL13 to, e.g., an IL13 receptor such as IL13Rα2.

[0070] As used herein, “fusion” or “fused”, in the context of a fusion protein refers to the combining of two or more amino acid sequences or polypeptides in a single amino acid chain. The amino acid sequences or polypeptides joined in the fusion protein may be the same, but typically are different and may have different activities, as in the case of a single amino acid sequence comprising both an IL13-derived targeting moiety and a cytotoxin moiety, as described herein. A fusion protein may be expressed from a single gene comprising a single open reading frame encoding the fusion protein.

[0071] As used herein, “determining the level of marker (or biomarker) expression” is meant as an assessment of the degree of expression or presence of a marker in a sample at the nucleic acid or protein level, using technology available to the skilled artisan to detect a sufficient portion of any marker expression product, such as “determining the level of IL13Rα2.”

[0072] As used herein, the “level” of one or more marker (or biomarker) means the absolute or relative amount or concentration of the marker (or biomarker) in the sample.

[0073] The present disclosure provides a radiolabeled mutagenized interleukin-13 (rmIL13) targeting agent comprising: a detectable label, such as a radiolabel detectable by PET or other methods; a polypeptide comprising a IL13 or a mutagenized interleukin-13 (mIL13); and, optionally, a cytotoxin that can be a PE amino acid sequence in a fusion protein comprising an amino acid sequence of IL13 or a mIL13 and a PE cytotoxin amino acid sequence. In one example, the mIL13 is IL13.E13K- PE4E.

[0074] The rmIL13 may comprise an mIL13. The mIL13 may be engineered to have increased affinity for IL13Rα2 compared to wild-type human IL13. The affinity of the mIL13 for IL13Rα2 may be increased compared to a wild-type IL13, such as a 2-foldAttorney Docket No. TPI-7GCA1WO increase, a 3-fold increase, a 5-fold increase, a 10-fold increase, a 100-fold increase, a 1000-fold increase, or more of kinetic KD.

[0075] The mIL13 also may be engineered to have decreased affinity for the interleukin 13 receptor α 1 (IL13Rα1) compared to wild-type human IL13. For example, the mIL13 may have a reduced affinity to the IL13Rα1 compared to a wild-type IL13, for example, a 2-fold decrease, a 3-fold decrease, a 5-fold decrease, a 10-fold decrease, a 100-fold decrease, or more of kinetic KD while at least retaining binding or activation of the IL13Rα2.

[0076] In examples, a polypeptide, such as IL13.E13K-PE4E, IL13.E13K, or comprising an mIL13 amino acid sequence may be produced by any useful method known to those of skill in the art. IL13 Mutations may be obtained by random or site- directed mutagenesis methods. Proteins, such as fusion proteins described herein may be produced in any gene expression system or by solid-phase synthesis and may be purified or enriched by affinity to IL13Rα2, using, e.g., column or magnetic bead affinity methods.

[0077] The terms “homology” and “homologous” refer to the subunit sequence identity between two molecules, such as two protein or peptide molecules. When a subunit position in both molecules is occupied by the same subunit, then the 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, if half of the positions in two sequences are homologous, then the two sequences are 50% homologous and if 70% of the positions (7 of 10) are matched or homologous, then the two sequences are 70% homologous. Homologs can be the result of natural allelic variation, including natural mutation. Homologs of the present disclosure can be the result of natural allelic variation, including natural mutation. Homologs of the present disclosure can also be produced using techniques known in the art, including direct modifications of nucleic acid sequences encoding the protein, using, for example, recombinant DNA techniques to effect random or targeted mutagenesis.

[0078] The mIL13 may be complexed with an extracellular vesicle (EV) to form an extracellular vesicle complex (EV complex). The extracellular vesicle may be obtained from a particular type of biological sample and / or may be derived from a particular typeAttorney Docket No. TPI-7GCA1WO of cell, such as glioma stem cells (see, e.g., Uwishema O, et al. Stem cell therapies and glioma stem cells in glioblastoma: a systematic review of current challenges and research directions. Int J Emerg Med.2025 Aug 6;18(1):144). In examples, the glioma stem cells may be mesenchymal glioma stem cells or proneural glioma stem cells. The extracellular vesicle may comprise an exosome, such as a tumor-associated exosome. The extracellular vesicles may be purified or concentrated from a biological sample using differential centrifugation, ultracentrifugation, and / or other methods known to those of skill in the art.

[0079] Subpopulations of extracellular vesicles may be isolated using a biological marker. The biological marker may be a receptor, such as a tumor-associated receptor. The tumor-associated receptor may be IL13Rα2. Each exosome may, for example, express 1, 2, 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000, or more biological markers.

[0080] The cytotoxin may comprise, consist essentially of, or consist of a bacterial- derived toxin. The bacterial-derived toxin may comprise, for example, Pseudomonas exotoxin A and derived cytotoxic mutations thereof (collectively, “a PE cytotoxin” or “PE”).

[0081] The rmIL13 may comprise a radiolabel, e.g., a radionuclide detectable by PET, such as89Zr. The radiolabel may be conjugated to one of the IL13 or mIL13 polypeptides disclosed herein. The conjugation may be accomplished using a chelating moiety linked to the mIL13 peptide. The linking agent may comprise, for example, a hydroxamic moiety, such as desferrioxamine B (“DFO”). The chelating moiety may be conjugated to the IL13- or mIL13-containing polypeptide in an approximately 1:1 ratio, such as an average of from 0.5 to 1.5, from 0.7-1.3, from 0.8 to 1.2, or from 0.9-1.1, including any increment therebetween, radiolabel-chelating moieties, such as hydroxamic or DFO moieties, per polypeptide chain.

[0082] The disclosure is further directed to a composition comprising any of the rmIL13 disclosed herein and a pharmaceutically acceptable carrier.

[0083] The rmIL13 binds to cells comprising IL13Rα2 (e.g., tumor cells). The rmIL13 binding may be detected using photographic film, scintillation counters, positron emission tomography (PET) imaging, or any other means for detection of a radiolabelAttorney Docket No. TPI-7GCA1WO known to a person of skill in the art. Without wishing to be bound by theory, it is believed that detection of the rmIL13 through imaging detects the biodistribution of the compositions disclosed herein following administration.

[0084] The composition may be formulated as a pharmaceutical composition.

[0085] The composition may be administered by a catheter. For example, the composition may be administered by convection enhanced delivery (CED). As used herein, “convection enhanced delivery” or “CED” refers to a method of drug administration wherein a pressure gradient is generated at the tip of an infusion catheter for direct delivery of therapeutics through the interstitial spaces of the central nervous system to the structure to be treated, e.g., into a resection cavity or into an intact tumor. The pressure gradient may be a positive pressure gradient, meaning that the composition may be administered under positive pressure. The composition also may be administered by CED at a constant pressure. Any suitable catheter known in the art may be used. The composition may be administered by convective infusion at a specified flow rate controlled by an external syringe pump.

[0086] The method comprises positioning the tip of a catheter within the area to be treated. An external pump may be connected to the catheter, which supplies a composition comprising a therapeutically effective dose of a therapeutic agent, e.g., rmIL13, while maintaining a positive pressure gradient through delivery.

[0087] The composition may be administered acutely. As used herein, “acute administration” and the like refers to an in-patient procedure in which the composition is administered to the patient by a healthcare professional.

[0088] Any type of tumor comprising cells that express IL13Rα2 may be detected or treated with the compositions disclosed herein. For example, the compositions disclosed herein may be used to detect or treat cancers of the brain and / or central nervous system (“CNS”), such as malignant gliomas. Examples of malignant gliomas that may be detected or treated with the method disclosed herein include but are not limited to an adult glioblastoma, a pediatric glioblastoma, an anaplastic astrocytoma, such as a diffuse midline glioma, an anaplastic oligodendroglioma, an anaplastic oligoastrocytoma, an anaplastic ependymoma, and / or an anaplastic ganglioma. InAttorney Docket No. TPI-7GCA1WO other examples, the compositions disclosed herein may be used to treat soft-tissue sarcomas.

[0089] Prior to administration of one of the compositions disclosed herein, expression of IL13Rα2 may be detected in a sample of the tumor. In examples, the sample of the tumor may be purified prior to detection of expression of IL13Rα2. Detecting expression of IL13Rα2 may be detected by mass spectrometry, analytical assay, immunostaining, and / or sequencing. In examples, the analytical assay may comprise an enzyme-linked immunosorbent assay (ELISA). In other examples, the immunostaining may comprise immunohistochemistry (IHC) and / or fluorescent cytochemistry. In other examples the sequencing comprises whole genome sequencing, exome sequencing, proteomic sequencing, and / or RNA sequencing. Expression of the IL13Rα2 by the tumor is indicative that the tumor will be responsive to treatment with the compositions disclosed herein.

[0090] It has been surprisingly discovered that IL13Rα2-positive cells demonstrated significantly higher uptake of rmIL13 than IL13Rα2-negative cells in diffuse midline gliomas and glioblastomas. Further, the described labeled agent exhibits excellent serum stability, target specificity, and cellular uptake. Labeling of the IL13-containing or mIL13-containing polypeptide with a radiolabel does not prevent targeting and target cell uptake of the labeled targeting agent. Use of the labeled targeting agent described herein enables clear, real-time determination of targeting of the therapeutic agent.

[0091] The present disclosure provides a new labeled targeting agent for use in in vivo imaging. The labeled therapeutic agent can be tracked in real time, which makes possible the successful evaluation of the distribution of the targeting agent, therapeutic monitoring, and patient selection.

[0092] GBM is the most common malignant brain and other central nervous system (CNS) tumors accounting for nearly half of all cases. GBM is an aggressive type of brain tumor that invades the nearby brain tissue but generally does not spread to distant organs. In adults, GBM occurs most often in the cerebral hemispheres, especially in the frontal and temporal lobes of the brain. The annual incidence of glioblastoma is approximately 35 per million individuals, with a male-to-female ratio ofAttorney Docket No. TPI-7GCA1WO 1.6:1. Survival is poor with approximately 40% survival in the first-year post diagnosis, and only about 5% of patients survive 5 years post-diagnosis.

[0093] DMGs are primary CNS tumors that begin in the brain or spinal cord. DMGs are a rare subtype of glial tumors characterized by aggressive, diffuse infiltrative growth of tissue affecting the brain stem. The high-risk neuroanatomical location of the tumor in the midline of the brain often renders surgical resection unfeasible. DMGs primarily affect children and young adults, occurring with an incidence of 0.8 in 100,000 children per year at a median age of 6–7 years old. DMG is characterized by a low median overall survival time, with over 50% of affected individuals surviving less than 12 months after diagnosis. Currently, there are no treatments that improve the prognosis of DMG.

[0094] For in vivo imaging, PET may be used to visualize tissue distribution of delivered labeled targeting agent as described herein. Referring now to FIG.1, a PET system 100 that can be used with the described labeled targeting agent comprises an imaging hardware system 110 that includes a detector ring assembly 112 about a central axis or bore 114. An operator workstation 116 including a commercially available processor running a commercially available operating system communicates through a communications link 118 with a gantry controller 120 to control operation of the imaging hardware system 110.

[0095] The detector ring assembly 112 is formed of a multitude of radiation detector units 122 that produce a signal responsive to detection of a photon on communications line 124 when an event occurs. A set of acquisition circuits 126 receive the signals and produce signals indicating the event coordinates (x, y) and the total energy associated with the photons that caused the event. These signals are sent through a cable 128 to an event locator circuit 130. Each acquisition circuit 126 also produces an event detection pulse that indicates the exact moment the interaction took place. Other systems utilize sophisticated digital electronics that can also obtain this information regarding the precise instant in which the event occurred from the same signals used to obtain energy and event coordinates.

[0096] The event locator circuits 130 in some implementations, form part of a data acquisition processing system 132 that periodically samples the signals produced byAttorney Docket No. TPI-7GCA1WO the acquisition circuits 126. The data acquisition processing system 132 includes a general controller 134 that controls communication on a backplane bus 136 and on the general communications network 118. The event locator circuits 130 assemble the information regarding each valid event into a set of numbers that indicate precisely when the event took place and the position in which the event was detected. This event data packet is conveyed to a coincidence detector 138 that is also part of the data acquisition processing system 132.

[0097] The coincidence detector 138 accepts the event data packets from the event locator circuit 130 and determines if any two of them are in coincidence. Coincidence is determined by a number of factors. First, the time markers in each event data packet must be within a predetermined time window, for example, 0.5 nanoseconds or even down to picoseconds. Second, the locations indicated by the two event data packets must lie on a straight line that passes through the field of view in the scanner bore 114. Events that cannot be paired are discarded from consideration by the coincidence detector 138, but coincidence event pairs are located and recorded as a coincidence data packet. These coincidence data packets are provided to a sorter 140. The function of the sorter in many traditional PET imaging systems is to receive the coincidence data packets and generate memory addresses from the coincidence data packets for the efficient storage of the coincidence data. In that context, the set of all projection rays that point in the same direction (θ) and pass through the scanner's field of view (FOV) is a complete projection, or “view”. The distance (R) between a particular projection ray and the center of the FOV locates that projection ray within the FOV. The sorter 140 counts all of the events that occur on a given projection ray (R, θ) during the scan by sorting out the coincidence data packets that indicate an event at the two detectors lying on this projection ray. The coincidence counts are organized, for example, as a set of two-dimensional arrays, one for each axial image plane, and each having as one of its dimensions the projection angle θ and the other dimension the distance R. This θ by R map of the measured events is called a histogram or, more commonly, a sinogram array. It is these sinograms that are processed to reconstruct images that indicate the number of events that took place at each image pixel locationAttorney Docket No. TPI-7GCA1WO during the scan. The sorter 140 counts all events occurring along each projection ray (R, θ) and organizes them into an image data array.

[0098] The sorter 140 provides image datasets to an image processing / reconstruction system 142, for example, by way of a communications link 144 to be stored in an image array 146. The image arrays 146 hold the respective datasets for access by an image processor 148 that reconstructs images. The image processing / reconstruction system 142 may communicate with and / or be integrated with workstation 116 or other remote workstations.

[0099] The PET system 100 provides an example of an emission tomography system for acquiring a series of medical images of a subject during an imaging process after administering a pharmaceutically acceptable composition including a labeled targeting agent as described herein. The system includes a plurality of detectors configured to be arranged about the subject to acquire gamma rays emitted from the subject over a time period relative to an administration of the composition to the subject and communicate signals corresponding to acquired gamma rays. The system also includes a reconstruction system configured to receive the signals and reconstruct therefrom a series of medical images of the subject. In one version of the system, a second series of medical images is concurrently acquired using an x-ray computed tomography imaging device. In one version of the system, a second series of medical images is concurrently acquired using a magnetic resonance imaging device.

[0100] Administration to the subject of a pharmaceutical composition including a labeled targeting agent as described herein for in vivo detection of the labeled targeting agent in a target region of the subject can be accomplished intravenously, intraarterially, intrathecally, intramuscularly, intradermally, subcutaneously, intracavitary, or by convection enhanced delivery. Sufficient time may be allowed after administration of a detectable amount of the radiolabeled targeting agent such that the radiolabeled targeting agent can accumulate in a target region of the subject. A "detectable amount" means that the amount of the detectable radiolabeled targeting agent that is administered is sufficient to enable detection of accumulation of the radiolabeled targeting agent in a subject by a medical imaging technique.Attorney Docket No. TPI-7GCA1WO

[0101] One non-limiting example method of imaging involves the use of a radiolabeled composition including a labeled targeting agent delivered by CED. A positron emitting atom of the radiolabeled composition gives off a positron, which subsequently annihilates and gives off coincidence gamma radiation. This high energy gamma radiation is detectable outside the body through the use of positron emission tomography imaging, or positron emission tomography concurrent with computed tomography imaging (PET / CT). With PET / CT, the location of the injected and subsequently accumulated labeled therapeutic agent within the body can be identified.

[0102] The labeled targeting agent may comprise a labeled therapeutic agent. A labeled therapeutic agent may comprise a targeting agent amino acid sequence coupled to a cytotoxic agent, such an amino acid sequence of a cytotoxin, which may be in the form of a single fusion protein comprising a targeting amino acid sequence and a cytotoxin amino acid sequence, such as a PE amino acid sequence. A labeled therapeutic agent can include a therapeutic moiety coupled to a chelating moiety and at least one of (i) a radionuclide and (ii) a paramagnetic metal ion or compound.

[0103] The therapeutic moiety can include a toxin conjugate (TC) such as a IL13 or mIL13 targeting group coupled to a toxin. The targeting group of the toxin conjugate can be a small molecule, peptide, protein, or any combination thereof. In particular, the targeting group can bind to or be specifically taken up by glioma cells, GBM cells or DMG cells. The IL13 or mIL13 targeting group can have specificity for the cytokine interleukin 13 receptor, IL-13Rα2. IL-13Rα2 is overexpressed in the majority of GBMs and absent in normal brain tissue (Knudson et al. 2022). For example, the targeting group can include at least a portion of interleukin 13. In other examples, the targeting group can include a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% sequence identity with interleukin 13. In some examples, the targeting group can include a substitution mutation of interleukin 13. For example, the targeting group can comprise any of IL13.K105R, IL13.R109K, IL13 (E13K.R109K), IL13 (E13K.R66D.S69D.R109K), or IL13-E13K. See for example, Muhammed, N. et al. “A novel TanCAR targeting IL13Rα2 and EphA2 for enhanced glioblastoma therapy” Molecular Therapy: Oncolytics Vol. 24 March 2022 and Madhankumar, A. B., et al.Attorney Docket No. TPI-7GCA1WO “Interleukin 13 mutants of enhanced avidity toward the glioma-associated receptor, IL13Ralpha2”, Neoplasia, 2004 Jan-Feb;6(1):15-22.

[0104] The mIL13 may be a modified full-length IL13 molecule, such as a human full-length IL13 molecule. In examples, the mIL13 may comprise amino acid changes relative to wild-type IL13 at a position corresponding to residue 13 of the human IL13 (SEQ. ID NO:1, FIG.32), 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. The mIL13 may comprise a substitution to the glutamic acid at a position 13 of the human IL13 (SEQ. ID NO:1). For example, lysine may be substituted for the glutamic acid at a position 13. The mIL13 may comprise a substitution to the arginine at position 66 of the human IL13 (SEQ. ID NO:1). For example, aspartic acid may be substituted for the arginine at position 66. The mIL13 may comprise a substitution to the serine at position 69 of the human IL13 (SEQ. ID NO:1). Aspartic acid may be substituted for the serine at position 69. The mIL13 may comprise a substitution of the lysine at position 105 of the human IL13 (SEQ. ID NO:1). Arginine may be substituted for the lysine at position 105. The mIL13 may comprise changes at positions E13, R66, S69, and / or K105. The mIL13 may be designated IL13.E13K.R66D.S69D.K105R. In an example, the mIL13 may comprise the amino acid sequence set forth in SEQ. ID NO:3 (FIG. 32). In other examples, the mIL13 may comprise any of SEQ. ID NOS:4 to 22 (FIG. 32), or may comprise a sequence sharing at least 80, 85, 90, 95, or 99% homology with any of SEQ. ID NOS:1 to 24 (FIG.32), or may comprise a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues from any of SEQ. ID NOS:1 to 24 (FIG.32).

[0105] The mIL13 and analogs thereof provide for selective binding to IL13 receptors. The mIL13 may be a highly selective antagonist and / or selective agonist of wild-type IL13 activity. The mIL13 may be engineered to have one or more of the following properties: (a) altered affinity (either decreased affinity or increased affinity) for IL13Rα2 relative to wild-type human IL13; (b) altered affinity (either decreased affinity or increased affinity) for IL13Rα1 relative to wild-type human IL13; and / or (c) a disruption in the binding site for IL4Rα1. The mIL13 may comprise amino acidAttorney Docket No. TPI-7GCA1WO modifications made at one or more of the amino acids within the set of contact residues that interact with IL13Rα1, IL13Rα2, or IL4Rα1, which 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, to which the numbering of amino acids refers). The mIL13 may comprise modifications at two or more, three or more, four or more, five or more, and not more than 14 amino acids within the combined set of contact residues defined herein.

[0106] The mIL13 may comprise one or more of the following amino acid substitutions (for reference purposes, relative SEQ. ID. NO:1, to which the numbering of amino acids refers): (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 an altered affinity for one or both of IL13Rα1 and IL13Rα2. The mIL13 may comprise modified residues at two or more, three or more, four or more, five or more, and not more than 14 amino acids within the combined set of contact residues defined herein.

[0107] Polypeptides comprising IL13 and mIL13, polypeptide cytotoxins such as PE, and fusion proteins thereof, may be produced and purified by any useful method. Typically, a coding sequence (e.g. open reading frame or ORF) is inserted into an expression vector for expression in a cell, such as bacterial, yeast, or mammalian cell, and the cell is cultured under conditions suitable for production of the polypeptide. Expression vectors suitable for producing a gene product polypeptide in a desired cell type are broadly available through a large variety of commercial and public sources, such as Addgene, and a desired coding sequence for a polypeptide can be inserted into the vector using any useful cloning method as are broadly known.

[0108] In some examples, the cytotoxin of the compounds or compositions described herein can include a small molecule, peptide, protein, or any combination thereof. For example, the cytotoxin can include at least a portion of PseudomonasAttorney Docket No. TPI-7GCA1WO exotoxin A (PE) which is broadly known to inhibit protein synthesis and induce apoptosis of cancer cells. In other examples, the cytototoxin can include a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with PE. In further examples, the cytotoxin can include a modified sequence of PE that includes additional amino acids. Modified PE sequence examples are PE38QQR,PE38, PE40, PE24, PE38KDEL, PE40KDEL, and PE4E. These modified PE-based polypeptides can be combined with IL13 or mIL13 in a fusion protein targeting IL-13Rα2-expressing cancer cells. See for example, Havaei SM, et al. Pseudomonas Exotoxin-Based Immunotoxins: Over Three Decades of Efforts on Targeting Cancer Cells With the Toxin. Front Oncol.2021 Dec 16;11:781800, Weldon JE, Pastan I. A guide to taming a toxin-recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer. FEBS J. 2011 Dec;278(23):4683-700, and Michalska M, Wolf P. Pseudomonas Exotoxin A: optimized by evolution for effective killing. Front Microbiol. 2015 Sep 15;6:963, describing PE and variants thereof, e.g., for use as a cytotoxin in cancer therapies. Amino acid sequences for PE and variants thereof, and methods for their selection, production and use in fusion proteins are therefore well-known See also, UniProt P11439, e.g., (www.uniprot.org / uniprotkb / P11439 / entry).

[0109] The cytotoxin conjugate can include any of the above toxins conjugated with any of the targeting groups in any combination, e.g., as a fusion protein. For example, any of IL13.K105R, IL13.R109K, IL13 (E13K.R109K), IL13 (E13K.R66D.S69D.R109K), and IL13-E13K can be conjugated with any of PE38QQR, PE38, PE40, PE24, PE38KDEL, PE40KDEL, and PE4E. In some examples, IL13- E13K can be conjugated with PE4E to form TC1. For purposes herein,”conjugated” includes any suitable chemical linkage, e.g., covalent linkage. Fusion proteins comprising an IL13 or IL13 analog sequence and a PE sequence are considered to be conjugated, via a peptide bond.

[0110] The chelating moiety of the labeled therapeutic agent binds to, or chelates, metals by coordinating groups. Coordinating groups in the chelating moiety can include carboxylic acids, hydroxamic acids, amines, and thiols. In some examples, the chelating moiety can be desferrioxamine (DFO). In other examples, the chelatingAttorney Docket No. TPI-7GCA1WO moiety can include diethylenetriaminepentaacetic acid (DTPA), ethylene- diaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-triacetic acid (NOTA).

[0111] The chelating moiety can include a radionuclide. The radionuclide can be a positron emitter. For example, the radionuclide can be11C,13N,15O,18F,34mCl,38K,45Ti,51Mn,52Mn,52mMn,52Fe,55Co,60Cu,61Cu,62Cu,64Cu,66Ga,68Ga,71As,72As,74As,75Br,76Br,82Rb,43Sc, 44Sc,86Y,89Zr,90Nb,94mTc,99mTc,145Tb,148Tb,150Tb,151Tb,154Tb,154mTb,158Tb,110mIn,111In,118Sb,120I,121I,122I,123I, or124I.

[0112] The chelating moiety can include a paramagnetic metal ion. For example. The chelating moiety can include Gd3+, Fe3+, Mn2+, and Y3+. Additionally, and alternatively, the therapeutic moiety can be coupled to a paramagnetic compound. For example, a paramagnetic compound can include one or more19F atoms.

[0113] The chelating moiety can be coupled to the targeting moiety by any useful method, resulting in, for example and without limitation, an amide bond, an ester bond, an ether bond, a disulfide bond, or a thiourea group to form the targeting agent. A linker can be included between the chelating moiety and the therapeutic moiety. The linker can include aliphatic bonds, double bonds, triple bonds, peptide bonds, aromatic rings, aliphatic rings, heterocyclic rings, ethers, esters, amides, and thioamides. The linker can have a rigid structure. Additionally, and alternatively, the linker can be flexible in nature. The linker can include, for example, a linear, branched, and / or cyclical, substituted or unsubstituted C1-C18 alkyl or hydrocarbyl moiety. In other examples, the linker can include a polyethylene glycol chain (-CH2-CH2-O-)n or a polypropylene glycol chain (-CH2-CH2-CH2-O-)n, where n is between 1 and 50. In further examples, the linker can include natural polyamines, including spermine, spermidine, and putrescine. The linker can include synthetic linear or cyclic polyamines, for example tris(2-aminoethyl)amine, cyclen, 1,4,7-triazacyclononane, 1,1,1-tris(aminomethyl)ethane, subunits or oligomers of polyethylenimine, and hexamethylenetetramine. In other examples, the linker can include a polythioether (e.g., (-CH2CH2-S-)).

[0114] A method for preparing a labeled targeting agent that optionally comprises a therapeutic, e.g. cytotoxic moiety, is provided. In the context of theAttorney Docket No. TPI-7GCA1WO present disclosure the targeting moiety or targeting moiety optionally combined with a therapeutic, e.g. cytotoxic, moiety may be in the form of a polypeptide or polypeptide fusion protein, respectively. The method may comprise: conjugating a targeting, and optionally a therapeutic polypeptide with a chelating moiety to create a pre- radiolabeled agent; contacting a radionuclide with the pre-radiolabeled agent to create a radiolabeled preparation; and purifying the radiolabeled preparation to prepare a labeled targeting agent.

[0115] The conjugation reaction results in covalently bonding at least one chelating moiety to the targeting agent. More than one chelating moiety can be coupled to each targeting agent, e.g., polypeptide, depending on the number and location of reactive groups on the targeting agent, though the ratio of the number of chelating moieties per polypeptide may be approximately 1 chelating moiety per polypeptide chain, e.g., , such as an average of from 0.5 to 1.5, from 0.7-1.3, from 0.8 to 1.2, or from 0.9-1.1, including any increment therebetween, chelating moieties, such as hydroxamic or DFO moieties, per polypeptide chain.

[0116] The conjugation reaction can form a bond between the chelating moiety and the targeting moiety, between the chelating moiety and a linker, or between a linker and the targeting agent. Linking the chelating moiety to the polypeptide comprising the targeting moiety and therapeutic moiety, may be accomplished by any suitable method for conjugation of compounds to polypeptides. In some examples, the conjugation reaction can link the chelating moiety to the therapeutic moiety by the formation of an amide, an ester, an ether, a disulfide, a carbonate, a urea, or a thiourea bond. In other examples, the conjugation reaction can be a thiol-ene reaction, a cycloaddition (e.g., click chemistry), a ring-opening reaction, or a photochemical reaction. In one example, an isothiocyanate amine reaction is used to conjugate the chelating moiety to the polypeptide.

[0117] The reaction conditions for the conjugation reaction between the targeting agent and chelating moiety (chelating moiety-containing compound) can include a ratio of targeting agent to the chelating moiety, time, temperature, and pH . In some examples of the method, the targeting agent is combined with the chelating moiety where the ratio of the targeting agent to the chelating moiety is between 1:0.1Attorney Docket No. TPI-7GCA1WO and 1:12. In other examples, the ratio is 1:3. In further examples, the ratio is 1:6. The temperature of the reaction can be from 0°C to 50°C. In some examples, the time of the reaction can be 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, or 120 minutes. In some examples, the time of the reaction can be greater than 120 minutes. The pH of the reaction can be between about 5 and about 10. In some examples, the pH of the reaction is about 9. The pre- radiolabeled agent can be characterized, for example using MALDI mass spectrometry to determine the conjugation results by providing the ratio of targeting agent to chelating moiety after the reaction. Examples of such results are shown in FIGS.3-5. A table showing example reaction conditions and results are shown in FIG.6.

[0118] The reaction conditions for radiolabeling can include variations of concentration of pre-radiolabeled agent, salts, buffers, pH, and time. The radionuclide can include any of11C,13N,15O,18F,34mCl,38K,45Ti,51Mn,52Mn,52mMn,52Fe,55Co,60Cu,61Cu,62Cu,64Cu,66Ga,68Ga,71As,72As,74As,75Br,76Br,82Rb,43Sc, 44Sc,86Y,89Zr,90Nb,94mTc,99mTc,145Tb,148Tb,150Tb,151Tb,154Tb,154mTb,158Tb,110mIn,111In,118Sb,120I,121I,122I,123I, or124I. The radionuclide may be a zirconium, copper, and iodine isotope. One example of suitable reaction conditions is provided in FIG.7. The radionuclide can be contacted with the pre-radiolabeled agent in a reaction mixture such that the radionuclide is chelated by the chelating group to form the labeled therapeutic agent. The pre-radiolabeled agent (e.g. chelating moiety-conjugated polypeptide) can be present in a concentration ranging from 1 pmol / µL to 100 pmol / µL.

[0119] The method can include contacting the compound with a solution of a halide salt including a radionuclide cation. The radionuclide cation can be89Zr4+. The halide can be chloride (Cl-). The method can comprise contacting the compound with89Zr-chloride in a hydrochloride solution. The method can comprise contacting the compound with89Zr-chloride in a hydrochloride solution at a pH in a range of from 7 to 9. The buffer conditions can include 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium acetate and gentisic acid. The pH can range from 4 and 10. The time can range from 5 minutes to 60 minutes. The temperature can range from 5°C to 50°C. FIG.8 shows a scheme for conducting the reaction steps.Attorney Docket No. TPI-7GCA1WO

[0120] The method can include creating the labeled targeting agent at a radiolabeling yield of at least 15%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. FIG.9 shows the radiolabeling yields achieved for different concentrations of the pre-radiolabeled agent. The radiolabeling yield can be enhanced by increasing the relative protein concentration in the reaction mixture as demonstrated in FIG.9.

[0121] The labeled targeting agent can be purified by size exclusion column or HPLC. This results in separation of the targeting agent from any unreacted radiolabel or radiometal ion, pre-radiolabeled excess buffer salts, and any other impurity formed during the reaction. FIG. 10 shows one example of the separation between an example labeled targeting agent and free89Zr using silica gel and 0.1 M sodium citrate. FIGS.11-13 show examples of r-TLC of89Zr-containing labeled targeting agents.

[0122] The method can comprise purifying the radiolabeled preparation using reverse phase chromatography. The method can comprise purifying the radiolabeled preparation using gradient elution. The gradient elution may use at least two different solvents. One of the solvents can comprise water and trifluoroacetic acid, and another of the solvents can comprise acetonitrile and trifluoroacetic acid.

[0123] The labeled targeting agent produced by the method can have a radiochemical stability greater than 85%, greater than 90%, or greater than 95% in mouse serum or formulation (0.25 M sodium acetate with 5 mg / mL gentisic acid, pH 5.4-5.6) measured at 24 hours after purifying the labeled targeting agent.

[0124] Administration to the subject of a pharmaceutical composition including a labeled therapeutic agent as described herein for in vivo imaging of cancer cells in a target region of the subject can be accomplished intravenously, intraarterially, intrathecally, intramuscularly, intradermally, subcutaneously, intracavitarily, or by using convection-enhanced delivery with a cannula in the brain. The labeled therapeutic agent can be formulated with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to any carrier, diluent or excipient that is compatible with the other ingredients of the formulation and not deleterious to the subject. The pharmaceutically acceptable carrier may be chosen in accordance with the selected route of administration and standard pharmaceutical practice for eachAttorney Docket No. TPI-7GCA1WO agent. For example, for administration, the active agent may be mixed with a suitable carrier or diluent such as water, an oil, ethanol, saline solution (e.g., phosphate buffer saline or saline), aqueous dextrose (glucose) and related sugar solutions, glycerol, or a glycol such as propylene glycol or polyethylene glycol. Stabilizing agents, antioxidant agents and preservatives may also be added. Suitable antioxidant agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl-paraben, propyl-paraben, and chlorbutanol. The composition for parenteral administration may take the form of an aqueous or nonaqueous solution, dispersion, suspension or emulsion.

[0125] In the method described herein, sufficient time may be allowed after administration of a detectable amount of the labeled targeting agent such that the labeled targeting agent can accumulate in a target region of the subject, such as the brain or in a tumor. A “detectable amount” means that the amount of the detectable labeled targeting agent that is administered is sufficient to enable detection of accumulation of the labeled therapeutic agent in a subject by a non-invasive medical imaging technique.

[0126] The non-invasive imaging technique may be selected from positron emission tomography imaging, positron emission tomography with computed tomography imaging, or positron emission tomography with magnetic resonance imaging.

[0127] One non-limiting example method of imaging involves the use of a PET active composition such as including the labeled targeting agent, with or without additional contrast agents such as for x-ray techniques or magnetic resonance imaging, delivered by convection-enhanced delivery (CED). Without intent to be bound by this theory, a positron emitting atom of the labeled therapeutic agent gives off a positron, which subsequently annihilates and gives off coincident gamma radiation. This high energy gamma radiation is detectable outside the body through the use of positron emission tomography imaging, or positron emission tomography concurrent with computed tomography imaging (PET / CT). With PET / CT, the location of the injected and subsequently accumulated labeled targeting agent within the body can be identified. Additionally, and alternatively, magnetic resonance imaging (MRI) can beAttorney Docket No. TPI-7GCA1WO used in conjunction with PET and the labeled targeting agent to image the body. The MRI can be performed with or without an MRI contrast agent, for example gadopentetate dimeglumine (MagnevistTM), gadodiamide (OmniscanTM), gadoteridol (ProHanceTM), gadoterate meglumine (DotaremTM, ArtiremTM), or gadobenate dimeglumine (MultiHanceTM) or Galbumin.

[0128] A method for in vivo imaging of a subject can include administering a labeled targeting agent to the subject, waiting a time sufficient to allow the labeled targeting agent to accumulate at a tissue site to be imaged and imaging the tissue site with a non-invasive imaging technique. The tissue site to be imaged can be in the brain or brain stem.

[0129] A method of imaging a subject by emission tomography can include administering to the subject the labeled targeting agent, using a plurality of detectors to detect gamma rays emitted from the subject and to communicate signals corresponding to the detected gamma rays; and reconstructing a series of medical images of a region of interest of the subject from the signals. The labeled targeting agent, once administered, may accumulate in a tumor or other types of cells in the region of interest based on the nature of the targeting moiety. The accumulation of the labeled targeting agent increases the PET signal in the region of interest. The subject can be monitored over time to evaluate any changes in the region of interest, for example the response to a therapeutic can be observed.

[0130] A kit also is provided herein. The kit comprises a first container (for example and without limitation a vial, tube, filled syringe, or bag), comprising an unlabeled cell targeting agent as described herein, and a second container comprising a radiolabel or a paramagnetic ion or compound capable of binding to the binding moiety of the unlabeled cell targeting agent to produce a labeled cell targeting agent as described herein. This may be a way to distribute the described agent and the radiolabel or a paramagnetic ion or compound, for example in the case of a radiolabel, such that the radiolabel may be newly produced and has not substantially decayed. EXAMPLES

[0131] The following Examples are provided for illustration and are not to be construed as limiting the scope of this disclosure.Attorney Docket No. TPI-7GCA1WO Example 1

[0132] As disclosed herein, desferrioxamine (DFO) is conjugated to a peptide, TC1 (IL13.E13K-PE4E), an immunotoxin conjugate, resulting in a compound referred to as [89Zr]Zr-DFO-TC1 as shown in FIG. 2. Briefly, DFO is conjugated to the TC1 polypeptide (shorthand for IL13.E13K-PE4E or GB13) by reacting isocyanate groups of P-SCN-Bn-DFO with amines, such as primary amines of the TC1 polypeptide. T he MALDI-TOF mass spectrometry characterization of [89Zr]Zr-DFO-TC1 is shown in FIG. 3.

[0133] This compound is characterized by its ability to be radiolabeled with Zirconium-89, a positron-emitting radionuclide. In this context, a quantitative and sensitive imaging modality like positron emission tomography (PET) could be used to assess the real-time tissue distribution of the administered drug following CED. To achieve this, an IL13-based PE immunotoxin conjugate, TC1 was radiolabeled with PET radioisotope89Zr using functionalized desferrioxamine (DFO) chelator. The half- life of89Zr (T1 / 2=78.41h) allows [89Zr]Zr-TC1 to be imaged for up to 7 days post-delivery.

[0134] The agent TC1 was chosen because TC1 is more selective for IL-13Rα2 than IL13. The radio-stability of [89Zr]Zr-DFO-TC1 has been tested in mouse and human serum. In addition, uptake of [89Zr]Zr-DFO-TC1 has been determined in IL- 13Rα2 expressing cell lines from glioblastoma, GBM (GBM59) and diffuse mid-line glioma, DMG (PED17). The GBM cell line, GBM6, and DMG cell line, SU-DIPG-XVII with low expression of IL-13Rα2 were used as negative controls. FIG.15 shows the expression levels of IL-13Rα2 in these cell lines. FIGS. 16-18 show the results of uptake of [89Zr]Zr-DFO-TC1 in SU-DIPG-XVII, PED17, GBM6 and GM59 as a % uptake per 106cells / h. The uptake for PED 17 and GBM 59 is greater than for SU- DIPG-XVII or GBM6.

[0135] The size of [89Zr]Zr-DFO-TC1 was confirmed by autoradiography of [89Zr]Zr-DFO-TC1 in an acrylamide gel, as shown in FIG.19. Example 2

[0136] A scheme for imaging IL-13Rα2 in diffuse midline glioma tumor model using [89Zr]Zr-DFO-TC1 is shown in FIG. 20. In this example, luciferin is injectedAttorney Docket No. TPI-7GCA1WO intraperitoneally. Fig.21 shows the bioluminescence imaging of luciferase expressing Luc-PED17 tumor using [89Zr]Zr-DFO-TC1 with and without Galbumin.

[0137] An additional scheme for imaging IL-13Rα2 in diffuse midline glioma tumor model using [89Zr]Zr-DFO-TC1 is shown in FIG.33. In this example, the [89Zr]Zr- DFO-TC1 is delivered using convection-enhanced delivery by a cannula as shown in FIG.22.

[0138] The biodistribution of [89Zr]Zr-DFO-TC1 in PED17 tumors with and without co-injection of Galbumin is shown in PET-CT images acquired at different time points post-injection (0 hours, 2 hours, and 24 hours). (See FIG.23.)

[0139] FIG.24 shows the bioluminescence imaging of an animal implanted with a luciferase-expressing Luc-DIPGXVII tumor.

[0140] PET-CT was used to compare the biodistribution of [89Zr]Zr-DFO-TC1 in DIPGXVII tumor with and without co-injection of Galbumin. (See FIG. 25.) Images were obtained at different time points post-injection (0 hours, 2 hours, and 24 hours).

[0141] PET / CT (0 hours, 2 hours, 24 hours, 48 hours) and PET / X-ray (72 hours, 5 days, and 7 days) imaging were used to compare the uptake of [89Zr]Zr-DFO-TC1 in PED17 and DIPGXVII tumors at various timepoints. IVIS images confirm the presence of the tumor prior to PET / CT or PET / X-ray imaging. (See FIG.26.)

[0142] FIG.27 shows a comparison of biodistribution of [89Zr]Zr-DFO-TC1 when co-infused with Galbumin in PED17 and DIPGXVII tumor models. Images show the biodistribution at 0 hours, 2 hours, and 24 hours.

[0143] MR imaging was used to show the clearance of the Gd-Albumin contrast agent in mice with DIPGXVII tumors (FIG.28) and PED17 tumors (FIG.29) at 1 hour, 2 hours, and 24 hours.

[0144] Surface plasma resonance (SPR) data demonstrates that TC1-DFO has a significantly higher binding affinity for IL13Rα2 over IL13Rα1 for both human and mouse proteins. (FIG.30) This data shows that after conjugation of the DFO chelator, the TC1 retains its function and selectivity towards the target IL13Rα2 receptor.Attorney Docket No. TPI-7GCA1WO Table 1 Surface plasma resonance (SPR) data for binding affinity for TC1 versus TC1-DFO for IL13Rα2 and IL13Rα1 (human and mouse). The chelator conjugated TC1 (TC1- DFO) maintains high specificity for IL13Rα2 over IL13Rα1. Substrate Receptor Binding Affinity

[0145] FIG. 33 shows the determination of Vd for89Zr-DFO-TC1 alone, or in combination with magnetic resonance imaging (MRI) contrast agents.

[0146] The [89Zr]Zr-DFO-TC1 compound provides a method for the targeted imaging of IL13Rα2-positive tumors, which improves the accuracy of diagnosis and monitoring of treatment efficacy in GBM and DMG patients. The specificity of [89Zr]Zr- DFO-TC1 for IL13Rα2-expressing cells and its stability in formulation and mouse serum suggest [89Zr]Zr-DFO-TC1 can be a reliable diagnostic tool. A dditionally, and alternatively, [89Zr]Zr-DFO-TC1 can be a therapeutic agent.

[0147] Although the disclosure has been described in considerable detail with reference to certain embodiments, one skilled in the art will appreciate that the present disclosure can be practiced by other than the described embodiments, which have been presented for purposes of illustration and not of limitation.

Claims

Attorney Docket No. TPI-7GCA1WO CLAIMS What Is Claimed Is:

1. A cell targeting agent, comprising: a binding moiety that binds to a radiolabel or a paramagnetic ion or compound; a polypeptide comprising a naturally occurring IL13 (IL13) amino acid sequence or a mutagenized IL13 (mIL13) amino acid sequence that binds IL13Rα2; and optionally a radiolabel or a paramagnetic ion or compound bound to the binding moiety.

2. The cell targeting agent of claim 1, further comprising a cytotoxin linked to the polypeptide.

3. The cell targeting agent of claim 2, wherein the polypeptide comprising an IL13 or mIL13 amino acid sequence is a fusion protein also comprising an amino acid sequence of a polypeptide cytotoxin.

4. The cell targeting agent of claim 2 or 3, wherein the cytotoxin comprises a Pseudomonas exotoxin A (PE) cytotoxin.

5. The cell targeting agent of claim 4, wherein the cytotoxin comprises PE4E.

6. The cell targeting agent of any one of claims 1-5, wherein the binding moiety comprises a chelating moiety linked to the polypeptide that chelates the radiolabel or paramagnetic ion or compound.

7. The cell targeting agent of claim 6, wherein the chelating moiety comprises a hydroxamic group, such as desferrioxamine B (“DFO”), or one of diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), or 1,4,7- triazacyclononane-triacetic acid (NOTA).Attorney Docket No. TPI-7GCA1WO 8. The cell targeting agent of any one of the preceding claims, wherein the IL13 or mIL13 amino acid sequence has at least 80% sequence identity with IL13.

9. The cell targeting agent of any one of the preceding claims, wherein the cell targeting agent is a labeled cell targeting agent comprising a radiolabel or a paramagnetic ion or compound.

10. The cell targeting agent of claim 9, comprising11C,13N,15O,18F,34mCl,38K,45Ti,51Mn,52Mn,52mMn,52Fe,55Co,60Cu,61Cu,62Cu,64Cu,66Ga,68Ga,71As,72As,74As,75Br,76Br,82Rb,43Sc, 44Sc,86Y,89Zr,90Nb,94mTc,99mTc,145Tb,148Tb,150Tb,151Tb,154Tb,154mTb,158Tb,110mIn,111In,118Sb,120I,121I,122I,123I,124I, Gd+3, Fe+3, Mn+2, Y+3, or a paramagnetic compound comprising one or more19F atoms.

11. The cell targeting agent of claim 9, comprising a zirconium, copper, or iodine isotope 12. The cell targeting agent of claim 9, comprising89Zr.

13. The cell targeting agent of claim 1, wherein the polypeptide comprises, consists essentially of, or consists of IL13.E13K-PE4E.

14. The cell targeting agent of claim 1, comprising, consisting essentially of, or consisting of [89Zr]Zr-DFO-IL13.E13K-PE4E.

15. A composition comprising the cell targeting agent of any one of the preceding claims and a pharmaceutically acceptable carrier.Attorney Docket No. TPI-7GCA1WO 16. A method of in vivo imaging, comprising: administering a labeled cell targeting agent of any one of claims 9-14 to a subject such that an IL13Rα2-expressing cell, such as a diffuse midline glioma (DMG) cell or a glioblastoma (GBM) cell, of the subject becomes labeled for in vivo imaging.

17. A method of in vivo imaging, comprising: labeling a cell targeting agent of any one of claims 1-8 with a radiolabel or a paramagnetic ion or compound; and administering the labeled cell targeting agent to a subject such that an IL13Rα2- expressing cell, such as a diffuse midline glioma (DMG) cell or a glioblastoma (GBM) cell, of the subject becomes labeled for in vivo imaging.

18. The method of claim 16 or 17, further comprising imaging a tissue site of the subject to visualize, localize, and optionally quantify IL13Rα2-expressing cells at the site of the subject using a non-invasive imaging technique.

19. The method of any one of claims 16-18, wherein the IL13Rα2-expressing cell and / or tissue site is in the brain, the frontal lobe of the brain, the temporal lobe of the brain, the midline of the brain, the brain stem, or the spinal cord of the subject.

20. The method of any one of claims 16-19, wherein the cell is a DMG cell.

21. The method of any one of claims 16-19, wherein the cell is a GBM cell.

22. The method of any one of claims 16-21, wherein the binding moiety comprises a chelating moiety that chelates the radiolabel or paramagnetic ion or compound.

23. The method of claim 22, wherein the chelating moiety is a hydroxamic group, such as desferrioxamine B (“DFO”), or one of diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-Attorney Docket No. TPI-7GCA1WO tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), or 1,4,7-triazacyclononane- triacetic acid (NOTA).

24. The method of any one of claims 16-23, wherein the radiolabel or paramagnetic ion or compound comprises11C,13N,15O,18F,34mCl,38K,45Ti,51Mn,52Mn,52mMn,52Fe,55Co,60Cu,61Cu,62Cu,64Cu,66Ga,68Ga,71As,72As,74As,75Br,76Br,82Rb,43Sc, 44Sc,86Y,89Zr,90Nb,94mTc,99mTc,145Tb,148Tb,150Tb,151Tb,154Tb,154mTb,158Tb,110mIn,111In,118Sb,120I,121I,122I,123I,124I, Gd+3, Fe+3, Mn+2, Y+3, or a paramagnetic compound comprising one or more19F atoms.

25. The method of any one of claims 16-23, wherein radiolabel or paramagnetic ion or compound comprises a zirconium, copper, or iodine isotope.

26. The method of any one of claims 16-23, wherein the radiolabel or paramagnetic ion or compound comprises89Zr.

27. The method of any one of claims 16-26, comprising after administering the labeled cell targeting agent to the subject, waiting a time sufficient to allow the labeled cell targeting agent to accumulate at a tissue site to be imaged.

28. The method of any one of claims 16-27, wherein the non-invasive imaging technique is selected from positron emission tomography imaging, positron emission tomography with computed tomography imaging, or positron emission tomography with magnetic resonance imaging.

29. The method of any one of claims 16-27, comprising imaging the subject by emission tomography, and further comprising using a plurality of detectors to detect gamma rays emitted from the subject and to communicate signals corresponding to the detected gamma rays; and reconstructing from the signals a series of medical images of a region of interest of the subject.Attorney Docket No. TPI-7GCA1WO 30. The method of any one of claims 16-27, for use in detecting or ruling out diffuse midline glioma or glioblastoma in a subject.

31. The method of any of claims 16-30, wherein the labeled cell targeting agent comprises, consists essentially of, or consists of [89Zr]Zr-DFO-IL13.E13K-PE4E.

32. The method of any one of claims 16-31, wherein the labeled IL13 agent is administered by convection enhanced drug delivery.

33. A method of preparing the cell targeting agent of any one of claims 1-14, comprising conjugating a compound comprising the binding moiety that binds to a radiolabel or a paramagnetic ion or compound with a polypeptide comprising the IL13 or mIL13 amino acid sequence, and optionally a PE cytotoxin amino acid sequence, to produce a pre-radiolabeled cell targeting agent.

34. The method of claim 33, further comprising mixing the pre-radiolabeled cell targeting agent with a radiolabel or a paramagnetic ion or compound to produce a labeled cell targeting agent.

35. The method of claim 33 or 34, wherein the binding moiety comprises a hydroxamic group.

36. The method of claim 35, wherein the hydroxamic group comprises a desferrioxamine group.

37. The method of claim 35 or 36, wherein the polypeptide is linked to the compound comprising the binding moiety by an isothiocyanate-amine reaction.

38. The method of claim 35 or 36, wherein the polypeptide is linked to the compound comprising the binding moiety by formation of an amide bond, reaction of a thiol with a maleimide, or by click chemistry.Attorney Docket No. TPI-7GCA1WO 39. The method of any one of claims 33-38, wherein the polypeptide comprises an IL13.E13K-PE4E amino acid sequence.

40. A kit comprising: a first container comprising a cell targeting agent, the cell targeting agent comprising: a. a binding moiety that binds to a radiolabel or a paramagnetic ion or compound; and b. a polypeptide comprising a naturally occurring IL13 (IL13) amino acid sequence or a mutagenized IL13 (mIL13) amino acid sequence that binds IL13Rα2; and a second container, comprising a radiolabel or a paramagnetic ion or compound capable of binding to the binding moiety.

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