Her2-based suicide switch

Abstract

The present application relates to a suicide gene system and its use as part of immunotherapy. In some embodiments, the suicide gene system is expressed in a CAR T-cell, optionally along with armoring proteins. The present application also discloses uses of the suicide gene system, such as in cell tracking, immune regulation, rapid elimination of CAR T-cells, and as part of a therapeutic treatment.

Description

CHMRS.006WO PATENT TARGETED BINDING DOMAINSRELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims the benefit of U. S. Provisional Ser. No. 63 / 726,104, filed November 27, 2024, which is hereby incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence lasting in electronic format. The Sequence Listing is provided as a file entitled CHMRS.006WO.xml, which was created and last modified on November 25, 2025, and is 21,556 bytes in size. The information in the electronic Sequence Listing is hereby incorporated by reference in its entirety.RELATED FIELD

[0003] Aspects of the present disclosure relate to polypeptide constructs and their uses as part of an immunotherapy. In some embodiments, the constructs have use as part of chimeric antigen receptor (CAR) T-cell therapy.BACKGROUND

[0004] Chimeric antigen receptor (CAR) T-cell therapy has emerged as a revolutionary approach for treating certain cancers by redirecting immune cells to target tumour-associated antigens. However, this promising treatment is often associated with severe toxicities, including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and on- target, off-tumour effects, where CAR T cells attack healthy tissues expressing the target antigen.SUMMARY

[0005] Disclosed herein are systems designed to enhance the safety and functionality of CAR T-cell therapy. In some embodiments, the systems include a suicide gene system. In some embodiments, the suicide gene encodes a single polypeptide with three keydomains, including an extracellular domain derived from human HER2 protein (domain 4), a transmembrane domain derived from either HER2 or CD5, and an intracellular domain derived from HER2 or CD5, with kinase-inactive HER2 as an alternative. Also disclosed herein are uses of the polypeptide as a surface marker for identifying transduced cells through binding with antibodies such as trastuzumab or pertuzumab, and uses of the polypeptide as a suicide protein that can induce cell death upon engagement with an antibody-drug conjugate (ADC), such as trastuzumab emtansine (TDM-1). The present disclosure describes systems for controlling CAR T cells by facilitating both precise monitoring of transduction efficiency and rapid elimination of the cells in response to toxi cities.

[0006] In some embodiments, the suicide switch is co-expressed with a CAR in a single vector separated by a self-cleaving 2A peptide. In some embodiments, the system is configured to and / or capable of simultaneous expression of the CAR and the suicide protein on the T-cell surface. In some embodiments, a populati on of transduced CAR T cells is stained with trastuzumab and their expression of the HER2-derived suicide protein acts as a surrogate for CAR expression and enables quality control and determination of transduction rates. In some embodiments, in the event of intolerable toxicity, administration of TDM-1 triggers internalization of the suicide protein, releasing the cytotoxic DM-1 toxin into the cytosol, leading to selective CAR T-cell death. In some embodiments, the systems described herein provide a versatile and clinically translatable tool to mitigate the risks of CAR T-cell therapy while maintaining therapeutic efficacy.

[0007] Aspects of the present disclosure relate to methods of inducing death in a cell. In some embodiments, the methods include introducing into the cell a polypeptide encoding a protein that is expressed on the surface of the cell, and administering an antibodydrug conjugate to the cell. In some embodiments, the protein is capable of binding to the antibody. In some embodiments, the drug has activity in inducing cell death. In some embodiments, the protein comprises an extracellular domain and a transmembrane domain. In some embodiments, the protein includes an intracellular domain. In some embodiments, the extracellular domain is derived from HER2. In some embodiments, the extracellular domain is derived from HER2 domain 4. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a T cell. In some embodiments, the cell expresses a chimeric antigen receptor (CAR). In some embodiments, the antibody is trastuzumab or pertuzumab. In someembodiments, the drug is a tubulin inhibitor. In some embodiments, the drug is mertansine enitansine, auristatin, ravtansine, deruxtecan, or any combination thereof.

[0008] Also disclosed herein are kits for inducing targeted cell death in a subject. In some embodiments, the kits include a vector encoding a protein that can be incorporated onto the surface of a targeted cell in the subject, and an antibody-drug conjugate. In some embodiments, the antibody is capable of binding to the protein. In some embodiments, the drug is capable of inducing cell death m the targeted cell. In some embodiments, the vector includes any protein as described herein. In some embodiments, the antibody-drug conjugate includes any antibody, drug, or antibody-drug conjugate as described herein. In some embodiments, the antibody-drug conjugate is trastuzumab emtansine (TDM-1).

[0009] Also disclosed herein are polypeptides for use in inducing cell death. In some embodiments, the polypeptide includes an extracellular domain derived from a human HER2 protein, and a transmembrane domain. In some embodiments, the extracellular domain includes a sequence with at least about 80, 85, 90, 95, 99, or 100% identity with the sequence of HER2 domain 4 (SEQ ID NO: 18). In some embodiments, the transmembrane domain includes a sequence with at least about 80, 85, 90, 95, 99, or 100% identity with the transmembrane domain sequence of HER2 or CD5. In some embodiments, the transmembrane domain includes the transmembrane domain of HER2 or CD5 (SEQ ID NO: 11), or a variant thereof. In some embodiments, the polypeptide further includes an intracellular domain. In some embodiments, the intracellular domain includes at least one of: a truncated HER2 intracellular domain (SEQ ID NO: 12), a kinase-inactive mutant of HER2, a human CD 5 intracellular domain, a variant thereof, or any combination thereof. In some embodiments, the intracellular domain includes a sequence with at least about 80, 85, 90, 95, 99, or 100% identity with at least one of: a truncated HER2 intracellular domain (SEQ ID NO: 12), a kinase- inactive mutant of HER2, a human CD5 intracellular domain, or any combination thereof. In some embodiments, the extracellular domain is capable of binding to trastuzumab and / or pertuzumab. In some embodiments, the extracellular domain is capable of binding to TDM-1.

[0010] Some embodiments provided herein relate to uses of any of the polypeptides described herein as a surface marker for identifying a cell, tissue, organ, or organism.

[0011] Some embodiments provided herein relate to nucleic acid sequences encoding any of the polypeptides described herein.

[0012] Some embodiments provided herein relate to vectors that include any of the nucleic acid sequences described herein. In some embodiments, the vectors further include a nucleic acid sequence encoding a CAR. In some embodiments, the vectors further include an at least one self-cleaving 2A peptide. In some embodiments, the vectors further include a nucleic acid sequence encoding an at least one armouring protein. In some embodiments, the at least one armouring protein comprising a cytokine secretion or membrane tethering protein, dominant negative receptor, a checkpoint inhibitor, a cytokine receptor, a chemokine receptor, an internal signaling protein for a cytokine and / or chemokine receptor, a variant, or any combination thereof. In some embodiments, the cytokine secretion or membrane tethering protein comprises at least one of IL12, IL15, IL18, IL21, a variant, or any combination thereof. In some embodiments, the dominant negative receptor comprises at least one of TGFb, CTLA4, PD1, a variant, or any combination thereof. In some embodiments, the checkpoint inhibitor comprises CTLA4 and / or PD1.

[0013] Also disclosed herein are cells that include any of the polypeptides described herein, any of the nucleic acid sequences described herein, or any of the vectors described herein. In some embodiments, at least one of: the CAR, polypeptide, armouring protein, or any combination thereof, is expressed on the cell’s surface. In some embodiments, the at least one armouring protein comprising a cytokine secretion or membrane tethering protein, dominant negative receptor, a checkpoint inhibitor, a cytokine receptor, a chemokine receptor, an internal signaling protein for a cytokine and / or chemokine receptor, a variant, or any combination thereof. In some embodiments, the cytokine secretion or membrane tethering protein comprises at least one of IL12, IL15, IL18, IL21, a variant, or any combination thereof. In some embodiments, the dominant negative receptor comprises at least one of TGFb, CTLA4, PD1, a variant, or any combination thereof. In some embodiments, the checkpoint inhibitor comprises CTLA4 and / or PD1. In some embodiments, the cell is an immune cell, preferably wherein the cell is a T cell.

[0014] Some embodiments described herein relate to methods of identifying transduced cells. In some embodiments, the methods include transducing a population of cells with any of the polypeptides described herein, any of the nucleic acid sequences described herein, or any of the vectors described herein. In some embodiments, the methods further include contacting the population of cells with an antibody specific to the polypeptide, anddetecting antibody binding. In some embodiments, positive detection of the antibody binding to the polypeptide signifies a transduced cell.

[0015] Also disclosed herein are methods for inducing death or suicide in a cell. In some embodiments, the methods include introducing into the cell any of the polypeptides, the nucleic acid sequences, and / or the vectors described herein. In some embodiments, the methods further include administering an antibody-drug conjugate to the cell. In some embodiments, the polypeptide is capable of binding to the antibody. In some embodiments, the drug has activity in inducing cell death. In some embodiments, the antibody-drug conjugate is trastuzumab emtansine (TDM-1).

[0016] Also disclosed herein are uses of any of the polypeptides, nucleic acid sequences, or vectors described herein in the preparation of a medicament.

[0017] Some embodiments provided herein relate to methods for preparing CAR T-cells. In some embodiments, the methods include transducing a T-cell population with any of the polypeptides, nucleic acid sequences, or vectors described herein. In some embodiments, the methods further include selecting a transduced cell using an antibody capable of binding to the polypeptide of any one of the embodiments of the present disclosure.

[0018] Some embodiments provided herein relate to methods for treating a disease or disorder in a subject in need thereof. In some embodiments, the methods include administering to the subject any of the polypeptides, nucleic acid sequences, vectors, or cells described herein. In some embodiments, the subject is a mammal and / or a human. In some embodiments, the disease or disorder is a tumor and / or cancer. In some embodiments, the methods further include administering an antibody-drug conjugate capable of binding to the polypeptide of any one of the embodiments of the present disclosure. In some embodiments, the antibody-drug conjugate is trastuzumab emtansine (TDM-1).

[0019] Some embodiments provided herein relate to methods of preventing, reducing, or ameliorating an at least one adverse effect of CAR T-cell therapy in a subject in need thereof. In some embodiments, the methods includeadminist ering to the subject any of the polypeptides, nucleic acid sequences, vectors, and / or cells described herein. In some embodiments, the methods further include monitoring for when the at least one adverse effect in the subject is occurring or will occur and / or administering to the subject an antibody-drug conjugate capable of binding to the polypeptide of any one of the embodiments of the presentdisclosure. In some embodiments, the antibody-drug conjugate is trastuzumab emtansine (TDM-1).BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 depicts a non-limiting example cartoon graphic of a cell expressing a “sort-safety” switch, and the process of internalizing and ligating an antibody drug conjugate.

[0021] Figure 2A depicts non-limiting example cartoon schematics of single vector cassettes, comprising GFP and a variant of the sort-safety switch.

[0022] Figure 2B depicts non-limiting representative dot plots showing the expression of GFP and detection of different versions of the sort-safety switch using trastuzumab in non-transduced (“NT”) or transduced (“VI -V7”) cells. Detection is quantified by flow cytometry.

[0023] Figure 2C depicts non-limiting representative dot plots showing the expression of GFP and detection of different versions of the sort-safety switch using pertuzumab in non-transduced (“NT”) or transduced (“VI -V7”) cells. Detection is quantified by flow cytometry.

[0024] Figure 3A depicts a non-limiting example cartoon graphic detailing the ligation mechanism following a receptor binding to an antibody conjugated with a pH sensitive dye. In this example, a fluorescent signal is emitted from the dye upon the internalization of receptor-ligand complex when exposed to the endosome.

[0025] Figure 3B depicts a non-limiting representative image of fluorescence detection from non-transduced (“NT”) cells (left panel) or cells expressing the sort-safety switch (right panel) when exposed to a ligand conjugated with a pH-sensitive dye. Detection is visualized by microscopy.

[0026] Figure 3C depicts a non-limiting example quantification of TDM-1 internalization by cells expressing variants of the cell safety switch. Internalization is quantified by the conjugation of TDM- 1 with enzyme sensitive fluorescent beads.

[0027] Figure 4 depicts a non-limiting example quantification of cell viability from cells expressing different versions of the sort-safety switch when exposed to severalconcentrations of the antibody drug conjugate, TDM-1. Viability is quantified by flow cytometry.

[0028] Figure 5 A depicts a non-limiting example cartoon schematic of a single vector cassette comprising a transgene marker (HA), CAR, and a sort-safety switch.

[0029] Figure 5B depicts non-limiting representative dot plots showing the expression of the HA transgene marker and CAR (left panel), as well as detection of the soft safety switch using trastuzumab (right panel).

[0030] Figure 6 depicts a non-limiting example of quantification of cell viability from cells expressing CAR alone, or CAR and the sort-safety switch, in the presence of the antibody drug conjugate TDM-1.

[0031] Figure 7 A depicts a non-limiting example quantification of the cytotoxicity of IL13Ra2 binding non-transduced cells (“NT”), cells expressing CAR alone, or cells expressing CAR and a sort-safety switch towards IL13Ra2 expressing targets, following exposure to increasing concentrations of TDM- 1.

[0032] Figure 7B depicts a non-limiting example quantification of the fold-change in IFN-g, following IL13Ra2 binding cells expressing CAR alone, or cells expressing CAR + the sort- safety switch towards IL13Ra2 expressing targets. Quantification is measured through ELISA in the presence or absence of TDM- 1.

[0033] Figure 8A depicts a non-limiting example cartoon graphic of an in vivo model showing a timeline for an experiment in mice and the concurrent tumour engraftment, CAR T-cell injections, and TDM1 injections as described in Example 8 below.

[0034] Figure 8B depicts a non-limiting example quantification of tumour growth, as measured through bioluminescence imaging, of mice injected with non-transduced (“NT”) cells, or CAR T cells with and without TDM1.

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

[0036] Aspects of the present disclosure relate to a suicide gene system and its use as part of immunotherapy. In some embodiments, the gene system has use as part of chimericantigen receptor (CAR) T-cell therapy. In some embodiments, the systems encode a polypeptide comprising segments from a HER2 protein. In some embodiments, the suicide gene system encodes a polypeptide comprising: (i) an extracellular domain derived from human HER2 protein (domain 4), and (ii) a transmembrane domain derived from HER2 or human CD5. In some embodiments, the polypeptide further comprises an intracellular domain derived from HER2 or CD5, such as a kinase- inactive HER2

[0037] Also disclosed herein are uses of the polypeptide as a surface marker for identifying transduced cells through antibody binding, and as a suicide protein to selectively induce CAR T-cell death upon engagement with an antibody-drug conjugate (ADC), such as trastuzumab emtansine (TDM-1). In some embodiments, the polypeptide has use for cell tracking and / or for the rapid elimination of CAR T cells. In some embodiments, the polypeptide targets cells as part of a method of preventing severe toxicities or side effects of immunotherapy, such as cytokine release syndrome (CRS), neurotoxicity, or on-target, off-tumor effects. This system also provides an efficient, scalable solution for enhancing the safety and efficacy of CAR T-cell therapy in cancer and other diseases.

[0038] Toxicities associated with CAR T-cell therapy can be life-threatening and limit the therapeutic potential of CAR T-cell therapy. To mitigate these risks, suicide switches have been developed to selectively eliminate CAR T cells when toxicities become intolerable. While existing suicide mechanisms allow for CAR T-cell elimination, they often lack dual functionality, such as enabling tracking of transduced cells for quality control or treatment monitoring. Thus, there is a critical need for an integrated suicide switch that combines precise cell tracking with effective elimination capabilities, enhancing the safety and controllability of CAR T-cell therapies.

[0039] The suicide gene system of the present disclosure allows for the co¬ expression of a suicide protein with a CAR. In some embodiments, the suicide protein is expressed along with an at least one armoring protein. In some embodiments, the suicide protein, the CAR, and optionally the at least one armoring protein is encoded in a single vector separated by self-cleaving 2A peptides. In some embodiments, the at least one armouring protein comprising a cytokine secretion or membrane tethering protein, dominant negative receptor, a checkpoint inhibitor, a cytokine receptor, a chemokine receptor, an internal signaling protein for a cytokine and / or chemokine receptor, a variant, or any combinationtliereof. In some embodiments, the cytokine secretion or membrane tethering protein comprises at least one of IL12, IL15, IL18, IL21, a variant, or any combination thereof. In some embodiments, the dominant negative receptor comprises at least one of TGFb, CTLA4, PD1, a variant, or any combination thereof. In some embodiments, the checkpoint inhibitor comprises CTLA4 and / or PD1.Terms

[0040] 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.

[0041] 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.

[0042] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0043] 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.

[0044] 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%.

[0045] 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 stepor 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.

[0046] 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.

[0047] 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 m 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 ofother components). As used herein, the term “isolated cell” may refer to a cell not contained in a multi-cellular organism or tissue.

[0048] 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.

[0049] 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.

[0050] 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,

[0051] 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 be affected 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.

[0052] 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 refersto 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.

[0053] 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 is escalated 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.

[0054] 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.

[0055] 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 canbe 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.

[0056] 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 in an 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.

[0057] “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.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] 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 and absorption 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, glycerolmonostearate, 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.

[0062] 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 of pharmaceutically 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.

[0063] The term “% w / w” or “% wt / wf ’ 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.

[0064] 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,

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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. In some embodiments, a CAR is a synthetic receptor expressed on the surface of immune cells, such as T cells, that enables the cells to recognize and bind to specific antigens on target cells, typically cancer cells.

[0069] 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.

[0070] The term “transmembrane domain” refers to a domain of a protein that spans the cell membrane. In some embodiments, the transmembrane domains allows protein anchoring on the cell surface. A non-limiting example transmembrane domain is the cell membrane-spanning region of HER2 or CD5.

[0071] The term “intracellular domain” refers to the cytoplasmic domain of a protein. A non-limiting example intracellular domain is a truncated or kinase-inactive form of the intracellular domain of HER2 or CD5.

[0072] The terms “armoring element” or “armoring module” refers to a genetic element and / or protein that enhances the functionality, persistence, or therapeutic efficacy of a cell. In some embodiments, the armoring element / module is a modification to a CAR T-cell. In some embodiments, the armoring element / module enhances T cell persistence, proliferation, or resistance to immunosuppressive factors in the tumor microenvironment. Non-limiting examples include cytokines, cytokine receptors, or immune- modulatory proteins that are encoded within the same vector cassette, such as GM-CSF receptor components or IL-12 constructs. In some embodiments, the at least one armouring protein comprising a cytokine secretion or membrane tethering protein, dominant negative receptor, a checkpoint inhibitor, a cytokine receptor, a chemokine receptor, an internal signaling protein for a cytokine and / or chemokine receptor, a variant, or any combination thereof. In some embodiments, the cytokine secretion or membrane tethering protein comprises at least one of IL12, IL15, IL18, IL2I, a variant, or any combination thereof. In some embodiments, the dominant negative receptor comprises at least one of TGFb, CTLA4, PD1, a variant, or any combination thereof. In some embodiments, the checkpoint inhibitor comprises CTLA4 and / or PD1.

[0073] 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 some embodiments, the suicide gene is activated upon exposure to a specific compound, such as an antibody-drug conjugate (ADC). In some embodiments, a suicide gene is a nucleic acid sequence that encodes a polypeptide capable of selectively inducing cell death in transduced cells under specific conditions, such as engagement with an antibody-drug conjugate (ADC).

[0074] The term “suicide protein” refers to a polypeptide expressed from the suicide gene that includes an extracellular domain, a transmembrane domain, and optionallyan intracellular domain. The protein serves dual functions as a transduction marker and as a mechanism for inducing cell death.

[0075] 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.

[0076] 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.

[0077] 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 protein expression in T cells. In some embodiments, vector comprises at least one sequence encoding a: CAR, suicide protein, armoured module, at least one self-cleaving 2A peptide, or any combination thereof.

[0078] 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.

[0079] The term “extracellular domain” refers to the portion of a protein that is located outside of a cell. In some embodiments, the extracellular domain is capable of binding with antibodies and / or ADCs. A non-limiting example extracellular domain is the extracellular domain derived from human HER2 domain 4.

[0080] The term “antibody drug conjugate” or “ADC” refers to a therapeutic agent that combines an antibody with a cytotoxic drug. A non-limiting example ADC is trastuzumab emtansine (TDM-1), which binds to the extracellular domain of the suicide protein and induces cell death upon internalization.

[0081] The term “self-cleaving peptide” refers to a sequence that, when included as part of a larger sequence, results in a break in expression. In some embodiments, a selfcleaving peptide is a short peptide sequence that enables the co-expression of multiple proteins from a single transcript by mediating cleavage between linked polypeptides during translation. Non-limiting examples of a self-cleaving peptide include P2A, E2A, F2A, and T2A.

[0082] The term “transduction” refers to the process by which foreign DNA is introduced into a cell. In some embodiments, transduction is performed through electroporation or a viral vector. Given that, the term “transduction marker” refers to anyfeature that can be used to identify and / or quantify an aspect of transduction. In some embodiments, a transduction marker allows for identification and quantification of transduced cells, such as by staining with an antibody specific to the extracellular domain of the protein. It will also be understood that “transduced cells” refer to cells that have successfully underwent transduction. In some embodiments, transduced cells have successfully incorporated and expressed the genetic material delivered by a vector.

[0083] The term “on-target, off-tumor toxicity” refers to a type of adverse event where CAR T cells attack healthy tissues expressing the target antigen, leading to unintended damage.

[0084] The term “cytokine release syndrome” or “CRS” refers to a systemic inflammatory response resulting from the activation of immune cells, commonly observed in CAR T-cell therapies.

[0085] The term “Immune Effector Cell-Associated Neurotoxicity Syndrome” or “ICANS” refers to a form of neurotoxicity associated with CAR T-cell therapies, characterized by symptoms such as confusion, seizures, or cerebral edema.

[0086] The term “antigen” refers to a ligand that is recognized by the immune system. For example, an antigen may bind to an antibody or an immune cell. A “tumor- associated antigen” refers to an antigen that is found on the surface of or in the presence of a tumor. In some embodiments, a tumor-associated antigen is expressed on the surface of a tumor cell and is a target for CAR T-cells.

[0087] Embodiments provided herein are described by way of the following numbered alternatives:

[0088] 1. A polypeptide capable of function in inducing cell death, comprising: (a) an extracellular domain derived from a human HER2 protein; and (b) a transmembrane domain.

[0089] 2. The polypeptide of alternative 1, wherein the extracellular domain comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity with the sequence of HER2 domain 4 (SEQ ID NO: 18).

[0090] 3. The polypeptide of alternative 1 or 2, wherein the transmembrane domain comprises the transmembrane domain of HER2 and / or CD5 (SEQ ID NO: 11).

[0091] 4. The polypeptide of any one of alternatives 1-3, further comprising an intracellular domain.

[0092] 5. The polypeptide of alternative 4, wherein the intracellular domain comprises a truncated HER2 intracellular domain (SEQ ID NO: 12), a kinase- inactive mutant of HER2, a human CD5 intracellular domain, or any combination thereof.

[0093] 6. The polypeptide of any one of alternatives 1-5, wherein the extracellular domain is capable of binding to trastuzumab and / or pertuzumab.

[0094] 7. The polypeptide of any one of alternatives 1-6, wherein the function as the suicide protein is activated by an antibody-drug conjugate (ADC) binding to the extracellular domain.

[0095] 8. The polypeptide of alternative 7, wherein the ADC is trastuzumab emtansine (TDM-1).

[0096] 9. The polypeptide of any one of alternatives 1-8, wherein the polypeptide is capable of inducing death in a cell it is expressed in (i.e. a “suicide protein”).

[0097] 10. A use of the polypeptide of any one of alternatives 1-9 as a surface marker for identifying transduced cells.

[0098] 11. A first nucleic acid sequence encoding the polypeptide of any one of alternatives 1-9.

[0099] 12. A vector comprising: (a) the first nucleic acid sequence of alternative 11; and (b) a second nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the first and second nucleic acid sequences are separated by an at least one selfcleaving 2A peptide.

[0100] 13. The vector of alternative 12, further comprising a third nucleic acid sequence encoding an armouring protein, separated by a self-cleaving 2A peptide.

[0101] 14. A cell comprising the polypeptide of any one of alternatives 1-9, the first nucleic acid sequence of alternative 11, or the vector of any one of alternatives 12-13.

[0102] 15. A cell comprising a CAR and (i) the polypeptide of any one of alternatives 1-9, or (ii) the first nucleic acid sequence of alternative 11.

[0103] 16. The cell of alternative 15, further comprising an at least one armouring protein.

[0104] 17. The cell of any one of alternatives 14-16, wherein the CAR, polypeptide, armouring protein, or any combination thereof, is expressed on the cell’s surface.

[0105] 18. The cell of any one of alternatives 14-17, wherein the cell is a T cell.

[0106] 19. A method of identifying transduced cells, comprising: transducing a population of cells with the polypeptide of any one of alternatives 1-9, the first nucleic acid sequence of alternative 11, or the vector of any one of alternatives 12-13; contacting the population of cells with an antibody specific to the extracellular domain of the polypeptide; and detecting antibody binding, wherein positive detection of the antibody binding to the polypeptide signifies a transduced cell,

[0107] 20, A method for inducing cell death or suicide, comprising: contacting a population of cells expressing the polypeptide of any one of alternatives 1-9 with an antibodydrug conjugate, wherein the contact induces cytotoxicity7.

[0108] 21. The method of alternative 21, wherein the antibody-drug conjugate is trastuzumab emtansine (TDM-1)

[0109] 22. Use of the polypeptide of any one of alternatives 1 -9, the first nucleic acid sequence of alternative 11, or the vector of any one of alternatives 12-13, in the preparation of a medicament.

[0110] 23. A method for preparing CAR T-cells, comprising: (a) transducing a T- cell population with the polypeptide of any one of alternatives 1-9, the first nucleic acid sequence of alternative 11, or the vector of any one of alternatives 12-13; and (b) selecting a transduced cell using an antibody capable of binding to the polypeptide of any one of alternatives 1-9.

[0111] 24. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the polypeptide of any one of alternatives 1-9, the first nucleic acid sequence of alternative 11, the vector of any one of alternatives 12-13, or the cell of any one of alternatives 14-18.

[0112] 25. The method of alternative 24, wherein the subject is a mammal and / or a human.

[0113] 26. The method of alternative 24 or 25, wherein the disease or disorder is a tumor and / or cancer.

[0114] 27. The method of any one of alternatives 24-26, further comprising administering an antibody-drug conjugate capable of binding to the polypeptide of any one of alternatives 1-9.

[0115] 28. The method of alternative 21, wherein the antibody-drug conjugate is trastuzumab emtansine (TDM-1).

[0116] 29. A method of preventing, reducing, or ameliorating an at least one adverse effect of CAR T-cell therapy in a subject in need thereof, the method comprising: administering to the subject the polypeptide of any one of alternatives 1 -9, the first nucleic acid sequence of alternative 11, the vector of any one of alternatives 12-13, or the cell of any one of alternatives 14-18; monitoring for when the at least one adverse effect in the subject is occurring or will occur; and administering to the subject an antibody-drug conjugate capable of binding to the polypeptide of any one of alternatives 1 -9.

[0117] 30. The method of alternative 29, wherein the antibody-drug conjugate is trastuzumab emtansine (TDM-1)

[0118] 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.

[0119] 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 SequencesSEQ ID NO: Name Sequence1 VI Full GCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVL1length AHN VRQVPLQRLRIVRGTQLFEDN YALA VLDNGD HER2 PLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQR w / o NPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRAC kinase HPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCAR domain CKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCV TACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRC EKCS KPC AR VC YGLGMEHLREVRA VTS ANI QI 1F A GCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFE TLEEITGYLYIS AWPDSLPDLS VFQNLQVIRGR1 LI -IN GAYSLTLQGLGISWLGLRSLRELGSGLALIHIINTHL CFVIITVPWDQLFRNPIIQALLIITANRPEDECVGEG LACHQLCARGHCWGPGPTQCVNCSQFLRGQECVE ECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCF GPEADQCVACAHYKDPPFCVARCPSGVKI’DLSYM PIWKF'PDEEGACQPCPINCTHSCVDLDDKGCPAEQ RASPLTSnSAVVGILLVVVLGVVFGILIKRRQQKIR KYTMRRLLQETELVEPLTPSGAMPNQAQMRILKET EV2 D-IV PEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQ (vl) w / o FLRGQECVEECRVLQGLPREYVNARHCLPCHPECQ kinase PQNGSVTCFGPEADQCVACAIIYKDPPFCVARCPSG domain VKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLD DKGCP AEQRASPLTSII S A WGILL V WLG VVFG1 LI KRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQA QMRILKETEV3 D-IV QCVNCSQFLRGQECVEECRVI> QGLPREYVNARHC (v2) w / o LECHPECQPQNGSVTCFGPEADQCVACAHYKDPPF kinase CVARCPSG\7KPDI.JS\rMPIWKFPDEEGACQPCPINC domain TIISC / VDL. DDKCICrEAEQRASPLTSHSAVVCiILL. VVV I> G\A'TGIUKRRQQKIRKYTMRRIJ> QETEIATEPLTP SGAMPNQAQMRILKETEV4 Full GCQWQGNLELTYLPTNASLSFLQDIQEVQGYVLI length AHN VRQ VPLQRLR1 VRGTQLFED NYALAVLDNGD F1ER2 PLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQR CD5TM NPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRAC w / kinase HPCSPMCKGSRCWGESSEDCQSLIRTVCAGGCAR domain CKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHS GICELHCPALVTYNTDTFESMPNPEGRYTFGASCV TACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRC EKCSKPCARV C YGLGMEHLRE VRAVTS AN1QEF A GCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFE TLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHN GAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHL CFVH1VPWDQLFRNPHQALLHTANRPEDECVGEG LACHQLCARGHCWGPGPTQCVNCSQFLRGQECVE ECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCF GPEADQCVACAHYKDPPFCVARCPSGVKPDLSYM PIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQ RASPLTAGLAAGWASIILAL\nXWLLVVCGPLAY KRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQA QMRILKETELRKVKX^GSGAFGWYKGIWIPDGEN VKIPVAIKVI., RENTSPKANKEn. DEAYVMAGVGSP YX^SRTXGICIYSWQLVTQLMPYGCLLDHVRENRG RLGSQDLLNWCMQIAKGMSYIT. DMiLVHRDLAA R.maA7KSPNHVKITDFGLARLLDroETEYHADGGK VPIKyWAIESIL, RRRFTHQSDVU^SYGVTVrWE, LMT FGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYNn YlVKCWYHDSECRPRFRELVSEFSRMARDPQRFVVl QNEDLGPASPLDSTFYRSLLEDDDM(jDL\rDAEEYL VPQQGFFCPDPAPGAGGMVHIIRHRSSSTRSGGGD LTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMG AAKGLQSLPTIIDPSPLQRYSEDPTVPLPSETD(jYA'rAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAG ATLERPKTLSPGKNGVX’KDVFAFGCiAVENPEYLTP QGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPP STFKGTPTAENPEYLGLDVPVV5 Full GCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLI length AHNVRQVPLQRLR1VRGTQLFEDNYALAVLDNGD HER2 PLNN TTPV TGASPGGLRELQLRSLTEILKGGVLIQR CD5TM NPQLCYQDTILWKDIFHKNNQLALILIDTNRSKAC w / o HPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCAR kinase CKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHS domain GICELHCPALVTYNTDTFESMPNPEGRYTFGASCV TACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRC EKCSKPCARVCYGLGMEHLREVRAVTSANIQEFA GCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFE TLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHN GAYSLTI., QGLGISWI> GLRSLRELGSGLALIHHNTHE> CFVHTVPWDQLFRNPHQALLHTANRPEDECVGEG LA CHQLC AR GHCWGPGPTQC WCSQFI. R GQEC VE ECR\7I. QGI> PREWNARHCI.JPCHPECQPQNGSVrTCF GPEADQCVACAHYKDPPFCVARCPSGVKPDLSYM PIWKFPDEEGACQrA'PINCTHSCVDLDDKGCPAEQ RASPLTAGIAAGWASm. ALVL> LVVIXWCGPLAY KRRQQKIRKYTMRRLLQETELVEPLTPSCJAMPNQA QMRILKETEV6 D-IV PEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQ (vl) w / C FLRGQECVEECRVLQGLPREYVNARHCLPCHPECQ D5TM / Tr PQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGuncated VKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLD HER2 DKGCPAEQRASPLTAGLAAGTVASIILALVLLVVLL endo WCGPLAYKRRQQKIRKYTMRRLLQETELVEPLTP SGAMPNQAQMRILKETEV7 D-IV QCWCSQFLRGQECVEECRVLQGLPREYVNARHC (v2) LPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPF w / CD5T CVARCPSGWPDLSYWIWKFPDEEGACQPCPINC M / Trunca THSCVDLDDKGCPAEQRASPLTAGLAAGTVASnL ted HER2 AIA / IJAATJA / VCGPLAYKRRQQKIRKYTMRRLI. Q endo ETEIATiPLTPSGAAIPNQAQMRIIXETE Leader METDTL1LWVLLLLVPGSTGseqHER2 SIIS AWGILL\AAH. GWFGILITM HER 2 LRKVTCVLGSGAFGWYKGIWIPDGENVKIPVAIKV Kinase LRENTSPKANKEILDEAYVMAGVGSPYVSRLLGIC domain LTSWQLVTQLMPYGCLLDHVRENRGRLGSQDLL bAVCMQIAKGMSYLEDVRLVHRDLAARNVLVKSP NHVKITDFGLARLLDIDETEYHADGGKVPIKWMAL ESILRRRFTHQSDVAVSYGVTAAVELMTFGAKPYDGI PAREIPDLLEKGERLPQPPICHDVYAtMVKCWMID SECRPRFRELVSEFSRMARDPQRFV CD5 I'M P1NCTHSCVDLDDKGCPAEQRASPLTAGLAAGTVA SIILALVLLVVLLVVCGPLAYTruncated KRRQQKIRKYTMRRLLQETEIATsPLTPSGAJVIPNQA HER2 QMRUXETEendodomainTrastuzu EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH mab VH WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDG FYAMDYWGQGTLVTVSSTrastuzu DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW mab VL YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDF TLHSSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKPertuzutn EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMD ab VI I \WRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFT LSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPS FYFD Y WGQGTLV TV S SPertuzum DIQMTQSPSSLSASVGDRVUTCKASQDVSIGVAW ab VL YQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDF TimSSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK HA tag + YPYDVPDYASGGGGSPTTTPAPRPPTPAPTIASQPL IL13Ra2 SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT CAR + CGVLLLSLVITLYCNHRNRRRVCKCPRPVVEGRGS sort LLTCGDVEENPGPSMALPVTALLLPLALLLHAARP suicide EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMR switch WVRQAPGKGLEWVSSINSGGGSTSYVDSVKGRFTI SRDNAKNTAYLQMNSLRPEDTAVYYCNAALETNR GQNYWGQGTQVTVSSIEVMYPPPYLDNEKSNGTII HVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSL LYWAFllFWVRSKRSRIXHSDY^INlVrrPRRPGPTR KHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQ NQLYNELNI.. GRREEYDVI. DKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGI. YQGL. STATKDTVTDAI. HMQALPPRATNFSIJ. KQAGDVEENPGPJVffiTOTIJL^7VI., LLLVPGSTGQEF AQCVNCSQFLRGQECVEECRVL> QGLPREYVNARH CLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPP FCVARCPSGVKPDLSYMPIWKFPDEEGCQPCPINCT HSCVDLDDKGCPAEQRASPLTAGLAAGTVASnLALVLLVVLLWCGPLAYKRRQQKIRKYTMRRLLQE TELVEPLTPSGAMPNQAQMRILKETE18 Domain QCVNCSQFLRGQECVEECRVLQGLPREYVNARIIC IV v2 LPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCEXAMPLES

[0120] The non-limiting example methodology as disclosed herein are used in the following working Examples.Example 1: Mechanism of action of the sort-safety switch component.

[0121] Disclosed herein is a polypeptide with a “sort-safety switch.” As depicted schematically in Figure 1, a suicide polypeptide (encoded by a suicide gene) is expressed on the surface of a cell. The polypeptide comprises an extracellular domain, a transmembrane domain, and optionally an intracellular domain. In some embodiments, the extracellular domain is a variant derived from the HER2 domain 4. Upon bind ing of the extracellular domain to an antibody-drug conjugate (ADC) (such as trastuzumab emtansine (TDM-1)), the polypeptide-antibody complex undergoes internalization into the cell. The internalization of the ADC releases the cytotoxic payload, which inhibits the cell’s microtubules and induces ceil death.Example 2: Detection of sort-safety switch by HER2 targeting antibodies

[0122] A single gene cassette was designed to encode GFP and different versions of the-sort-safety switch separate by a self-cleaving 2A peptide (Figure 2A). Jurkat cells were transduced with y-retro viruses produced following the transfection of HEK293T 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 and transduction efficiency established based on GFP expression by flow cytometry.

[0123] Expression of the different versions of the sort-safety switch was verified based on the binding of Trastuzumab or Pertuzumab to GFP expressing cells. Results showed variants VI, V2, V3, V4, V5, V6 and V7 of the sort-safety switch bind to trastuzumab (Figure 2B), and VI, V5 and V6 also showed binding to Pertuzumab (Figure 2C). The correlation between GFP and Trastuzumab or Pertuzumab indicate that these safety switches can be used as a surrogate marker for transduction efficiency.Example 3: HER2 antibody internalization when bound to sort-safety' switch

[0124] To establish receptor mediated antibody internalization, the trastuzumab antibody was conjugated with a hydrophilic pH sensitive dye (ThermoFisher, P35372) and incubated with cells transduced to express the sort-safety switch. The conjugated dye only fluoresces when the ligated receptor is internalized and trafficked to endosomal vesicles. The acidic environment maintained in the endosomes enables the dye to fluoresce (Figure 3 A), This fluorescent signal was visualized via microscopy. NT and sort-safety switch expressing cells are incubated with the labelled antibody, a red fluorescent signal is emitted from cells expressing the sort-safety switch suggesting receptor ligation and internalization (Figure 3B). To further evaluate internalization, TDM-1 was conjugated with pH sensitive fluorescent dye and exposed to cells expressing versions of the sort-safety switch. All versions show fluorescent signal suggesting internalization, with maximum signal seen with V7 (Figure 3C).Example 4: Sort-safety switch expressing cells are switched off when exposed to HER2 antibody drug conjugate TDM-1

[0125] To evaluate the suicide function of sort-safety switch variants, Jurkat cells were transduced with y-retroviruses produced following the transfection of HEK293T 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 and transduction efficiency established based on GFP expression by flow cytometry. Cells expressing variants of the sort-safety switch were cultured in the presence of varying concentrations of Trastuzumab Emtansme (TDM-1) (Selleck Biotechnology, D4003) for 72 hours and viability assessed using flow cytometry.

[0126] All versions tested demonstrated decreased cellular viability when in the presence of TDM- 1. Some versions showed greater susceptibility at lower concentrations of TDM-1 (Figured).Example 5: Construction and detection of a CAR expression cassette with sort-safety switch

[0127] A gene cassette was designed to encode a transgene marker, a CAR, and the safety sort switch, each separated by a self-cleaving 2A peptide (Figure 5A). 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 expression levels of HA, CAR and sort-safety switch established via flow cytometry. Transduction data shows equal expression of transgene marker, CAR marker and the sort-safety switch in T cells (Figure 5B).Example 6: Exposure to the antibody drug conjugate TDM-1 decreases the viability of sort¬ safety' switch expressing CAR T cells

[0128] To evaluate the function in cells, CAR T cells expressing the sort-safety switch were generated. 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. Transduced cells were then cultured in the presence of Trastuzumab Emtansine (TDM-1) (Selleck Biotechnology, D4003) at varying concentrations.

[0129] When exposed to TDM1, there was a dose-dependent decrease in viability of CAR T cells expressing the sort-safety switch compared to CAR cells that did not express the switch (Figure 6).Example 7: Modulation of CAR T-cell cytotoxicity by the activation of the suicide protein through ligation with an antibody drug conjugate

[0130] Co-cultures were set up using CAR PBMCs generated as described in Example 6, and SupTl IL13Ra2 target cells. Trastuzumab Emtansine (TDM-1) (Selleck Biotechnology, D4003) was added at 2.5mg / ml or 5mg / ml and target cell survival and cytokine production measured after 72 hrs.

[0131] CAR T cells expressing the sort-safety switch demonstrated a decrease in cytotoxicity upon exposure to TDM-1, suggesting that these cells were successfully switched off and non-functional. In contrast, CAR T cells that lacked the sort-safety switch remained unaffected by the presence of TDM- 1, and cleared target cells (Figure 7A).

[0132] Cytokine production was also evaluated in the supernatants from these cultures via ELISAs (Biolegend, 430104). When CAR + sort-safety switch T cells are exposed to TDM-1, the levels of IFN-g secreted by these cells decreased compared to T cells expressing only a CAR construct (Figure 7B).Example 8: In vivo efficacy of CAR T cells with sort-safety switch components in a glioblastoma model.

[0133] 6-8 week old, female, NOG mice (Charles River Laboratory) were injected intracranially with U87-MG cells overexpressing IL13Ra2. Tumour cells were engrafted for 8 days prior to the intracranial injection of previously frozen CAR T cells. 7 days post CAR T cell injection, mice were treated with 2.5ug of Trastuzumab Emtansine (TDM-1) (Selleck Biotechnology, D4003). Tumour engraftment and clearance was measured by biolumin escent imaging utilizing the IVIS spectrum system, in addition to measuring changes in body weight.

[0134] NOG mice bearing intracranial IL13Ra2-expressing glioblastoma xenografts were treated with a known high toxic dose of CAR T cells engineered with the sortsafety component. After 7 days, T-DM1 was administered to activate the safety switch (Figure 8A), Tumour clearance was measured based on bioluminescence imaging over time (Figure 8B). Toxicity was assessed by tracking body weight changes over time (Figures 8C). CAR T cells were injected into mice bearing tumours followed by the addition of TDM1.

[0135] Mice that did not receive TDM1 showed tumour clearance, but exhibited decreased body weight. The introduction of TDM1 switched off CAR T cells, enabling successful tumour clearance whilst minimizing toxicity, as evidenced by the lack of change in body weight.

[0136] The results demonstrated the effective tumor clearance m mice receiving CAR T cells co-expressing a sort-safety switch to control I' cell activity and reduce toxicity.

Claims

WHAT IS CLAIMED IS:

1. A method of inducing death in a cell, the method comprising:introducing into the cell a polypeptide encoding a protein that is expressed on the surface of the cell; andadministering an antibody-drug conjugate to the cell;wherein the protein is capable of binding to the antibody,wherein the drug has activity in inducing cell death.

2. The method of claim 1, wherein the protein comprises an extracellular domain and a transmembrane domain.

3. The method of claim 1, wherein the protein comprises an intracellular domain.

4. The method of claim 2, wherein the extracellular domain is derived from HER2, 5. The method of claim 4, wherein the extracellular domain is derived from HER2 domain 4.

6. The method of claim 1, wherein the cell is an immune cell.

7. The method of claim 1, wherein the cell is a T cell.

8. The method of claim 1, wherein the cell expresses a chimeric antigen receptor (CAR).

9. The method of claim 1, wherein the antibody is trastuzumab or pertuzumab.

10. The method of claim 1, wherein the drug is a tubulin inhibitor.

11. The method of claim 10, wherein the drug is mertansine, emtansme auristatin, ravtansine, deruxtecan, or any combination thereof.

12. A kit for inducing targeted cell death in a subject, the kit comprising:a vector encoding a protein that can be incorporated onto the surface of a targeted cell in the subject; andan antibody-drug conjugate,wherein the antibody is capable of binding to the protein,wherein the drug is capable of inducing cell death in the targeted cell.

13. A polypeptide for use in inducing cell death, comprising:an extracellular domain derived from a human HER2 protein; anda transmembrane domain.

14. The polypeptide of claim 13, wherein the extracellular domain comprises a sequence with at least 80, 85, 90, 95, 99, or 100% identity with the sequence of HER2 domain 4 (SEQ ID NO: 18).

15. The polypeptide of claim 13, wherein the transmembrane domain comprises the transmembrane domain of HER2, CD5 (SEQ ID NO: 11), or a variant thereof.

16. The polypeptide of claim 13, further comprising an intracellular domain.

17. The polypeptide of claim 16, wherein the intracellular domain comprises a truncated HER2 intracellular domain (SEQ ID NO: 12), a kinase-inactive mutant of HER2, a human CD5 intracellular domain, a variant thereof, or any combination thereof,18. The polypeptide of claim 16, wherein the extracellular domain is capable of binding to trastuzumab and / or pertuzumab,19. The polypeptide of claim 18, wherein the extracellular domain is capable of binding to trastuzumab emtansine (TDM-1).

20. A use of the polypeptide of claim 13 as a surface marker for identifying a cell.

21. A nucleic acid sequence encoding the polypeptide of claim 13.

22. A vector comprising the nucleic acid sequence of claim 21.

23. The vector of claim 22, further comprising a nucleic acid sequence encoding a CAR.

24. The vector of claim 22, further comprising an at least one self-cleaving 2A peptide.

25. The vector of claim 22, further comprising a nucleic acid sequence encoding an at least one armouring protein.

26. A cell comprising the polypeptide of claim 13, the nucleic acid sequence of claim 21, or the vector of claim 22.

27. The cell of claim 26, wherein at least one of: the CAR, polypeptide, armouring protein, or any combination thereof, is expressed on the cell’s surface.

28. The cell of claim 26, wherein the cell is an immune cell, preferably wherein the cell is a T cell.

29. A method of identifying transduced cells, comprising:(a) transducing a population of cells with the polypeptide of claim 13, the nucleic acid sequence of claim 21, or the vector of claim 22;(b) contacting the population of cells with an antibody specific to the polypeptide; and(c) detecting antibody binding, wherein positive detection of the antibody binding to the polypeptide signifies a transduced cell.

30. A method for inducing death or suicide in a cell, comprising:(a) introducing into the cell the polypeptide of claim 13, the nucleic acid sequence of claim 21, or the vector of claim 22; and(b) administering an antibody-drug conjugate to the cell;wherein the polypeptide is capable of binding to the antibody,wherein the drug has activity in inducing cell death.

31. The method of claim 30, wherein the antibody-drug conjugate is trastuzumab emtansine (TDM-1).

32. Use of the polypeptide of claim 13, the nucleic acid sequence of claim 21, or the vector of claim 22, in the preparation of a medicament.

33. A method for preparing CAR T-cells, comprising:(a) transducing a T-cell population with the polypeptide of claim 13, the nucleic acid sequence of claim 21, or the vector of claim 22; and(b) selecting a transduced cell using an antibody capable of binding to the polypeptide of claim 13.

34. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the polypeptide of claim 13, the nucleic acid sequence of claim 21, the vector of claim 22, or the cell of claim 26.

35. The method of claim 34, wherein the subject is a mammal and / or a human.

36. The method of claim 34, wherein the disease or disorder is a tumor and / or cancer.

37. The method of claim 34, further comprising administering an antibody-drug conjugate capable of binding to the polypeptide of claim 13.

38. The method of claim 37, wherein the antibody-drug conjugate is trastuzumab emtansine (TDM-1).

39. A method of preventing, reducing, or ameliorating an at least one adverse effect of CAR T-cell therapy in a subject in need thereof, the method comprising:(a) administering to the subject the polypeptide of claim 13, the nucleic acid sequence of claim 21, the vector of claim 22, or the cell of claim 26;(b) monitoring for when the at least one adverse effect in the subject is occurring or will occur; and(c) administering to the subject an antibody-drug conjugate capable of binding to the polypeptide of claim 13.

40. The method of claim 39, wherein the antibody-drug conjugate is trastuzumab emtansine (TDM-1).

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