Armored chimeric antigen receptor t cells

Abstract

The present application relates to sequences for expression in engineered chimeric antigen receptor (CAR) T cells for targeting IL13Ra2. In some embodiments, the sequences include a humanized VHH binder specific to IL13Ra2, an intracellular signaling domain, armoring elements to support cell persistence and functionality, or any combination thereof. Also disclosed herein are T cells that include a suicide gene for controlled cell elimination, and therapeutic uses thereof.

Description

CHMRS.005WO PATENT ARMORED CHIMERIC ANTIGEN RECEPTOR T CELLSRELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims the benefit of U.S. Provisional Ser. No. 63 / 721,361, filed November 15, 2024, which is hereby incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The present application is filed with a Sequence Listing in Electronic format. The Sequence Listing is provided as a file entitled CHMRS005WO.xml created November 13, 2025, which is 27,761 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.RELATED FIELD

[0003] Disclosed herein are chimeric antigen receptor (CAR) T cell therapies specifically engineered to target a receptor of interest, such as interleukin- 13 receptor alpha 2 (IL13Ra2). In some embodiments, the CAR T cells are used in the treatment of glioblastoma and other solid tumors. Also disclosed herein are anti-IL13Ra2 sequences, methods of administration, and viral vectors encoding the CAR T cells described herein.BACKGROUND

[0004] The treatment of solid tumors, including glioblastoma, presents significant challenges due to factors like tumor heterogeneity, immune suppression within the tumor microenvironment, and the high risk of off-target toxicity. Chimeric antigen receptor (CAR) T cell therapy has demonstrated success in treating certain hematological cancers by enabling T cells to target specific tumor antigens, yet CAR T cell therapies for solid tumors have been less effective. This limitation has spurred the need for CAR T cells that can specifically and robustly target solid tumor antigens while remaining viable and active in the tumor environment.SUMMARY

[0005] Disclosed herein is an engineered T cell therapy targeting a receptor of interest, such as IL13Ra2, a tumor-associated antigen frequently overexpressed in glioblastoma and other solid tumors. Also disclosed herein are T cells modified to express a chimeric antigen receptor (CAR) with a humanized single-domain antibody (VHH) binder specific to IL13Ra2 over IL13Ral, enhancing the CAR’s selectivity and effectiveness in tumor targeting. In some embodiments, the CAR includes a flexible spacer derived from human CD28, a CD28 transmembrane domain, an intracellular signaling domain containing either a CD28-zeta or a 4-lBB-zeta motif, or any combination thereof, enabling robust activation of T cells upon antigen engagement.

[0006] Also disclosed herein are methods of treating glioblastoma and other IL13Ra2-expressing tumors through the administration of CAR T cells, optionally in combination with checkpoint inhibitors or agents that activate the suicide gene. Pharmaceutical compositions and kits are also included, supporting safe, controlled, and effective delivery of CAR T cell therapy in clinical settings.

[0007] Aspects of the present disclosure relate to a chimeric antigen receptor (CAR) polypeptide comprising: (1) a binding sequence specific to IL13Ra2; (2) an optional spacer region; (3) a transmembrane domain; and (4) an intracellular endodomain. In some embodiments, the binding sequence is a humanized, single domain antibody (VHH) binder. In some embodiments, the binding sequence does not significantly bind to IL13Ral. In some embodiments, the spacer region comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of a human CD28 hinge domain as set forth in SEQ ID NO: 5. In some embodiments, the transmembrane domain comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of a human CD28 transmembrane domain as set forth in SEQ ID NO: 6. In some embodiments, the intracellular endodomain comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of a CD28-zeta signaling domain as set forth in SEQ ID NO: 7 or a 4-lBB-zeta signaling domain. In some embodiments, the binding sequence comprises: (1) a CDR1 sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 24 (SYYMR); (2) a CDR2 sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 25 (SINSGGGSTSYVDSVKG); (3) a CDR3 sequence with at least about 80, 85, 90, 95,99, or 100% identity to the sequence of SEQ ID NO: 26 (ALETNRGQNY); or (4) any combination of ( 1 )-(3) thereof. In some embodiments, the CAR polypeptide further comprises an at least one additional costimulatory signaling domain, optionally wherein the at least one additional costimulatory signaling domain is selected from 4-1BB, 0X40, ICOS, or any combination thereof. In some embodiments, the CAR polypeptide is configured to target a glioblastoma.

[0008] Also disclosed herein is a nucleotide sequence encoding the CAR polypeptide of any one of the embodiments of the present disclosure.

[0009] Also disclosed herein is a vector encoding the CAR polypeptide of any one of the embodiments of the present disclosure. In some embodiments, the vector is a viral vector, optionally wherein the vector is a self-inactivating lentiviral or a retroviral vector. In some embodiments, the vector is capable of integrating into a cell’ s genome. In some embodiments, the vector further comprises an at least one self-cleaving 2A peptide. In some embodiments, the at least one self-cleaving 2A peptide is between at least two of the following: (1) the CAR polypeptide of any one of the embodiments of the present disclosure; (2) a suicide gene; (3) an armoring element; (4) an IL- 12 construct; or (5) any combination of (l)-(4) thereof.

[0010] Also disclosed herein is a cell capable of expressing the CAR polypeptide of any one of the embodiments of the present disclosure, optionally wherein the cell encodes the nucleotide of any one of the embodiments of the present disclosure, and / or the vector of any one of the embodiments of the present disclosure. In some embodiments, the cell is a T cell. In some embodiments, the cell further comprises an armoring element, wherein the armoring element comprises an antibody binder with a heavy and light chain on the extracellular domain, and the endodomain of the GM-CSF receptor. In some embodiments, the cell further comprises (1) a light chain polypeptide comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of a GM-CSF beta receptor endodomain as set forth in SEQ ID NO: 11; and / or (2) a heavy chain polypeptide comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence for a GM-CSF alpha receptor endodomain as set forth in SEQ ID NO: 15. In some embodiments, the armoring element is capable of stabilizing and / or enhancing T cell persistence and / or proliferation. In some embodiments, the armoring element includes a dimerization interface capable of forming one or more disulfide bonds, thereby stabilizing the heavy and light chains. In some embodiments,the dimerization interface is specific for TGF-betaR2 and inhibits TGF-beta signaling upon ligand interaction. In some embodiments, the cell comprises: (1) a TGF-betaR2 specific light chain comprising a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of SEQ ID NO: 9; and or (2) a TGF-betaR2 specific heavy chain comprising a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of SEQ ID NO: 13. In some embodiments, the intracellular endodomain of the CAR polypeptide is capable of activating cytokine signaling in the cell following the CAR polypeptide binding to IL13Ra2. In some embodiments, cytokine signaling promotes upregulation of IL-2, IFN-y, and / or INFIX. In some embodiments, the cell further comprises a suicide gene that is capable of activating selective apoptosis upon administration of a compound. In some embodiments, the suicide gene comprises a truncated HER2 extracellular domain (SEQ ID NO: 17). In some embodiments, the suicide gene comprises a transmembrane domain and / or an endodomain sequence of CD5 as set forth in SEQ ID NO: 18 and 19, respectively. In some embodiments, the suicide gene is capable of activating selective apoptosis upon administration of trastuzumab emtansine. In some embodiments, the cell further comprises an IL- 12 construct. In some embodiments, the IL-12 construct further comprises: (1) an N-terminal p35 subdomain sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of SEQ ID NO: 20; (2) a linker sequence; and (3) a C-terminal p40 subdomain sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of SEQ ID NO: 22. In some embodiments, the IL-12 linker sequence comprises a sequence having at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of SEQ ID NO: 21. In some embodiments, the IL-12 linker sequence is between 4-15 amino acids in length. In some embodiments, expression of the CAR polypeptide is regulated by an inducible promoter. In some embodiments, the cell is genetically modified to decrease PD-1 expression.

[0011] Also disclosed herein is a use for the CAR polypeptide of any one of the embodiments of the present disclosure as part of a medicament.

[0012] Also disclosed herein is a method of treating a cancer and / or a tumor in a subject. In some embodiments, the method comprises administering to the subject the CAR polypeptide of any one of the embodiments of the present disclosure, the nucleotide of any one of the embodiments of the present disclosure, the vector of any one of the embodiments of the present disclosure, the cell of any one of the embodiments of the present disclosure, or anycombination thereof. In some embodiments, the administration is conducted intracranially, or via intratumoral or intravenous injection. In some embodiments, the method further comprises administering a compound post-treatment to selectively activate the suicide gene.

[0013] Also disclosed herein is a method of treating glioblastoma in a subject. In some embodiments, the method comprises administering to the subject the CAR polypeptide of any one of the embodiments of the present disclosure, the nucleotide of any one of the embodiments of the present disclosure, the vector of any one of the embodiments of the present disclosure, the cell any one of the embodiments of the present disclosure, or any combination thereof. In some embodiments, the administration is conducted intracranially, or via intratumoral or intravenous injection. In some embodiments, the method further comprises administering a compound post-treatment to selectively activate the suicide gene.

[0014] Also disclosed herein is a pharmaceutical composition comprising the CAR polypeptide of any one of the embodiments of the present disclosure, the nucleotide of any one of the embodiments of the present disclosure, the vector of any one of the embodiments of the present disclosure, the cell any one of the embodiments of the present disclosure, or any combination thereof, and a pharmaceutically acceptable carrier, wherein the composition is optimized for administration in a mammal.

[0015] Also disclosed herein is a kit comprising the CAR polypeptide of any one of the embodiments of the present disclosure, the nucleotide of any one of the embodiments of the present disclosure, the vector of any one of the embodiments of the present disclosure, the cell any one of the embodiments of the present disclosure, or any combination thereof, a suicide gene activator compound, and instructions for administering the CAR T cells in treating glioblastoma.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A depicts a non-limiting example schematic of a single vector cassette including an IL13Ra2 CAR, a suicide switch component (AHER2CD3) and multiple armoured elements (TFGb / CCR and IL 12).

[0017] Figure IB depicts a non-limiting example cartoon graphic of each component of the cassette of Figure 1A expressed on the cell surface.

[0018] Figure 1C depicts non-limiting representative dot plots showing the expression of the VHH sequence (top row), the IL13Ra2 binder sequence (middle row), and the detection of the suicide switch using trastuzumab (bottom row) in non-transduced (“NT”, left column), CAR T cells (middle column), and T cells expressing CAR, Armour, and Suicide genes (right column). Detection of each is quantified by flow cytometry

[0019] Figure 2 depicts a non-limiting example quantification of the cytotoxicity of IL13Ra2 binding CAR T cells towards IL13Ral and IL13Ra2 expressing target cells

[0020] Figure 3A depicts a non-limiting example quantification of cytotoxic function of cells expressing CAR or CAR + Armour + Suicide when exposed to target cells endogenously expressing IL13Ra2, and co-cultured at different effector : target (E:T) ratios.

[0021] Figure 3B depicts a non-limiting example quantification of IFN-y detection from cells expressing CAR or CAR + Armour + Suicide when exposed to target cells endogenously expressing IL13Ra2, and co-cultured at different effector : target (E:T) ratios. IFN-y was quantified by ELISA.

[0022] Figure 3C depicts a non-limiting example quantification of IL2 detection from cells expressing CAR or CAR + Armour + Suicide when exposed to target cells endogenously expressing IL13Ra2, and co-cultured at different effector : target (E:T) ratios. IL2 was quantified by ELISA.

[0023] Figure 4A depicts a non-limiting example cartoon schematic outlining an assay to measure function following TGF-0 exposure.

[0024] Figure 4B depicts a non-limiting example quantification of cytotoxicity of CAR and CAR + Armour + Suicide cells towards IL13Ra2 expressing target cells following prior stimulation in the presence or absence of TGF0.

[0025] Figure 5 depicts a non-limiting example quantification of the changes in cell counts over multiple days when CAR and CAR + Armour + Suicide. Cells are cultured in the absence of cytokine or stimulation.

[0026] Figure 6A depicts a non-limiting example cartoon schematic of a transwell migration assay where media is transferred from transduced T cells to NK cells or CD8 T cells that are separated by a permeable membrane.

[0027] Figure 6B depicts a non-limiting example quantification of STAT4 phosphorylation in NK cells, as detected by flow cytometry, when cultured via permeable inserts with CAR and CAR + Armour + Suicide cells.

[0028] Figure 6C depicts a non-limiting example quantification of STAT4 phosphorylation in CD8 cells, as detected by flow cytometry, when cultured via permeable inserts with CAR and CAR + Armour + Suicide cells.

[0029] Figure 7A depicts non-limiting representative dot plots of suicide switch detection, by trastuzumab binding, and CAR detection via VHH binding in cells transduced to express CAR or CAR + Armour + Suicide. Expression by flow cytometry.

[0030] Figure 7B depicts a non-limiting example quantification of cell viability when CAR and CAR + Armour + Suicide cells are exposed to varying concentrations of T-DM1.

[0031] Figure 7C depicts a non-limiting example cartoon schematic outlining an assay to measure function following T-DM1 exposure.

[0032] Figure 7D depicts a non-limiting example quantification of cytotoxic function when CAR and CAR + Armour + Suicide cells are exposed to T-DM1.

[0033] Figure 8A depicts a non-limiting example cartoon graphic of an in vivo model showing tumour, CAR and T-DM1 injections.

[0034] Figure 8B depicts a non-limiting example quantification of detection of tumour growth by bioluminescence imaging of mice injected with non-transduced (NT) cells, or CAR T cells with and without .

[0035] Figure 8C depicts a non-limiting example quantification of bodyweight changes of mice injected with non-transduced (NT) cells, or CAR T cells with and without TDM1.

[0036] Figure 9A depicts a non-limiting example cartoon schematic of an in vivo model comparing CAR and CAR + Armour + Suicide cells.

[0037] Figure 9B depicts a non-limiting example quantification of bioluminescence images of mice over the duration of the study.

[0038] Figure 9C depicts a non-limiting example quantification of quantitative analysis of bioluminescence signal from tumour cells injected in mice in a graphical format.DETAILED DESCRIPTION

[0039] Disclosed herein are engineered CAR T cells for enhanced anti-tumor activity targeting a receptor, such as IL13Ra2. In some embodiments, the CAR T cells incorporate a humanized VHH binder specific to IL13Ra2, an intracellular signaling domain, armoring elements to support cell persistence and functionality, or any combination thereof. In some embodiments, the humanized VHH binder is a single-domain antibody. In some embodiments, the intracellular signaling domain includes the intracellular signaling domain of CD28-zeta or 4-lBB-zeta. Also disclosed herein are cells that include a suicide gene for controlled cell elimination. In some embodiments, the CAR is delivered via a viral vector. The present disclosure offers an advanced therapeutic approach for treating IL13Ra2-expressing cancers, including glioblastoma, with improved safety, stability, and efficacy.

[0040] Interleukin- 13 receptor alpha 2 (IL13Ra2) has emerged as a promising target for CAR T cell therapy due to its overexpression in several malignancies, including glioblastoma, and its low expression in normal tissues. Targeting IL13Ra2 with CAR T cells could enhance the specificity and efficacy of treatment for tumors that express this receptor.

[0041] Traditionally, CAR T cell constructs employ single-chain variable fragments (scFvs) to bind target antigens. However, scFv-based CARs can present challenges related to immunogenicity, stability, and aggregation. To address these limitations, VHH domains, derived from camelid antibodies, have been explored as alternative binders due to their small size, high stability, and lower immunogenicity. Humanized VHH binders, specifically tailored to minimize immune rejection in patients, offer a further advantage for therapeutic applications.

[0042] To optimize CAR T cell therapy targeting IL13Ra2, embodiments described herein introduce a CAR construct containing a humanized VHH binder for selective binding to IL13Ra2, an intracellular signaling domain such as CD28-zeta or 4-lBB-zeta to drive effective T cell activation, and armoring elements to enhance T cell persistence and resistance to immunosuppression. The CAR also includes a suicide gene to provide a mechanism for controlled elimination of the modified T cells, improving safety by allowing for the selective apoptosis of CAR T cells if adverse events occur. Furthermore, a lentiviral vector encoding the CAR is used for stable integration, with optional additional armoring elements such as IL-12constructs or checkpoint inhibitors to further promote efficacy and durability of response in the hostile tumor microenvironment.

[0043] Disclosed herein is a CAR T cell therapy targeting IL13Ra2 for the treatment of solid tumors, with a particular focus on glioblastoma. The design addresses key challenges in solid tumor CAR T therapy, offering an innovative approach to improving tumor specificity, persistence, and therapeutic effectiveness.

[0044] To improve the CAR T cell’s durability and efficacy within the immunosuppressive tumor microenvironment, in some embodiments the CAR construct includes one or more armoring elements. In some embodiments, the armoring elements include GM-CSF receptor components that enhance T cell persistence and IL-12 constructs that stimulate anti-tumor immune responses. Additionally, in some embodiments the cell includes a suicide gene which functions as a safety mechanism, allowing selective elimination of the CAR T cells upon administration of a specific compound, such as an antibody-drug conjugate (ADC).

[0045] In some embodiments, the CAR T cells are encoded within a viral vector, such as a self-inactivating lentiviral or retroviral vector, designed for stable genome integration and reliable CAR expression. In some embodiments, the vector may also incorporate selfcleaving 2A peptides, facilitating seamless expression of the CAR, suicide gene, and armoring elements.Terms

[0046] In the present disclosure, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in this disclosure, including the drawings and claims, are not meant to be limiting. Some embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs when read in light of the current disclosure.

[0048] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0049] The articles “a” and “an” are used herein to refer to one or to more than one (for example, at least one) of the grammatical object of the article, unless the context dictates otherwise. By way of example, “an element” means one element or more than one element.

[0050] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that is approximately the recited value. Where it is not clear from the context what is encompassed by “about,” it will mean the value recited + / -10%.

[0051] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0052] The terms “individual”, “subject”, or “patient” as used herein have their plain and ordinary meaning as understood in light of the specification, and mean a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a nonhuman primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. The term “mammal” is used in its usual biological sense. Thus, it includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.

[0053] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and / or spanning the aforementioned values). In some embodiments, isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and / or spanning the aforementioned values). As used herein, a substance that is “isolated” may be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multi-cellular organism or tissue.

[0054] As used herein, “in vivo” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.

[0055] As used herein, “ex vivo” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside a living organism with little alteration of natural conditions.

[0056] As used herein, “in vitro” has its plain and ordinary meaning as understood in light of the specification and refers to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.

[0057] The term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may beaffected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents. Purity can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0058] The term “yield” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and refers to the actual overall amount of the substance, compound, or material relative to the expected overall amount. For example, the yield of the substance, compound, or material is, is about, is at least, is at least about, is not more than, or is not more than about, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including all decimals in between. Yield may be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.

[0059] The terms “effective amount” or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to that amount of a recited composition or compound that results in an observable effect. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that is effective to achieve the desired response for a particular subject and / or application. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose isescalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0060] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to a biological, enzymatic, or therapeutic function.

[0061] The term “inhibit” as used herein has its plain and ordinary meaning as understood in light of the specification, and may refer to the reduction or prevention of a biological activity. The reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values. As used herein, the term “delay” has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of a biological event, to a time which is later than would otherwise be expected. The delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay may not necessarily indicate a 100% inhibition or delay. A partial inhibition or delay may be realized.

[0062] As used herein, the terms “treating” or “treatment” have their plain and ordinary meaning as understood in light of the specification, and refer to an approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (e.g., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the recurrence of disease, and remission, whether partial or total and whether detectable or undetectable. “Treating” and “treatment” as used herein also include prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may include a single administration or may include a series of administrations. The compositions are administered to the subject inan amount and for a duration sufficient to treat the subject. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age and genetic profile of the subject, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.

[0063] Tumor,” as used herein, has its plain and ordinary meaning as understood in light of the specification, and refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein. The term “neoplasia” encompasses the term tumor.

[0064] The terms “cancer” and “cancerous” plain and ordinary meaning as understood in light of the specification, and refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include lung cancer including small-cell lung cancer, non-small cell lung cancer and lung adenocarcinomas with neuroendocrine features; neuroendocrine prostate cancer, melanoma, gliomas, low-grade gliomas and glioblastoma, medullary thyroid cancer, carcinoid tumors, neuroendocrine tumors in the pancreas, bladder cancer, testicular cancer squamous cell cancer (e.g. epithelial squamous cell cancer), neuroendocrine neoplasms, such as neuroendocrine tumors of unknown primary, neuroendocrine neoplasms of the small bowel, carotid body, adrenal gland, colorectal gynecological organ, abdomen, esophagus, GI tract, bile duct, nervous system, appendix, liver, anal, thymus, ileocecal junction, head and neck, breast, peritoneum and retroperitoneum, kidney, thyroid, stomach, bone,; adenocarcinomas, such as adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma,salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, bone cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases. The term cancer includes adult and pediatric solid cancers. In some embodiments, the cancer can be a solid tumor. In some embodiments, the cancer is a highly fibrotic tumor or cancer. In some embodiments, the cancer is a desmoplasia.

[0065] As used herein, the term "therapeutic target" has its plain and ordinary meaning as understood in light of the specification and refers to a gene or gene product that, upon modulation of its activity (e.g., by modulation of expression, biological activity, and the like), can provide for modulation of the disease phenotype. As used throughout, "modulation" is meant to refer to an increase or a decrease in the indicated phenomenon (e.g., modulation of a biological activity refers to an increase in a biological activity or a decrease in a biological activity).

[0066] The term “administering” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intra-tumoral, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intra-tumoral, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “coadminister” it is meant that a first compound described herein is administered at the same time, just prior to, or just after the administration of a second compound described herein.

[0067] As used herein, “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and / or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or carrier can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical formulation is administered. Such pharmaceutical diluent, excipient, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include sugars, starch, glucose, fructose, lactose, sucrose, maltose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, salts, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. The physiologically acceptable carrier may also include one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, isomalt, maltitol, or lactitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The formulation, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These formulations can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0068] The term “pharmaceutically acceptable salts” has its plain and ordinary meaning as understood in light of the specification and includes relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples ofpharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; amino acids, including glycine, arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl aminoethane.

[0069] The term “% w / w” or “% wt / wt” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100. The term “% v / v” or “% vol / vol” as used herein has its plain and ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.

[0070] The terms “first,” “second,” and “third” used in combination with substances are intended to designate distinguishable features to similar substances and do not imply any particular order unless otherwise specified.

[0071] The term “antibody” is used in the broadest sense and includes various antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies, antibody fragments, and any other constructs that retain antigen-binding activity.

[0072] The term “single-domain antibody” or “VHH” refers to an antibody fragment derived from camelid antibodies, including a single variable domain that retains the antigen-binding specificity of the full antibody. In embodiments described herein, the VHH may be humanized to reduce immunogenicity.

[0073] The term “humanized” refers to a modification of the antibody or antibody fragment to reduce immunogenicity by replacing some non-human amino acid sequences with human sequences while retaining antigen-binding properties.

[0074] The term “chimeric antigen receptor” or “CAR” refers to an engineered receptor expressed on immune cells, which typically includes an antigen-binding domain (e.g., a VHH), a spacer or hinge region, a transmembrane domain, and one or more intracellular signaling domains, often including costimulatory signaling domains.

[0075] The term “spacer” or “hinge” as used herein refers to a flexible polypeptide region connecting the antigen-binding domain and the transmembrane domain, providing structural flexibility. In one embodiment, the spacer includes a sequence derived from human CD28.

[0076] The term “transmembrane domain” refers to a domain in a CAR construct that spans the cell membrane, allowing receptor anchoring on the cell surface. In embodiments described herein, the transmembrane domain may include a sequence derived from human CD28.

[0077] The term “intracellular endodomain” refers to the cytoplasmic signaling domain(s) of the CAR. This domain transmits activation signals upon target engagement. In some embodiments, it includes CD28-zeta or 4-lBB-zeta sequences for signal transduction.

[0078] The term “armoring element” refers to a CAR modification that enhances T cell persistence, proliferation, or resistance to immunosuppressive factors in the tumor microenvironment. This can include GM-CSF receptor components or IL-12 constructs, among others.

[0079] The term “suicide gene” refers to a genetic element included in the CAR T cell construct that allows for controlled elimination of the CAR T cells. In one embodiment, the suicide gene is activated upon exposure to a specific compound, such as an antibody-drug conjugate (ADC).

[0080] The term “IL- 13 receptor alpha 2” or “IL13Ra2” refers to a tumor-associated antigen targeted by the CAR as described herein. IL13Ra2 is overexpressed in glioblastoma and other cancers, making it an ideal therapeutic target.

[0081] The term “interleukin- 12 construct” or “IL- 12 construct” refers to an armoring element including subdomains such as p35 and p40 connected by a linker, which may support immune activation in the tumor microenvironment.

[0082] The term “epitope” refers to the part of an antigen recognized by the antibody or antigen-binding region of a CAR, which facilitates targeted binding.

[0083] The term “vector” refers to a nucleic acid molecule capable of delivering a gene into cells. As described herein, the vector may be a lentiviral or retroviral vector for stable CAR expression in T cells.

[0084] The term “pharmaceutical composition” refers to a formulation that includes the CAR T cells in a carrier suitable for administration, providing biological activity of the CAR T cells when administered to a subject.

[0085] Embodiments provided herein are described by way of the following numbered alternatives:

[0086] 1. A chimeric antigen receptor (CAR) polypeptide comprising: a binding sequence specific to IL13Ra2; a spacer region; a transmembrane domain; and an intracellular endodomain.

[0087] 2 The CAR polypeptide of alternative 1, wherein the binding sequence is a humanized, single domain antibody (VHH) binder.

[0088] 3 The CAR polypeptide of alternative 1 or 2, wherein the binding sequence does not significantly bind to IL13Ral.

[0089] 4. The CAR polypeptide of any one of alternatives 1-3, wherein the spacer region comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of the human CD28 hinge domain (SEQ ID NO: 5).

[0090] 5 The CAR polypeptide of any one of alternatives 1-4, wherein the transmembrane domain comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of the human CD28 transmembrane domain (SEQ ID NO: 6).

[0091] 6 The CAR polypeptide of any one of alternatives 1-5, wherein the intracellular endodomain comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of the CD28-zeta signaling domain (SEQ ID NO: 7) or the 4-lBB-zeta signaling domain.

[0092] 7 The CAR polypeptide of any one of alternatives 1 -6, wherein the binding sequence comprises: a CDR1 sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 24 (SYYMR); a CDR2 sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 25 (SINSGGGSTSYVDSVKG); a CDR3 sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 26 (ALETNRGQNY); or any combination thereof.

[0093] 8. The CAR polypeptide of any one of alternatives 1-7, further comprising an at least one additional costimulatory signaling domain, optionally wherein the at least one additional costimulatory signaling domain is selected from 4-1BB, 0X40, ICOS, or any combination thereof.

[0094] 9. The CAR polypeptide of any one of alternatives 1-8, for use in glioblastoma targeting.

[0095] 10. A nucleotide sequence encoding the CAR polypeptide of any one of alternatives 1-8.

[0096] 11. A vector encoding the CAR polypeptide of any one of alternatives 1-8.

[0097] 12. The vector of alternative 11, wherein the vector is a viral vector, optionally wherein the vector is a self-inactivating lentiviral or a retroviral vector.

[0098] 13. The vector of alternative 11 or 12, wherein the vector is capable of integrating into a cell’s genome.

[0099] 14. The vector of any one of alternatives 11-13, further comprising an at least one self-cleaving 2A peptide.

[0100] 15. The vector of alternative 14, wherein the at least one self-cleaving 2A peptide is between at least two of the following: the CAR polypeptide of any one of alternatives 1-8; a suicide gene; an armoring element; an IL-12 construct; or any combination thereof of (a)-(d).

[0101] 16. A cell capable of expressing the CAR polypeptide of any one of alternatives 1-8, optionally wherein the cell encodes the nucleotide of alternative 10, and / or the vector of any one of alternatives 11-15.

[0102] 17. The cell of alternative 16, wherein the cell is a T cell.

[0103] 18. The cell of any one of alternatives 16-17, further comprising an armoring element, wherein the armoring element comprises an antibody binder with a heavy and light chin on the extracellular domain, and the endodomain of the GM-CSF receptor.

[0104] 19. The cell of alternative 18, wherein: the light chain polypeptide comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of the GM-CSF beta receptor endodomain (SEQ ID NO: 11); and / or the heavy chain polypeptide comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence for the GM-CSF alpha receptor endodomain (SEQ ID NO: 15).

[0105] 20. The cell of any one of alternatives 18-19, wherein the armoring element stabilizes and enhances T cell persistence and / or proliferation.

[0106] 21. The cell of any one of alternatives 18-20, wherein the armoring element includes a dimerization interface capable of forming one or more disulfide bonds, thereby stabilizing the heavy and light chains.

[0107] 22. The cell of alternative 21, wherein the dimerization interface is specific for TGF-betaR2 and inhibits TGF-beta signaling upon ligand interaction.

[0108] 23. The cell of alternative 22, wherein: the TGF-betaR2 specific light chain comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 9; and or the TGF-betaR2 specific heavy chain comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 13.

[0109] 24. The cell of any one of alternatives 16-23, wherein the intracellular endodomain of the CAR polypeptide is capable of activating cytokine signaling in the cell following the CAR polypeptide binding to IL13Ra2.

[0110] 25. The cell of alternative 24, wherein cytokine signaling promotes upregulation of IL-2, IFN-y, and / or TNF-a.[0U1] 26. The cell of any one of alternatives 16-25, further comprising a suicide gene that is capable of activating selective apoptosis upon administration of a compound.

[0112] 27. The cell of alternative 26, wherein the suicide gene comprises a truncated HER2 extracellular domain (SEQ ID NO: 17).

[0113] 28. The cell of alternative 26 or 27, wherein the suicide gene comprises a transmembrane domain and / or an endodomain sequence of CD5 (SEQ ID NO: 18 and 19, respectively).

[0114] 29. The cell of any one of alternatives 26-28, wherein the suicide gene is capable of activating selective apoptosis upon administration of trastuzumab emtansine.

[0115] 30. The cell of any one of alternatives 16-29, further comprising an IL- 12 construct.

[0116] 31. The cell of alternative 30, wherein the IL- 12 construct further comprises: an N-terminal p35 subdomain sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 20; a linker sequence; and a C-terminal p40 subdomain sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 22.

[0117] 32. The cell of alternative 31, wherein the IL-12 linker sequence comprises a sequence having at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 21, and is between 4-15 amino acids in length.

[0118] 33. The cell of any one of alternatives 16-32, wherein expression of the CAR polypeptide is regulated by an inducible promoter.

[0119] 34. The cell of any one of alternatives 16-33, wherein the cell is genetically modified to decrease PD-1 expression.

[0120] 35. A use of the CAR polypeptide of any one of alternatives 1-8 as part of a medicament.

[0121] 36. A method of treating a cancer and / or a tumor in a subject, the method comprising administering to the subject the CAR polypeptide of any one of alternatives 1-8, the nucleotide of alternative 10, the vector of any one of alternatives 11-15, the cell of any one of alternatives 16-34, or any combination thereof.

[0122] 37. A method of treating glioblastoma in a subject, the method comprising administering to the subject the CAR polypeptide of any one of alternatives 1-8, the nucleotide of alternative 10, the vector of any one of alternatives 11-15, the cell of any one of alternatives 16-34, or any combination thereof.

[0123] 38. The method of alternative 36 or 37, wherein the administration is conducted intracranially, or via intratumoral or intravenous injection.

[0124] 39. The method of any one of alternatives 36-38, further comprising administering a compound post-treatment to selectively activate the suicide gene.

[0125] 40. A pharmaceutical composition comprising the CAR polypeptide of any one of alternatives 1-8, the nucleotide of alternative 10, the vector of any one of alternatives 11-15, the cell of any one of alternatives 16-34, or any combination thereof, and a pharmaceutically acceptable carrier, wherein the composition is optimized for administration in a mammal.

[0126] 41. A kit comprising the CAR polypeptide of any one of alternatives 1-8, the nucleotide of alternative 10, the vector of any one of alternatives 11-15, the cell of any one of alternatives 16-34, or any combination, a suicide gene activator compound, and instructions for administering the CAR T cells in treating glioblastoma.

[0127] Although the present disclosure has been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of embodiments of the disclosure as defined in the appended claims.

[0128] The present disclosure will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the disclosure in any way.SequencesTable 1. Non-limiting Example Sequences*CDR sequences within a larger sequence are shown in underline.EXAMPLES

[0129] The non-limiting example methodology as disclosed herein are used in the following working Examples.Example 1 : Construction of a CAR Expression Cassette with Armored and Safety Components

[0130] A single gene cassette was designed to encode a chimeric antigen receptor (CAR) targeting IL13Ra2, with additional armoring components to promote T cell persistence, sustained cytotoxicity, and a safety mechanism (suicide gene) (Figure 1 A). Armoring elements included in the cassette enable the CAR T cells to secrete a modified IL-12 protein and block TGF-P signaling, providing a proliferative and anti -suppressive activation signal (Figure IB). Expression of the CAR construct was validated by measuring IL13Ra2 binding and VHH expression, while the presence of the suicide gene allowed differentiation between T cells expressing CAR-only and T cells expressing a CAR polypeptide, an armoring element, and a suicide gene (“CAR + Armoring + Suicide”) configurations (Figure 1C).Example 2: Target Specificity and Cytotoxic Function of CAR T Cells

[0131] To assess the specificity and cytotoxic function of the CAR T cells, target cells expressing either IL13Ral or IL13Ra2 on their surface were utilized. PBMCs were isolated from buffy cones from healthy donors (National health service, blood and transplant) via density centrifugation. Isolated PBMCs were stimulated with anti-CD3 (Miltenyi Biotech, 130-093-387) and anti-CD28 (Miltenyi Biotech, 130-093-375) + recombinant human IL-2 (Miltenyi Biotech, 130-097-746) for 48 hours. Cells were transduced with y-retroviruses produced following the transfection of HEK 293T cells. Cells and viruses were added to retronectin (Takara, T100B) coated plates and spinoculated (Thermo Fisher, 17171925). Cultures were maintained for 48 hours at 37°C and 5% CO2 prior to harvesting with transduction efficiencies established via flow cytometry.

[0132] SupTl CAR T cells with three different IL13Ra2-specific VHH binders were generated and transduced into primary T cells. Specificity for IL13Ra2 over IL13Ral was evaluated by co-culturing CAR T cells with each target cell line for 72 hours. Cytotoxicitywas obtained as a measure of target cell survival via flow cytometry (Figure 2). Results showed that CAR constructs 1 and 2 exhibited partial cross-reactivity with IL13Ral, while CAR 3 selectively targeted IL13Ra2, and did not significantly bind IL 13Ral -expressing cells (Figure 2).Example 3: Functional Assessment of Armored and Non-Armored CAR T Cells

[0133] To evaluate the cytotoxic function of both armored and non-armored CAR T cells, co-cultures with target cells that endogenously express IL13Ra2 were established. PBMCs were isolated from buffy cones from healthy donors (National health service, blood and transplant) via density centrifugation. Isolated PBMCs were stimulated with anti-CD3 (Miltenyi Biotech, 130-093-387) and anti-CD28 (Miltenyi Biotech, 130-093-375) + recombinant human IL-2 (Miltenyi Biotech, 130-097-746) for 48 hours. Cells were transduced with y-retroviruses produced following the transfection of HEK 293T cells. Cells and viruses were added to retronectin (Takara, T100B) coated plates and spinoculated (Thermo Fisher, 17171925). Cultures were maintained for 48 hours at 37°C and 5% CO2 prior to harvesting with transduction efficiencies established and normalised between constructs. Co-cultures were set up using SNB-19 target cells and CAR T cells in a 1:1, 1:2 and 1 :4 effector to target cell (E:T) ratio. Cultures were maintained for 6 days and function assessed based on target cell clearance by flow cytometry.

[0134] Cytotoxic activity of armoured and non-armoured CAR T cells was measured by flow cytometry from co-cultures set up against target cells expressing IL13Ra2. Cultures were maintained for up to 6 days and cytotoxicity measured via flow cytometry. Proportion of cytotoxicity was determined as a percentage of target cells recovered compared to non-transduced T cells co-cultured with the same target cell line (Figure 3 A). Culture supernatants were collected at 72 hours, and cytokine production was analyzed by ELISA to quantify IFN-y and IL-2 levels (Figures 3B and 3C, respectively). Armored CAR T cells showed greater cytotoxicity compared to non-armored CAR T cells at lower E:T ratios as well as increased IFN-y and IL-2 production.Example 4: Resistance of Armored CAR T Cells to TGF-B Suppression

[0135] PBMCs were isolated from buffy cones from healthy donors (National health service, blood and transplant) via density centrifugation. Isolated PBMCs were stimulated with anti-CD3 (Miltenyi Biotech, 130-093-387) and anti-CD28 (Miltenyi Biotech, 130-093-375) + recombinant human IL-2 (Miltenyi Biotech, 130-097-746) for 48 hours. Cells were transduced with y-retroviruses produced following the transfection of HEK 293T cells. Cells and viruses were added to retronectin (Takara, T100B coated plates and spinoculated (Thermo Fisher, 17171925). Cultures were maintained for 48 hours at 37°C and 5% CO2 prior to harvesting with transduction efficiencies established and normalised between constructs. CAR T cells were cultured with lug / ml of IL13Ra2 protein (Aero Biosystems, IL2-H5256) in the presence or absence of recombinant human TGF-P (BioTechne, 7754-BH) for 96 hours, T cells were recovered and cultured with SupTl IL13Ra2 target cells for an additional 96 hours and function assessed based on target cell clearance by flow cytometry.

[0136] The function of the anti-TGF-P armoring element was evaluated by stimulating CAR T cells with IL13Ra2-coated plates in the presence or absence of recombinant TGF-p. CAR T cells were cultured under these conditions for 96 hours, then re-challenged with IL13Ra2-expressing target cells for an additional 96 hours. Cytotoxic function was assessed by flow cytometry, measuring the percentage of target cells recovered compared to non-transduced cells cultured with the same target cell (Figures 4A-4B). Results showed that armored CAR T cells retained cytotoxic function after TGF-P exposure, whereas non-armored CAR T cells exhibited reduced cytotoxicity due to TGF-P suppression.Example 5: Expansion and Persistence of Armored CAR T Cells in the Absence of Stimulation

[0137] PBMCs were isolated from buffy cones from healthy donors (National health service, blood and transplant) via density centrifugation. Isolated PBMCs were stimulated with anti-CD3 (Miltenyi Biotech, 130-093-387) and anti-CD28 (Miltenyi Biotech, 130-093-375) + recombinant human IL-2 (Miltenyi Biotech, 130-097-746) for 48 hours. Cells were transduced with y-retroviruses produced following the transfection of HEK 293T cells. Cells and viruses were added to retronectin (Takara, T100B coated plates and spinoculated (Thermo Fisher, 17171925). Cultures were maintained for 48 hours at 37°C and 5% CO2 prior to harvesting with transduction efficiencies established and normalised between constructs.1x106CAR T cells were cultured in complete media in the absence of IL2, cell were counted every 3-4 days and changes in cell numbers tracked overtime by flow cytometry. Cell counts are normalised to counting beads.

[0138] CAR T cells, with or without armoring components, were cultured in the absence of antigen stimulation or exogenous cytokines to assess persistence and expansion. Cell counts were monitored over two weeks by flow cytometry (Figure 5). Armored CAR T cells demonstrated sustained expansion and remained viable over time, even without antigen or exogenous cytokine support. In contrast, non-armored CAR T cells showed limited persistence and failed to expand in the absence of stimulation.Example 6: Assessment of IL- 12 Armoring Element Activity in a Co-Culture System

[0139] PBMCs were isolated from buffy cones from healthy donors (National health service, blood and transplant) via density centrifugation. Isolated PBMCs were stimulated with anti-CD3 (Miltenyi Biotech, 130-093-387) and anti-CD28 (Miltenyi Biotech, 130-093-375) + recombinant human IL-2 (Miltenyi Biotech, 130-097-746) for 48 hours. Cells were transduced with y-retroviruses produced following the transfection of HEK 293T cells. Cells and viruses were added to retronectin (Takara, T100B coated plates and spinoculated (Thermo Fisher, 17171925). Cultures were maintained for 48 hours at 37°C and 5% CO2 prior to harvesting with transduction efficiencies established and normalised between constructs.

[0140] NK cells were generated from PBMCs using a human NK cell expansion kit (BioTechne, CDK015). CAR T cells and NK or T cells were cultured but separated using inserts (Thermo Fisher, 10421761) for 72 hours. pSTAT4 expression was assessed by flow cytometry in NK and CD8+ T cell populations.

[0141] The function and production of an armoured IL12 element was established using cultures where CAR T cells were separated from NK cells or CD8 T cells using a Transwell system porous membrane. This membrane enables migration of factors secreted by cells (Figure 6A). IL12 ligates the IL12 receptor to activate downstream signaling pathways via the phosphorylation of STAT4 protein. The phosphorylation of STAT4 was measured in NK cells (Figure 6B) and CD8 T cells (Figure 6C) that were cultured in the presence of CAR T cells. Factors secreted by armoured CAR T cells migrate via the membrane to activate NKand CD8 T cells which causes STAT4 phosphorylation. This activation of STAT4 was absent when NK and CD8 T cells were cultured in the presence non-armoured CAR T cells.Example 7: Functionality of the Suicide Gene Component

[0142] PBMCs were isolated from huffy cones from healthy donors (National health service, blood and transplant) via density centrifugation. Isolated PBMCs were stimulated with anti-CD3 (Miltenyi Biotech, 130-093-387) and anti-CD28 (Miltenyi Biotech, 130-093-375) + recombinant human IL-2 (Miltenyi Biotech, 130-097-746) for 48 hours. Cells were transduced with y-retroviruses produced following the transfection of HEK 293T cells. Cells and viruses were added to retronectin (Takara, T100B coated plates and spinoculated (Thermo Fisher, 17171925). Cultures were maintained for 48 hours at 37°C and 5% CO2 prior to harvesting with transduction efficiencies established and normalised between constructs. Transduced cells are cultured in the presence of Trastuzumab Emtansine (TDM-1) (Selleck Biotechnology, D4003) at varying concentrations between 0 and 5ug / ml for 48 hours, viability is determined based on DAPI exclusion (WVR, A1001.0010) in CAR+ cells by flow cytometry.

[0143] CAR T cells were also cultured with lug / ml of IL13Ra2 protein (Aero Biosystems, IL2-H5256) in the presence of TDM-1 for 96 hours, harvested and then cultured with SupTl IL13Ra2 target cells for an additional 96 hours and function assessed based on target cell clearance by flow cytometry.

[0144] To evaluate the functionality of the suicide gene, T cells expressing either the CAR alone or the CAR + Armour + Suicide system with a HER2 -based suicide gene were cultured with increasing concentrations of the antibody-drug conjugate Trastuzumab Emtansine (T-DM1). The expression of the suicide switch based on trastuzumab detection was assed via flow cytometry (Figure 7A). The function of the suicide switch was established by measuring the viability, based on DAPI exclusion in flow cytometry, of transduced cells in the presence of varying concentrations of TDM- 1 (Figure 7B). Additionally, changes in cytotoxic function was also assessed following prior exposure of transduced cells to T-DM1 in the presence of antigenic stimulation before assessing cytotoxicity towards IL13Ra2 expressing target cells (Figure 7C and 7D).

[0145] Armored CAR T cells expressing the suicide gene HER2 on their cell surface displayed positive binding to trastuzumab and trastuzumab related antibody drug conjugates such as TDM1. These cells also exhibited reduced viability upon exposure to T-DM1, while CAR T cells that did not express the suicide gene remained unaffected. T-DM1 exposure also deminised cytotoxic function when co-cultured with antigen positive target cells whereas CAR T cells that did not express the suicide gene remained unaffected. This confirms that the suicide gene enables selective elimination of CAR + Armour + Suicide T cells upon administration of T-DM1.Example 8: In Vivo Efficacy of CAR T Cells with Armoring and Suicide Gene Components in a Glioblastoma Model

[0146] PBMCs were isolated from buffy cones from healthy donors (National health service, blood and transplant) via density centrifugation. Isolated PBMCs were stimulated with anti-CD3 (Miltenyi Biotech, 130-093-387) and anti-CD28 (Miltenyi Biotech, 130-093-375) + recombinant human IL-2 (Miltenyi Biotech, 130-097-746) for 48 hours. Cells were transduced with y-retroviruses produced following the transfection of HEK 293T cells. Cells and viruses were added to retronectin (Takara, T100B coated plates and spinoculated (Thermo Fisher, 17171925). Cultures were maintained for 48 hours at 37°C and 5% CO2 prior to harvesting with transduction efficiencies established and normalised between constructs.

[0147] 6-8 week old, female, NOG mice (Charles River Laboratory) were injected intracranially with U87-MG cells overexpressing IL13Ra2. Tumour cells are engrafted for 8 days prior to the intracranial injection of previously frozen CAR T cells. 7 days post CAR T cell injection, mice are treated with 2.5ug of Trastuzumab Emtansine (TDM-1) (Selleck Biotechnology, D4003). Tumour engraftment and clearance was measured by bioluminescent imaging utilizing the IVIS spectrum system in addition to measuring changes in bodyweight.

[0148] The NOG mice bearing intracranial IL13Ra2-expressing glioblastoma xenografts were treated with a known high toxic dose of CAR T cells engineered with armoring and suicide gene components. After 7 days, T-DM1 was administered to activate the suicide switch (Figure 8A). Tumour clearance is measured based on bioluminescence imaging and measured in mice over time (Figure 8B). Toxicity was assessed by tracking body weight changes overtime (Figures 8C). Armoured CAR T cells are injected into mice bearing tumoursfollowed by the addition of TDM1. Mice that did not receive TDM1 show tumour clearance but exhibit decreased body weight. The introduction of TDM1 switches off CAR T cells enabling successful tumour clearance whilst minimizing changes in bodyweight thus limiting toxicity.

[0149] The results demonstrated effective tumor clearance in mice receiving the armored CAR T cells with suicide gene activation, with minimal weight loss observed, indicating controlled CAR T cell activity and reduced toxicity.Example 9: Evaluation of Tumor Clearance in Mice Treated with Armored CAR T Cells

[0150] IL13Ra2 expressing tumour U87-MG cells were injected intracranially into 6-8 week old, female, NOG mice (Charles River Laboratory) and allowed to engraft for 8 days. Then, the NOG mice were treated intracranially with 0.025x106transduced CAR T cells or an equivalent number of non-transduced T cells containing either the armor and suicide gene constructs or the CAR alone. Tumor burden was monitored by bioluminescent imaging utilizing the IVIS spectrum system over 14 days (Figure 9A). Tumour clearance was measured based on bioluminescence imaging (Figure 9B) and over time (Figure 9C). Mice treated with armored CAR T cells demonstrated significant tumor clearance, while non-armored CAR T cells showed limited anti-tumor effects, confirming that armoring components enhance CAR T cell efficacy in vivo.

Claims

WHAT TS CLAIMED IS:

1. A chimeric antigen receptor (CAR) polypeptide comprising:a binding sequence specific to IL13Ra2;a spacer region;a transmembrane domain; andan intracellular endodomain.

2. The CAR polypeptide of claim 1, wherein the binding sequence is a humanized, single domain antibody (VHH) binder.

3. The CAR polypeptide of claim 1, wherein the binding sequence does not significantly bind to IL13Ral.

4. The CAR polypeptide of claim 1, wherein the spacer region comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of a human CD28 hinge domain as set forth in SEQ ID NO: 5.

5. The CAR polypeptide of claim 1, wherein the transmembrane domain comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of a human CD28 transmembrane domain as set forth in SEQ ID NO: 6.

6. The CAR polypeptide of claim 1, wherein the intracellular endodomain comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of a CD28-zeta signaling domain as set forth in SEQ ID NO: 7 or a 4-lBB-zeta signaling domain.

7. The CAR polypeptide of claim 1, wherein the binding sequence comprises:a CDR1 sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 24 (SYYMR);a CDR2 sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 25 (SINSGGGSTSYVDSVKG);a CDR3 sequence with at least about 80, 85, 90, 95, 99, or 100% identity to the sequence of SEQ ID NO: 26 (ALETNRGQNY); orany combination thereof.

8. The CAR polypeptide of claim 1, further comprising an at least one additional costimulatory signaling domain, optionally wherein the at least one additional costimulatory signaling domain is selected from 4- IBB, 0X40, ICOS, or any combination thereof.

9. The CAR polypeptide of claim 1, configured to target a glioblastoma.

10. A nucleotide sequence encoding the CAR polypeptide of claim 1.

11. A vector encoding the CAR polypeptide of claim 1.

12. The vector of claim 11, wherein the vector is a viral vector, optionally wherein the vector is a self-inactivating lentiviral or a retroviral vector.

13. The vector of claim 11, wherein the vector is capable of integrating into a cell’s genome.

14. The vector of claim 11, further comprising an at least one self-cleaving 2A peptide.

15. The vector of claim 14, wherein the at least one self-cleaving 2A peptide is between at least two of the following:the CAR polypeptide of claim 1;a suicide gene;an armoring element;an IL- 12 construct; orany combination thereof.

16. A cell capable of expressing the CAR polypeptide of claim 1, optionally wherein the cell encodes the nucleotide of claim 10, and / or the vector of claim 11.

17. The cell of claim 16, wherein the cell is a T cell.

18. The cell of claim 16, further comprising an armoring element, wherein the armoring element comprises an antibody binder with a heavy and light chain on the extracellular domain, and the endodomain of the GM-CSF receptor.

19. The cell of claim 18, comprising:a light chain polypeptide comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of a GM-CSF beta receptor endodomain as set forth in SEQ ID NO: 11; and / ora heavy chain polypeptide comprises a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence for a GM-CSF alpha receptor endodomain as set forth in SEQ ID NO: 15.

20. The cell of claim 16, wherein the armoring element is capable of stabilizing and / or enhancing T cell persistence and / or proliferation.

21. The cell of claim 16, wherein the armoring element includes a dimerization interface capable of forming one or more disulfide bonds, thereby stabilizing the heavy and light chains.

22. The cell of claim 21, wherein the dimerization interface is specific for TGF-betaR2 and inhibits TGF-beta signaling upon ligand interaction.

23. The cell of claim 22, comprising:a TGF-betaR2 specific light chain comprising a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of SEQ ID NO: 9; and ora TGF-betaR2 specific heavy chain comprising a sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of SEQ ID NO: 13.

24. The cell of claim 16, wherein the intracellular endodomain of the CAR polypeptide is capable of activating cytokine signaling in the cell following the CAR polypeptide binding to IL13Ra2.

25. The cell of claim 24, wherein cytokine signaling promotes upregulation of IL-2, IFN-y, and / or TNF-a.

26. The cell of claim 16, further comprising a suicide gene that is capable of activating selective apoptosis upon administration of a compound.

27. The cell of claim 26, wherein the suicide gene comprises a truncated HER2 extracellular domain (SEQ ID NO: 17).

28. The cell of claim 26, wherein the suicide gene comprises a transmembrane domain and / or an endodomain sequence of CD5 as set forth in SEQ ID NO: 18 and 19, respectively.

29. The cell of claim 26, wherein the suicide gene is capable of activating selective apoptosis upon administration of trastuzumab emtansine.

30. The cell of claim 16, further comprising an IL-12 construct.

31. The cell of claim 30, wherein the IL-12 construct further comprises:an N-terminal p35 subdomain sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of SEQ ID NO: 20;a linker sequence; anda C-terminal p40 subdomain sequence with at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of SEQ ID NO: 22.

32. The cell of claim 1, wherein the IL-12 linker sequence comprises a sequence having at least about 80, 85, 90, 95, 99, or 100% identity to a sequence of SEQ ID NO: 21.

33. The cell of claim 31, wherein the IL- 12 linker sequence is between 4-15 amino acids in length.

34. The cell of claim 16, wherein expression of the CAR polypeptide is regulated by an inducible promoter.

35. The cell of claim 16, wherein the cell is genetically modified to decrease PD-1 expression.

36. A use of the CAR polypeptide of claim 1 as part of a medicament.

37. A method of treating a cancer and / or a tumor in a subject, the method comprising administering to the subject the CAR polypeptide of claim 1, the nucleotide of claim 10, the vector of claim 11, the cell of claim 16, or any combination thereof.

38. A method of treating glioblastoma in a subject, the method comprising administering to the subject the CAR polypeptide of claim 1, the nucleotide of claim 10, the vector of claim 11, the cell of claim 16, or any combination thereof.

39. The method of claim 38, wherein the administration is conducted intracranially, or via intratumoral or intravenous injection.

40. The method of claim 38, further comprising administering a compound posttreatment to selectively activate the suicide gene.

41. A pharmaceutical composition comprising the CAR polypeptide of claim 1, the nucleotide of claim 10, the vector of claim 11, the cell of claim 16, or any combination thereof, and a pharmaceutically acceptable carrier, wherein the composition is optimized for administration in a mammal.

42. A kit comprising the CAR polypeptide of claim 1, the nucleotide of claim 10, the vector of claim 11, the cell of claim 16, or any combination, a suicide gene activator compound, and instructions for administering the CAR T cells in treating glioblastoma.

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