Transmembrane signaling proteins that recognize brain-specific antigens

Abstract

Provided herein is a transmembrane signaling protein comprising: (a) an extracellular binding domain comprising a scFv having the CDRs of a selected antibody, (b) a transmembrane domain, and (c) an intracellular signaling domain. Cells expressing the same are also provided, as well as methods of treatment that employ the same.

Description

Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT TRANSMEMBRANESIGNALINGPROTEINS THATRECOGNIZEBRAIN-SPECIFICANTIGENSCROSS-REFERENCE TORELATEDAPPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 687,256, filed August 26, 2024, which application is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under D24AC00084 awarded by the Advanced Research Projects Agency for Health. The government has certain rights in the invention. INCORPORATION BYREFERENCE OFSEQUENCELISTINGPROVIDEDAS A SEQUENCE LISTING XML FILE

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “UCSF-798WO_SEQLIST” created on August 15, 2025, and having a size of 49,331 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety. INTRODUCTION

[0004] Most drugs act systemically, and even if they have perfect specificity for their molecular target, can show toxicities because most targets are expressed in both disease and normal tissues. In some cases, it would be desirable to restrict the activity of some drugs to specific, disease-relevant tissues. Targeting central nervous system (CNS) disorders, such as brain tumors, neuroinflammation and neurodegeneration, are a particularly challenging example. Achieving efficacy and minimizing toxicity is very difficult because of the barriers to delivering molecular therapeutics to the brain, and because drug targets can often also be expressed in tissues outside of the brain.

[0005] Cell therapies, however, potentially provide a unique solution to this conundrum, as it may be possible to engineer a cell to only act in a tissue-specific way. For example, using a cellAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT to selectively and autonomously deliver therapeutic payloads to the brain could reduce systemic off-target toxicity while also increasing local efficacy. Immune cells have evolved to infiltrate diverse tissues, respond to injury or infection, and reshape tissue ecosystems, properties that may make them suitable to act as local therapeutic delivery vehicles. T cells have the ability to cross the blood brain barrier (BBB), as in the case of intravenously infused CAR T cells programmed to recognize brain tumors. One way to harness T cells to deliver payloads selectively to the brain would be to engineer them to recognize normal (non-disease) CNS- specific antigens, and to use these to trigger production of a therapeutic payload. A cell-based CNS-specific delivery platform could potentially serve as a common disease-agnostic platform to treat diverse types of CNS diseases. SUMMARY

[0006] Provided herein is a transmembrane signaling protein comprising: (a) an extracellular binding domain comprising a scFv having the CDRs of an antibody selected from table 1; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the scFv comprises: a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain of the selected antibody; and a light chain variable domain that is at least 90% identical to the light chain variable domain of the selected antibody. In any embodiment, the scFv may comprise an amino acid sequence that is at least 90% identical to any of SEQ ID NOs. 41-45. In any embodiment, the transmembrane signaling protein may be, for example, a binding-triggered transcriptional switch (BTTS), an immune receptor such as a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR), or a synCAM.

[0007] A nucleic acid encoding the transmembrane signaling protein is also provided, as well as a cell comprising the same. Methods of treatment are also provided. This method may comprise administering the cell, where the subject may have a disease or disorder of the brain.

[0008] The antigens to which the binding domain binds are brain-selective and, as such, binding to any of these antigens limits or concentrates the cell’s effect to the brain. For example, in some embodiments, the transmembrane signaling protein may be a binding-triggered transcriptional switch. In these embodiments, the cell may further comprise a nucleic acid comprising: (i) a coding sequence encoding a therapeutic protein and (ii) a regulatory sequence, wherein the regulatory sequence is operably linked to the coding sequence and is responsive to activation ofAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT the binding-triggered transcriptional switch. In these embodiments, the therapeutic protein may be expressed or delivered to brain tissue and not other tissues because the binding-triggered transcription switch is preferentially activated in the brain. In other embodiments, the transmembrane signaling protein may cause therapeutic cells to localize the brain, thereby limiting migration of the therapeutic cells from the brain into other tissues.

[0009] These and other features may become apparent in view of the description that follows below. BRIEFDESCRIPTIONOFTHEDRAWINGS

[0010] Fig. 1: Screening for CNS-sensing synNotch receptors. Primary human CD4+ T cells expressing CNS-sensing synNotch and GFP reporter were cocultured with mouse primary neuronal / glial cultures or K562 cell engineered to overexpress the cognate antigen to test priming. Histograms show induction of GFP reporter only in the presence of the cognate antigen.

[0011] Figs. 2A-2F. SynNotch recognition of CNS-specific ECM molecule BCAN directs CAR anti-tumor activity specifically and potently to the intracerebral GBM PDX.

[0012] Fig. 2A: Design of brain-targeted CAR T cell. α-BCAN synNotch receptor is used to drive expression of anti-tumor CAR only in the brain. This tissue-specific priming circuit is designed to restrict the expression of CAR only to the CNS, preventing damage to normal, non- CNS tissues that express the CAR target antigens, EphA2 and IL13Rα2. This circuit should selectively identify GBM cells, which are the only cells expressing EphA2 or IL13Rα2 in the CNS.

[0013] Fig. 2B: Killing of GBM6 PDX tumors in vitro. Primary CD8+T cells transduced with α-BCAN synNotch^α-EphA2 / IL13Rα2 CAR circuit (or with the constitutively expressed α- EphA2 / IL13Rα2 CAR) were cocultured with GBM6 target cells, and K562 priming cells, either expressing or not expressing BCAN. Relative cell survival of target GBM6 cells is shown at 72h. (relative to untransduced T cell controls, n = 3, error bars indicate SEM).

[0014] Fig. 2C: In vivo clearance of GBM6 tumors. GBM6 tumors expressing mCherry and luciferase were orthotopically implanted in the brains of NCG mice. Ten days after tumor implantation, mice were infused i.v. with 2 million each of CD4+and CD8+T cells expressingAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT the α-BCAN synNotch–α-EphA2 / IL13Rα2 CAR circuit (n=5) or no construct (neg. control) (n = 5). Tumor size and survival were monitored over time by bioluminescence imaging. Thick line shows mean tumor size; thin lines show individual mice. Mixed-effects analysis, p = 0.018. Survival was analyzed over 80 days by log-rank (Mantel-Cox) test. p = 0.003. Data is representative of two independent experiments. BCAN knock out GBM6 tumors (See Fig. S4C) were also cleared with similar efficiency.

[0015] Fig. 2D: GBM6 tumor–bearing mice were euthanized 10 days after α-BCAN SynNotch- CAR T cell infusion (3 million each of CD4+and CD8+). Representative confocal fluorescent microscopy of brain sections shows synNotch activation (green) throughout the brain, and reveals that T cell–mediated killing (cleaved caspase 3 staining - purple) is restricted to the tumor (adjacent neurons are not apoptotic – grey NeuN stain). Scale bars, 1 mm (left) and 200 µm (right, enlargement of outlined region).

[0016] Fig. 2E: Flow cytometry of α-BCAN synNotch–α-EphA2 / IL13Rα2 CAR T cells isolated from blood, spleen and brain of GBM6-bearing mouse at day 6 after T cell injection demonstrates presence of GFP+primed T cells in the brain but not in the blood or the spleen.

[0017] Fig. 2F: Brain-Flank dual GBM6 tumor model. GBM6 tumor cells were implanted in the brain of NCG mice, while BCAN KO GBM6 cells were implanted in the flank of the identical mice. Both tumors express the CAR killing antigens (EphA2 and IL13Rα2), but BCAN is only expressed in the brain. Ten days after tumor implantation, mice were infused intravenously with 2 million each of CD4+and CD8+T cells expressing no construct (control) (n = 4) or α-BCAN synNotch–α-EphA2 / IL13Rα2 CAR circuit (n=5). Tumor size in the brain and in the flank were monitored over time by bioluminescence imaging. Only the tumor implanted in the brain was reduced over time while the flank tumor grew at the same rate as in the mice treated with non- transduced T cells. Data is representative of two independent experiments. Thick line shows mean, and shaded area SEM (studies showing efficient in vivo killing of brain-implanted GBM39 tumors – PDX line lacking BCAN expression were also performed).

[0018] Figs. 3A-3C: CNS-specific priming of synNotch-CAR T cells is generalizable tool for effective killing of brain metastases such as HER2+ and triple negative breast cancer brain metastases.Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT

[0019] Fig. 3A: Treatment for CNS metastases is a major unmet need, particularly for breast cancer. A CNS-specific priming circuit could be used as a general platform to target such metastases. Restricting CAR expression only to the brain could preventing damage to normal, nonbrain tissues that express target killing antigens. Tumor specific antigens are particularly hard to find for triple-negative breast cancer. This strategy could be applied to target brain metastases for HER2+breast cancer, or TROP2+triple negative breast cancer (TNBC).

[0020] Fig. 3B: Real-time in vitro killing of BT-474 breast cancer cell, a model of HER2+breast cancer. BT-474 cells express HER2, but are negative for BCAN. Therefore, to mimic brain priming, co-cultured BT-474 cells were co-cultured with K562 cells expressing the priming antigen BCAN. Killing of BT-474 cells is only observed in the presence of BCAN+K562 cells (n=3, error bars indicate SEM). Further in vitro killing studies of BT-20 breast cancer cells, a model of triple-negative breast cancer, were also performed.

[0021] Fig. 3C: In vivo tumor experiments with BT-474 (HER2+breast cancer) and BT-20 (TNBC) tumors. BT-474 or BT-20 tumors expressing GFP and luciferase were orthotopically implanted in brains of NSG mice. Seven days after tumor implantation, mice were infused intravenously with 3 million each of CD4+and CD8+T cells expressing no construct (control) (n = 5) or α-BCAN synNotch–CAR T cells (for BT-474: α-HER2 CAR; for BT-20: α-TROP2 CAR) (n=5). Tumor size and survival were monitored over time by bioluminescence imaging. Thick line shows mean + / - SEM (shaded area).

[0022] Figs 4A-4C: CNS-specific synNotch circuits can be programmed to produce anti- inflammatory cytokine IL-10.

[0023] Fig. 4A: CNS-specific synNotch cells could in principle be used to modulate neuroinflammation. For example, CNS-priming could be used to trigger expression of IL-10, a potent anti-inflammatory cytokine.

[0024] Fig. 4B: Primary human T cells were engineered with α-BCAN synNotch-mIL^10 circuit and co-cultured with K562 cells engineered to express BCAN. Supernatant was collected after 48h and IL10 was quantified by ELISA. Quantification shows the specific secretion of IL- 10 only when the engineered T cells are cultured with BCAN+K562 cells (n = 3).

[0025] Fig. 4C: In vitro inhibition assays of microglia and T cell activation. BV2 mouse microglia were cultured with control or therapeutic T cells (α-BCAN synNotch^IL-10) in theAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT presence of BCAN+K562 cells to induce payload expression. 2 hours later, IFN-γ and LPS were added to induce activation of the microglia. Cells were cultured for 24h and the supernatant was collected to assess inflammation by assaying for secretion of IL-6 and TNF-α by ELISA. n = 3, mean + / - std. Conventional CD25- TCR+CD4+T cells were sorted from MOG-specific TCR (2D2) transgenic mice and co-cultured with APCs presenting MOG peptide to induce their activation. To assay the inhibition of activation, MOG-specific TCR 2D2 CD4 T cells were co- cultured at a 1:1 ratio with control transduced or engineered with α-BCAN synNotch^IL-10 T cells for 4 days in presence of BCAN+ K562 cells (to stimulate IL-10 induction). Activation of the MOG-specific TCR 2D2 CD4 T cells was analyzed by flow cytometry using the activation marker CD25, or by ELISA to measure IFN-γ secretion (n = 3, mean + / - std).

[0026] Figs 5A-5D: CNS-targeted anti-inflammatory circuit ameliorates autoimmune encephalomyelitis model.

[0027] Fig. 5A: Schematic of adoptive transfer EAE model: RAG-1 - / - mice received an adoptive transfer of Th17 polarized CD4 T cells (20 x 106in B6x 10 in C) from P35-55 MOG immunized C57BL / 6J mice. At indicated days post adoptive transfer (arrows), mice received primary human CD4 T cells transduced with either control (no circuit, n=5) or α- BCAN synNotch^IL10 circuit (n=5) at the indicated times (10 x 106). The EAE neurological disease scoring scale is shown.

[0028] Fig. 5B: Treatment with α-BCAN synNotch-IL10 T cells yields improved EAE scores and increased survival. 106T cells were injected on each day indicated by a black arrow. A 2- way ANOVA was performed, p < 0.05. EAE severity was assessed by the area under the curve of each animal starting from the day of first treatment day 7 until day 25. n = 5, mean + / - standard error. Unpaired T-test, p <0.05. Survival curves shows improved protection by the α- BCAN synNotch^IL10 T cell treatment as analyzed by log-rank (Mantel-Cox) test.

[0029] Fig. 5C: Treatment with α-CDH10 synNotch-IL10 T cells yields improved EAE scores and increased survival. 106T cells were injected on each day indicated by a black arrow. EAE scores showed significant improvements with CNS-targeted α-CDH10 synNotch^IL10 T cells. A 2-way ANOVA was performed, p < 0.05. EAE severity was assessed by the area under the curve of each animal starting from the day of first treatment day 7 until day 25. n = 7, mean + / -Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT standard error. Unpaired T-test, p <0.05. Survival curves shows improved protection by the α- CDH10 synNotch^IL10 T cell treatment as analyzed by log-rank (Mantel-Cox) test.

[0030] Fig. 5D: Brain-sensing cells could be used as a general platform to treat a broad set of CNS diseases such as primary and secondary brain tumors, neuroinflammation, or even neurodegeneration. This customizable platform can be used to locally deliver any genetically encodable molecular therapy that is appropriate for a specific CNS disease, thereby improving on-target action and alleviating off-target toxicity. DEFINITIONS

[0031] As used herein, the terms "treatment," "treating," “treat” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect and / or a response related to the treatment. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0032] A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent (including biologic agents, such as cells), or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.

[0033] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g., rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), lagomorphs, etc. In some cases, the individual is a human. In some cases, the individual is a non-human primate. In some cases, the individual is a rodent, e.g., a rat or a mouse. In some cases, the individual is a lagomorph, e.g., a rabbit.

[0034] The term “refractory”, used herein, refers to a disease or condition that does not respond to treatment. With regard to cancer, “refractory cancer”, as used herein, refers to cancer thatAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT does not respond to treatment. A refractory cancer may be resistant at the beginning of treatment or it may become resistant during treatment. Refractory cancer may also called resistant cancer.

[0035] The term “histology” and “histological” as used herein generally refers to microscopic analysis of the cellular anatomy and / or morphology of cells obtained from a multicellular organism including but not limited to plants and animals.

[0036] The term “cytology” and “cytological” as used herein generally refers to a subclass of histology that includes the microscopic analysis of individual cells, dissociated cells, loose cells, clusters of cells, etc. Cells of a cytological sample may be cells in or obtained from one or more bodily fluids or cells obtained from a tissue that have been dissociated into a liquid cellular sample.

[0037] The terms “chimeric antigen receptor” and “CAR”, used interchangeably herein, refer to artificial multi-module molecules capable of triggering or inhibiting the activation of an immune cell which generally but not exclusively comprise an extracellular domain (e.g., a ligand / antigen binding domain), a transmembrane domain and one or more intracellular signaling domains. The term CAR is not limited specifically to CAR molecules but also includes CAR variants. CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero- dimerization of the two portions of the split CAR is pharmacologically controlled (e.g., as described in PCT publication no. WO 2014 / 127261 A1 and US Patent Application No. 2015 / 0368342 A1, the disclosures of which are incorporated herein by reference in their entirety). CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013) ;5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; BarrettAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Useful CARs also include the anti-CD19—4-1BB—CD3ζ CAR expressed by lentivirus loaded CTL019 (Tisagenlecleucel-T) CAR-T cells as commercialized by Novartis (Basel, Switzerland).

[0038] The terms “T cell receptor” and “TCR” are used interchangeably and will generally refer to a molecule found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR complex is a disulfide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (α) and beta (β) chains expressed as part of a complex with CD3 chain molecules. Many native TCRs exist in heterodimeric αβ or γδ forms. The complete endogenous TCR complex in heterodimeric αβ form includes eight chains, namely an alpha chain (referred to herein as TCRα or alpha), beta chain (referred to hereinas TCRβ or TCR beta), delta chain, gamma chain, two epsilon chains and two zeta chains. In some instance, a TCR is generally referred to by reference to only the TCRα and TCRβ chains, however, as the assembled TCR complex may associate with endogenous delta, gamma, epsilon and / or zeta chains an ordinary skilled artisan will readily understand that reference to a TCR as present in a cell membrane may include reference to the fully or partially assembled TCR complex as appropriate.

[0039] Recombinant or engineered individual TCR chains and TCR complexes have been developed. References to the use of a TCR in a therapeutic context may refer to individual recombinant TCR chains. As such, engineered TCRs may include individual modified TCRα or modified TCRβ chains as well as single chain TCRs that include modified and / or unmodified TCRα and TCRβ chains that are joined into a single polypeptide by way of a linking polypeptide.

[0040] As used herein, the term “binding-triggered transcriptional switch” or “BTTS” refers to any polypeptide or complex of the same that is capably of transducing a specific binding event on the outside of the cell (e.g. binding of an extracellular domain of the BTTS) to activation of a recombinant promoter within the nucleus of the cell. Many BTTSs work by releasing a transcription factor that activates the promoter. In these embodiments, the BTTS is made up of one or more polypeptides that undergo proteolytic cleavage upon binding to the antigen toAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT release a gene expression regulator that activates the recombinant promoter. For example, a BTTS may comprise (i) an extracellular domain comprising the antigen binding region of a antigen-specific antibody; (ii) a proteolytically cleavable sequence comprising one or more proteolytic cleavage sites; and (iii) an intracellular domain, wherein binding of the antigen binding region to the antigen induces cleavage of the sequence at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain and wherein the intracellular domain activates transcription of an expression cassette. A BTTS can be based on synNotch, A2, MESA, or force receptor, for example, although others are known or could be constructed.

[0041] As used herein, by “chimeric bispecific binding member” is meant a chimeric polypeptide having dual specificity to two different binding partners (e.g., two different antigens). Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BiTE), bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann. MAbs. (2012) 4(2): 182–197; Stamova et al. Antibodies 2012, 1(2), 172-198; Farhadfar et al. Leuk Res. (2016) 49:13-21; Benjamin et al. Ther Adv Hematol. (2016) 7(3):142-56; Kiefer et al. Immunol Rev. (2016) 270(1):178-92; Fan et al. J Hematol Oncol. (2015) 8:130; May et al. Am J Health Syst Pharm. (2016) 73(1):e6-e13; the disclosures of which are incorporated herein by reference in their entirety.

[0042] A “biological sample” encompasses a variety of sample types obtained from an individual or a population of individuals and can be used in various ways, including e.g., the isolation of cells or biological molecules, diagnostic assays, etc. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by mixing or pooling of individual samples, treatment with reagents, solubilization, or enrichment for certain components, such as cells, polynucleotides, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples. The term “biological sample”Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions such as plasma and serum, and the like. The term “biological sample” also includes solid tissue samples, tissue culture samples (e.g., biopsy samples), and cellular samples. Accordingly, biological samples may be cellular samples or acellular samples.

[0043] The terms "antibodies" and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, nanobodies, single-domain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.

[0044] "Antibody fragments" comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

[0045] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0046] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. Non-specific binding would refer to binding with an affinity of less than about 10-7M, e.g., binding with an affinity of 10-6M, 10-5M, 10-4M, etc.

[0047] A “orthogonal” or “orthogonalized” member or members of a binding pair are modified from their original or wild-type forms such that the orthogonal pair specifically bind one anotherAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT but do not specifically or substantially bind the non-modified or wild-type components of the pair. Any binding partner / specific binding pair may be orthogonalized, including but not limited to e.g., those binding partner / specific binding pairs described herein.

[0048] The terms “domain” and “motif”, used interchangeably herein, refer to both structured domains having one or more particular functions and unstructured segments of a polypeptide that, although unstructured, retain one or more particular functions. For example, a structured domain may encompass but is not limited to a continuous or discontinuous plurality of amino acids, or portions thereof, in a folded polypeptide that comprise a three-dimensional structure which contributes to a particular function of the polypeptide. In other instances, a domain may include an unstructured segment of a polypeptide comprising a plurality of two or more amino acids, or portions thereof, that maintains a particular function of the polypeptide unfolded or disordered. Also encompassed within this definition are domains that may be disordered or unstructured but become structured or ordered upon association with a target or binding partner. Non-limiting examples of intrinsically unstructured domains and domains of intrinsically unstructured proteins are described, e.g., in Dyson & Wright. Nature Reviews Molecular Cell Biology 6:197-208.

[0049] The terms “synthetic”, “chimeric” and “engineered” as used herein generally refer to artificially derived polypeptides or polypeptide encoding nucleic acids that are not naturally occurring. Synthetic polypeptides and / or nucleic acids may be assembled de novo from basic subunits including, e.g., single amino acids, single nucleotides, etc., or may be derived from pre- existing polypeptides or polynucleotides, whether naturally or artificially derived, e.g., as through recombinant methods. Chimeric and engineered polypeptides or polypeptide encoding nucleic acids will generally be constructed by the combination, joining or fusing of two or more different polypeptides or polypeptide encoding nucleic acids or polypeptide domains or polypeptide domain encoding nucleic acids. Chimeric and engineered polypeptides or polypeptide encoding nucleic acids include where two or more polypeptide or nucleic acid “parts” that are joined are derived from different proteins (or nucleic acids that encode different proteins) as well as where the joined parts include different regions of the same protein (or nucleic acid encoding a protein) but the parts are joined in a way that does not occur naturally.

[0050] The term "recombinant", as used herein describes a nucleic acid molecule, e.g., a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin, which, byAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide sequences with which it is associated in nature. The term recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression from a recombinant polynucleotide. The term recombinant as used with respect to a host cell or a virus means a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein to refer to, with reference to material (e.g., a cell, a nucleic acid, a protein, or a vector) that the material has been modified by the introduction of a heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).

[0051] The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Operably linked nucleic acid sequences may but need not necessarily be adjacent. For example, in some instances a coding sequence operably linked to a promoter may be adjacent to the promoter. In some instances, a coding sequence operably linked to a promoter may be separated by one or more intervening sequences, including coding and non-coding sequences. Also, in some instances, more than two sequences may be operably linked including but not limited to e.g., where two or more coding sequences are operably linked to a single promoter.

[0052] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0053] The terms “polypeptide,” “peptide,” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT

[0054] A "vector" or "expression vector" is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an "insert", may be attached so as to bring about the replication of the attached segment in a cell.

[0055] The term “heterologous”, as used herein, means a nucleotide or polypeptide sequence that is not found in the native (e.g., naturally-occurring) nucleic acid or protein, respectively. Heterologous nucleic acids or polypeptide may be derived from a different species as the organism or cell within which the nucleic acid or polypeptide is present or is expressed. Accordingly, a heterologous nucleic acids or polypeptide is generally of unlike evolutionary origin as compared to the cell or organism in which it resides.

[0056] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0057] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT

[0059] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the cell” includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0060] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0061] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. DETAILED DESCRIPTION

[0062] As summarized above, the present disclosure provides a transmembrane signaling protein comprising: (a) an extracellular binding domain comprising a scFv having the CDRs of an antibody selected from Table 1, (b) a transmembrane domain, and (c) an intracellular signaling domain. scFvsAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT Table 1 provides the targets and CDR sequences of five antibodies (designated 34, 60, 26, 49 and 58). Ab ID Target CDR SEQUENCE SEQ ID NO 34 BCANHC CDR1 LSTYWM 1

[0063] As such, in any embodiment, the scFv may be selected from the group consisting of: (i) a first scFv that binds BCAN and comprises: a VH domain comprising: a VH CDR1 of SEQ ID NO. 1;Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT a VH CDR2 of SEQ ID NO. 2; and a VH CDR3 of SEQ ID NO. 3; and a VL domain comprising: a VL CDR1 of SEQ ID NO. 4; a VL CDR2 of SEQ ID NO. 5; and a VL CDR3 of SEQ ID NO. 6; (ii) a second scFv that binds CDH10 and comprises: a VH domain comprising: a VH CDR1 of SEQ ID NO. 7; a VH CDR2 of SEQ ID NO. 8; and a VH CDR3 of SEQ ID NO. 9; and a VL domain comprising: a VL CDR1 of SEQ ID NO. 10; a VL CDR2 of SEQ ID NO. 11; and a VL CDR3 of SEQ ID NO. 12; (iii) a third scFv that binds CDH10 and comprises: a VH domain comprising: a VH CDR1 of SEQ ID NO. 13; a VH CDR2 of SEQ ID NO. 14; and a VH CDR3 of SEQ ID NO. 15; and a VL domain comprising: a VL CDR1 of SEQ ID NO. 16; a VL CDR2 of SEQ ID NO. 17; and a VL CDR3 of SEQ ID NO. 18; (iv) a fourth scFv that binds NrCAM and comprises: a VH domain comprising: a VH CDR1 of SEQ ID NO. 19; a VH CDR2 of SEQ ID NO. 20; and a VH CDR3 of SEQ ID NO. 21; and a VL domain comprising: a VL CDR1 of SEQ ID NO. 22;Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT a VL CDR2 of SEQ ID NO. 23; and a VL CDR3 of SEQ ID NO. 24 (v) a fifth scFv that binds PTPRZ and comprises: a VH domain comprising: a VH CDR1 of SEQ ID NO. 25; a VH CDR2 of SEQ ID NO. 26; and a VH CDR3 of SEQ ID NO. 27; and a VL domain comprising: a VL CDR1 of SEQ ID NO. 28; a VL CDR2 of SEQ ID NO. 29; and a VL CDR3 of SEQ ID NO. 30.

[0064] In some embodiments, the binding domain may comprise a heavy chain variable domain comprising CDR1, CDR2 and CDR3 regions that have up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions of the heavy chain variable domain of the selected antibody and / or a light chain variable domain comprising CDR1, CDR2 and CDR3 regions that are otherwise identical to the light chain CDR1, CDR2 and CDR3 regions of the selected antibody except for up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions in the collective CDR regions of the light chain variable domain of the selected antibody

[0065] In these embodiments, the scFv may comprise: a heavy chain variable domain that is at least 90% (e.g., at least 95%, at least 97%, at least 98%, at least 99% or 100%) identical to the heavy chain variable domain of the selected antibody and a light chain variable domain that is at least 90% identical (e.g., at least 95%, at least 97%, at least 98%, at least 99% or 100%) to the light chain variable domain of the selected antibody.

[0066] The sequence of the heavy and light chain variable domains of the selected antibodies are shown in Table 2. Table 2: Ab ID Target HC / LC SEQUENCE SEQ ID NOAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT VL DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQ 32 KPGKAPKLLIYSASDLYSGVPSRFSGSRSGTDFTLTISSLQ

[0067] As such, in any embodiment, the scFv may have a heavy and light chain variable domains that are independently at least at least 90% (e.g., at least 95%, at least 97%, at leastAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT 98%, at least 99% or 100%) identical to SEQ ID NOS. 31 and 32, SEQ ID NOS. 33 and 34, SEQ ID NOS. 35 and 36, SEQ ID NOS. 37 and 38, SEQ ID NOS. 39 and 40, respectively.

[0068] Heavy and light chain sequences each have three CDRs (CDR1, CDR2 and CDR3) that are flanked by framework. In some embodiments, these sequences (particularly the framework) may be humanized, i.e., modified so that it becomes more like a human antibody and therefore less immunogenic, methods for which are known.

[0069] The VH and VL sequences should be separated by a linker and can be in either order in the scFv. In some embodiments, the scFv comprise an amino acid sequence that is at least 90% identical (e.g., at least 95%, at least 97%, at least 98%, at least 99% or 100%) to any of SEQ ID NOs. 41-45, which are shown in Table 3 below. The underlined sequence in Table 3 is the linker sequence. Table 3: Ab ID Target SEQUENCE SEQ ID NOAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT IYSSGGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCARFGYRSSPSFALDYWGQGTLVTVSS [0070g g p y p s capable of transmitting a signal from the outside of the cell to the inside of the cell. In other words, binding of the extracellular binding domain to BCAN, CDH10, PTPRZ1 or NrCAM in the brain (which antigens may be on another cell or in the extracellular matrix, for example) generates a signal in the inside of the cell. In some embodiments, the transmembrane signaling protein may be a binding-triggered transcriptional switch (BTTS), an engineered immune receptor such as a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR), or a synCAM, the details of which are described in greater detail below. Binding-triggered transcriptional switches (BTTSs) A “binding-triggered transcriptional switch” or “BTTS” is a polypeptide or complex of the same that is capable of transducing a specific binding event on the outside of the cell (by binding of an extracellular domain of the BTTS to an antigen on another cell or extracellular matrix) that activates a recombinant promoter within the nucleus of the cell. Many BTTSs work by releasing a transcription factor that activates the promoter. In these embodiments, the BTTS is made up of one or more polypeptides that undergo proteolytic cleavage upon binding to the antigen to release a gene expression regulator that activates the recombinant promoter. For example, a BTTS may comprise (i) an extracellular domain comprising the antigen binding region of an antigen-specific antibody; (ii) a proteolytically cleavable sequence comprising one or more proteolytic cleavage sites; and (iii) an intracellular domain, wherein binding of the antigen binding region to the antigen induces cleavage of the sequence at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain and wherein the intracellular domain activates transcription of an expression cassette that encodes the immunosuppressive protein, growth factor and / or the anti-fibrotic agent. A BTTS can be basedAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT on Notch, A2, MESA, SNIPR or a force receptor, for example, although others are known (e.g., see Zhu et al 2022 Cell. 185: 1431-1443 and WO2021061856). In any embodiment, the BTTS may be a cleavable fusion protein that contains: (a) an extracellular binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising a transcriptional activator, where binding of the binding domain to an antigen in the brain (which may be on the surface of another cell or in the extracellular matrix) induces proteolytic cleavage of the BTTS to release the transcriptional activator that, in turn, induces the expression of a payload in the brain. In some embodiments, the BTTs may additionally contain an extracellular force sensing region between regions (a) and (b) and (d) one or more force- dependent cleavage sites in (c) that are cleaved when the force sensing region is activated. In this molecular switch, the fusion protein is cleaved to release the intracellular domain when the extracellular domain of the fusion protein engages with its cognate antigen. In some cases, the fusion protein may contain a force sensing region (which is typically in the extracellular domain) and one or more force-dependent cleavage sites that are cleaved when the force sensing region is activated. The position of the force-dependent cleavage sites may vary and, in some embodiments the fusion protein may contain at least two cleavage sites. In some cases, one of the cleavage sites may be extracellular and the other may be in the transmembrane domain or within 10 amino acids of the transmembrane domain in the intracellular domain. In any embodiment, the force sensing region and / or the one or more force-dependent cleavage sites may be from a Delta / Serrate / Lag2 (DSL) superfamily protein, as reviewed by Pintar et al (Biology Direct 20072: 1-13). For example, the force sensing region and / or the one or more force-dependent cleavage sites may be from Notch (see Morsut Cell. 2016164: 780-91), von Willebrand Factor (vWF), amyloid-beta, CD16, CD44 , Delta, a cadherin , an ephrin-type receptor or ephrin ligand, a protocadherin, a filamin, a synthetic E cadherin, interleukin-1 receptor type 2 (IL1R2), major prion protein (PrP), a neuregulin or an adhesion-GPCR. Several other examples of this type of protein are known and listed in Pintar, supra. Many members of this family appear to share a similar architecture a region that unfolds and opens up a protease cleavage site (e.g., EGF-like repeats; see Cordle et al Nat. Struct. Mol. Biol.200815: 849–857), a trans-membrane segment, and a relatively short (~100–150 amino acids) intracellular domain. These sequences permit the binding-triggered release of a transcriptional activator from the membrane in their natural environment and can be readily adapted herein.Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT In some cases, the one or more ligand-inducible proteolytic cleavage sites are selected from S1, S2, and S3 proteolytic cleavage sites. In some cases, the S1 proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid. In some cases, the S2 proteolytic cleavage site ADAM-17-type protease cleavage site comprising an Ala-Val dipeptide sequence. In some cases, the S3 proteolytic cleavage site is a γ-secretase cleavage site comprising a Gly-Val dipeptide sequence. The S3 proteolytic cleavage site is in the transmembrane domain. In many cases, the shear force generated by binding of the extracellular domain of this fusion protein to another cells unfolds the force sensing region (which, in the case of Notch contains EGF-like repeats whereas in other protein is made up of other sequences such as the A2 domain in vWF (see, e.g., J Thromb Haemost. 20097:2096-105, Lippok Biophys J. 2016110: 545-54, Lynch Blood. 2014123: 2585-92, Crawley, Blood. 2011118:3212-21 and Xy J Biol Chem. 2013288:6317-24) or modified A2 domain that has, e.g., the R1597W, E1638K and I1628T substitutions. The architecture of such proteins is described in, e.g., Morsut et al, Cell. 2016164: 780- 91, WO2016138034 and WO2019099689, among other places). In some cases, the fusion protein includes an S1 ligand-inducible proteolytic cleavage site. An S1 ligand-inducible proteolytic cleavage site can be located between the HD-N segment and the HD-C segment. In some cases, the S1 ligand-inducible proteolytic cleavage site is a furin-like protease cleavage site. A furin-like protease cleavage site can have the canonical sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid; the protease cleaves immediately C-terminal to the canonical sequence. For example, in some cases, an amino acid sequence comprising an S1 ligand-inducible proteolytic cleavage site can have the amino acid sequence GRRRRELDPM (SEQ ID NO:46), where cleavage occurs between the “RE” sequence. As another example, an amino acid sequence comprising an S1 ligand-inducible proteolytic cleavage site can have the amino acid sequence RQRRELDPM (SEQ ID NO:47), where cleavage occurs between the “RE” sequence. In some cases, the fusion protein polypeptide includes an S2 ligand-inducible proteolytic cleavage site. An S2 ligand-inducible proteolytic cleavage site can be located within the HD-C segment. In some cases, the S2 ligand-inducible proteolytic cleavage site is an ADAM-17-type protease cleavage site. An ADAM-17-type protease cleavage site can comprise an Ala-Val dipeptide sequence, where the enzyme cleaves between the Ala and the Val. For example, inAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT some cases, amino acid sequence comprising an S2 ligand-inducible proteolytic cleavage site can have the amino acid sequence KIEAVKSE (SEQ ID NO:48), where cleavage occurs between the “AV” sequence. As another example, an amino acid sequence comprising an S2 ligand-inducible proteolytic cleavage site can have the amino acid sequence KIEAVQSE (SEQ ID NO:49), where cleavage occurs between the “AV” sequence. In some cases, the fusion protein includes an S3 ligand-inducible proteolytic cleavage site. An S3 ligand-inducible proteolytic cleavage site can be located within the TM domain. In some cases, the S3 ligand-inducible proteolytic cleavage site is a gamma-secretase (γ-secretase) cleavage site. A γ-secretase cleavage site can comprise a Gly-Val dipeptide sequence, where the enzyme cleaves between the Gly and the Val. For example, in some cases, an S3 ligand- inducible proteolytic cleavage site has the amino acid sequence VGCGVLLS (SEQ ID NO:50), where cleavage occurs between the “GV” sequence. In some cases, an S3 ligand-inducible proteolytic cleavage site comprises the amino acid sequence GCGVLLS (SEQ ID NO:51). In some cases, the fusion protein polypeptide lacks an S1 ligand-inducible proteolytic cleavage site. In some cases, the BTTS lacks an S2 ligand-inducible proteolytic cleavage site. In some cases, the BTTS lacks an S3 ligand-inducible proteolytic cleavage site. In some cases, the BTTS lacks both an S1 ligand-inducible proteolytic cleavage site and an S2 ligand-inducible proteolytic cleavage site. In some cases, the BTTS includes an S3 ligand-inducible proteolytic cleavage site; and lacks both an S1 ligand-inducible proteolytic cleavage site and an S2 ligand- inducible proteolytic cleavage site. In some embodiments, the fusion protein may have an vWF A2 sequence or a variation thereof, an ADAMTS13 cleavage site (which may be described by the consensus sequence HEXXHXXGXXHD (SEQ ID NO: 52); Crawley, Blood. 2011118:3212-21), and an S3 or γ- secretase cleavage site, although many other arrangements exist. In some embodiments, the switch may contain components that are borrowed from Notch. In other embodiments, the switch may not contain components that are from Notch. For simplicity, BTTSs, including but not limited to chimeric notch receptor polypeptides, are primarily single polypeptide chains. However, BTTSs, including chimeric notch receptor polypeptides, may be divided or split across two or more separate polypeptide chains where the joining of the two or more polypeptide chains to form a functional BTTS, e.g., a chimeric notch receptor polypeptide, may be constitutive or conditionally controlled. ForAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT example, constitutive joining of two portions of a split BTTS may be achieved by inserting a constitutive heterodimerization domain between the first and second portions of the split polypeptide such that upon heterodimerization the split portions are functionally joined. Useful BTTSs that may be employed in the subject methods include, but are not limited to modular extracellular sensor architecture (MESA) polypeptides. A MESA polypeptide comprises: a) a ligand binding domain; b) a transmembrane domain; c) a protease cleavage site; and d) a functional domain. The functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor). In some cases, a MESA receptor comprises two polypeptide chains. In some cases, a MESA receptor comprises a single polypeptide chain. Non-limiting examples of MESA polypeptides are described in, e.g., U.S. Patent Publication No. 2014 / 0234851; the disclosure of which is incorporated herein by reference in its entirety. Useful BTTSs that may be employed in the subject methods include, but are not limited to polypeptides employed in the TANGO assay. The subject TANGO assay employs a TANGO polypeptide that is a heterodimer in which a first polypeptide comprises a tobacco etch virus (Tev) protease and a second polypeptide comprises a Tev proteolytic cleavage site (PCS) fused to a transcription factor. When the two polypeptides are in proximity to one another, which proximity is mediated by a native protein-protein interaction, Tev cleaves the PCS to release the transcription factor. Non-limiting examples of TANGO polypeptides are described in, e.g., Barnea et al. (Proc Natl Acad Sci USA. 2008 Jan. 8; 105(1):64-9); the disclosure of which is incorporated herein by reference in its entirety. Useful BTTSs that may be employed in the subject methods include, but are not limited to von Willebrand Factor (vWF) cleavage domain-based BTTSs, such as but not limited to e.g., those containing a unmodified or modified vWF A2 domain. A subject vWF cleavage domain- based BTTS will generally include: an extracellular domain comprising a first member of a binding pair; a von Willebrand Factor (vWF) cleavage domain comprising a proteolytic cleavage site; a cleavable transmembrane domain and an intracellular domain. Non-limiting examples of vWF cleavage domains and vWF cleavage domain-based BTTSs are described in Langridge & Struhl (Cell (2017) 171(6):1383-1396); the disclosure of which is incorporated herein by reference in its entirety. Useful BTTSs that may be employed in the subject methods include, but are not limited to chimeric Notch receptor polypeptides, such as but not limited to e.g., synNotch polypeptides,Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT non-limiting examples of which are described in PCT Pub. No. WO 2016 / 138034, U.S. Patent No. 9,670,281, U.S. Patent No.9,834,608, Roybal et al. Cell (2016) 167(2):419-432, Roybal et al. Cell (2016) 164(4):770-9, and Morsut et al. Cell (2016) 164(4):780-91; the disclosures of which are incorporated herein by reference in their entirety. The "SNIPR" switch is another example of a BTTS (see Zhu et al 2022 Cell. 185: 1431-1443 and WO2021061856), although others exist (see, e.g., WO2019099689) and / or can be readily designed. Expression of the BTTS in the cell may be constitutive or inducible, e.g., by binding of another BTTS to an antigen on another cell. As such, in some embodiments, the second promoter may be constitutive in the cell. For example, the second promoter may be a CMV, EF-1, hPGK or RPBSA promoter, although many other choices are available.In any embodiment, the BTTS comprises: i. an extracellular binding domain, ii. a force sensing region, iii. a transmembrane domain, iv. one or more force-dependent cleavage sites that are cleaved when the force sensing region is activated, and v. an intracellular domain comprising a transcriptional activator, where binding of the extracellular binding domain to the antigen induces proteolytic cleavage of the one or more force-dependent cleavage sites to release the transcriptional activator, and wherein the released transcriptional activator induces transcription of the first coding sequence which, in turn, results in expression of the payload. Examples of transcriptional activators that can be part of the fusion protein are numerous and include artificial transcription factors (ATFs) such as, e.g., Zinc-finger-based artificial transcription factors (including e.g., those described in Sera T. Adv Drug Deliv Rev. 200961(7- 8):513-26; Collins et al. Curr Opin Biotechnol. 200314(4):371-8; Onori et al. BMC Mol Biol. 201314:3. In some cases, the transcriptional activator may contain a GAL4 DNA binding domain, which binds to the Gal4 responsive UAS, which has been well characterized in the art. Examples of suitable transcriptional activators include GAL4-VP16 and GAL4-VP64, although many others could be used. As would be appreciated, the identity of the transcription activators may vary. In some embodiments, the transcription factor may have a DNA binding domain that binds to a corresponding promoter sequence and an activation domain. In many embodiments, the DNA binding domain transcription factor may be independently selected from Gal4-, LexA- , Tet-, Lac-, dCas9-, zinc-finger- and TALE-based transcription factors. TALE- and CRISPR / dCas9-based transcription factors are described in Lebar (Methods Mol Biol. 2018 1772: 191-203), among others. The binding sites for such domains are well known or can beAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT designed at will. The transcriptional activator can have any suitable activation domain, e.g., VP16, VP64, Ela, Sp1, VP16, CTF, GAL4 among many others. In these embodiments, the regulatory sequence to which the transcription activator binds should contain one or more copies of a binding site for the transcriptional activator, e.g., a GAL4 UAS. Engineered immune receptors In some embodiments, the transmembrane signaling protein may be an engineered immune receptor, e.g., a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR) (including an HLA Independent TCR (HIT)). CAR-Tregs and TCR-Tregs are described in a number of publications, including: Arjomandnejad et al (Biomedicines 202210: 287), Skuljec et al (Front Immunol. 20178: 1125) and Proics et al (Gene Therapy 202330: 309–322). In these embodiments, binding of the immune receptor to its antigen activates the Treg cell and, upon activation of a CAR-Treg and TCR-Treg, the cell dampens immune responses locally by secreting immune suppressive cytokines such as TGF-β, IL-10 or IL-35 and / or by interacting with other cells such as dendritic cells. In these embodiments, the receptor may be a chimeric antigen receptor (CAR), where the terms “chimeric antigen receptor” and “CAR”, used interchangeably herein, refer to artificial multi-module molecules capable of triggering the activation of an immune cell which generally but not exclusively comprise an extracellular domain (e.g., a ligand / antigen binding domain), a transmembrane domain and one or more intracellular signaling domains. Such cells may by generated by transduction of polyclonal Tregs with CAR construct or cotransduction of T cells with CAR construct and forkhead box P3 (FoxP3) gene, for example. The principles for designing CARs for Tregs is similar to the principle for designing CARs for cytotoxic T cells, which are described below. synCAMs Engineered cell adhesion molecules (synCAMs) are described in WO2022094416 and Stevens et al (Nature 2023614: 144–152), which are incorporated by reference herein for disclosure of such molecules. Engineered cell adhesion molecules generally comprises three domains: an extracellular binding domain, one or more transmembrane domains and an intracellular domain, where the wherein the extracellular binding domain and the intracellular binding domain of the fusion protein are “heterologous” i.e., not from the same native cell adhesion molecule. For example, the extracellular binding domain may comprise the antigen- binding domain of an antibody or a dimerization domain, for example. In some cases, the targetAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT for the engineered cell adhesion molecule on the surface another cell may be endogenous to the cell or added to the other cell by recombinant means. The intracellular domain of the engineered cell adhesion molecule, on the other hand, may be the intracellular domain of a naturally-occurring cell adhesion molecule or a variant thereof that retains its ability to engage with and apply local control over the cytoskeleton, e.g., to reinforce the linkage and / or locally control cytoskeletal filament polymerization. Thus, the intracellular domain of the cell adhesion molecule is capable of signaling to and reorganizing the cytoskeleton of the cell upon specific binding of the first binding moiety of the cell adhesion molecule to a second binding moiety. Because signaling of many cell adhesion molecules is triggered by physical force (e.g., a force that “tugs” at the molecule), the second binding moiety that activates signaling should be at least partially immobilized or tethered, i.e., not in solution. For example, the second binding moiety that triggers signaling may be on the surface of another cell or tissue scaffold, or tethered to another cell or tissue scaffold, for example. An engineered cell adhesion molecule also contains one or more transmembrane domains (which should be in between the extracellular and intracellular domains). In some embodiments, e.g., the engineered cell adhesion molecule may have a single transmembrane domain. In other embodiments, the engineered cell adhesion molecule may have multiple transmembrane domains. The transmembrane domain of an engineered cell adhesion molecule can be the transmembrane domain of a naturally-occurring cell adhesion molecule (e.g., the same naturally-occurring cell adhesion molecule as the intracellular domain). However, this is not necessary because the transmembrane domain can be readily designed using hydrophobic amino acids or a transmembrane domain from another transmembrane protein can be used. As would be apparent, the fusion protein may have other sequences, e.g., linkers, effector domains, signaling domains, etc., in addition to the domains that are specifically described herein. In addition to embodiments in which the cells adhere to each other directly, the cells may also adhere to each other indirectly via a soluble bridging molecule, e.g., a soluble protein, to which two engineered cell adhesion molecules bind. In these embodiments, the engineered cell adhesion molecules on the different cells may bind to different sites (e.g., epitopes) on the soluble bridging molecule. How cell adhesion proteins engage with and apply local control over the cytoskeleton, e.g., to reinforce the linkage and / or and recruit additional cytoskeleton to the area has been wellAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT studied. For example, Harburger et al (Journal of Cell Science 2009122: 159-163) provides a detailed review of the mechanism by which integrins are able to signal from extracellular binding to cytoskeletal reorganization; Liu et al (Journal of Cell Science 2000113: 3563-3571) describes the structure / function relationship of integrins, particularly details the known binding partners of the intracellular sequence of integrins; Hoffman et al (Quant. Cell Biology 201525: 803-814) describes the mechanism of cadherin intracellular signaling and how it is mechanically regulated; Beutel et al (Cell 2019179: 923-936) describes the phase-separation-based mechanism by which JAMs are able to recruit the scaffold protein ZO-1 and signal; and Lawson et al (Pharmacol Rep. 200961: 22-32) describes the intracellular signaling pathways that are activated by ICAM signaling. It is noted that some cadherin / integrin and protocadherin / e- cadherin chimeras retain their ability to signal (Geiger et al J. Cell Science 1992103, 943-951 and Obato et al J. Cell Science 1995108: 3765-3773). In any embodiment, the intracellular domain may be selected from Table 1 of WO2022094416, where the amino acid sequence of the intracellular domain may be at least 80%, at least 90%, at least 95% or identical to a human sequence found in that table. In these embodiments, the intracellular domain may be an intracellular domain of a cell adhesion molecule selected from Table 1 of WO2022094416, or a variant thereof that retains the ability to engage with the cytoskeleton. A nucleic acid encoding the transmembrane signaling protein as described above, is also provided, as well as a cell (e.g., an immune cell or cell) comprising the same. The recombinant nucleic acid may be in a vector, such as a viral vector, e.g., an adenoviral vector, a retroviral vector, a lentiviral vector, an adeno-associated virus vector, an herpes simplex virus vector, etc. Replication-defective viruses can also be advantageous. Some vectors become incorporated into the nuclear genome of the host cell, whereas others do not. The use of viral vectors (particularly retroviral and lentiviral vectors) for expressing CARs in immune cells is known (see, e.g., Lanne et al Viruses. 202113: 1528) and Moco et al Methods Mol Biol 20202086: 69-76 among many others). Cells and circuits A cell comprising a nucleic acid encoding the transmembrane signaling protein is also provided. In some embodiments, the transmembrane signaling protein is a BTTS and the cell further comprises: a coding sequence for a therapeutic protein (which will be referred to as aAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT “payload” below) and a regulatory sequence operably linked to the coding sequence. In these embodiments, the transcriptional activator released by the BTTS binds to the regulatory sequence and induces expression of the therapeutic protein. This arrangement may be referred to as a “circuit), where the BTTs induces a payload when it engages with a cognate antigen. Payloads Payloads (i.e., the therapeutic protein) may be secreted, intracellular or localized on the cell surface. For example, the payload may be a secreted peptide (e.g., a growth factor, pro- inflammatory cytokine, a cytokine sink, an anti-inflammatory cytokine), a secreted enzyme or secreted antibody. In other embodiments, the therapeutic protein may be intracellular (e.g., a transcription factor or enzyme). In other embodiments, the payload may be a cell surface protein (e.g., a cytokine sink or receptor such as a CAR, TCR, or another BTTS for example). In some embodiments, the payload may be an anti-cancer therapeutic. In these cases the payload may be a secreted protein (e.g., a pro-inflammatory cytokine or an antibody such as a multi-specific antibody or an immune checkpoint inhibitor for example) or a protein that is localized to the surface of the cell (e.g., may be a recombinant immune receptor such as a CAR or engineered T cell receptor). In some embodiments, activation of the BTTS may induce expression of both a secreted protein and an immune receptor (e.g., by way of a divergent promoter or by using an IRES, for example). If the anti-cancer therapeutic is not a recombinant immune receptor then the cell may further comprise a recombinant immune receptor e.g., a CAR or engineered TCR, that is activated by binding to a killing antigen expressed by the cancer cells. In these latter embodiments, the recombinant immune receptor may be inducibly expressed, or constitutive. In any embodiment, the antibody or immune receptor may bind to a killing antigen expressed by the cancer cells. Engineered immune receptors In some embodiments, the payload may be an engineered immune receptor, e.g., a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR) (including an HLA Independent TCR (HIT)). CARs can be designed in several ways (see, generally, e.g., Guedan et al, Methods and Clinical Development 201912: 145-156) and may include an extracellular domain that contains an antigen binding domain such as a scFv or nanobody, a hinge, a transmembrane region (which may be derived from CD4, CD8α, or CD28), a costimulatory signaling domain (which may beAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT derived from the intracellular domains of the CD28 family (e.g., CD28 and ICOS) CD2 (US9783591B2) or the tumor necrosis factor receptor (TNFR) family of genes (e.g., 4-1BB, OX40, or CD27)), and an ITAM domain, e.g., the signaling domain from the zeta chain of the human CD3 complex (CD3zeta). In practice, any of these domains may be a variation of a wild type sequence. In practice, any of these sequences may be a variant of a wild type sequence, e.g., a sequence that is at least 90%, 95%, or 98% identical to a sequence described in WO2014127261, for example. For example, a CAR may have a signaling domain from CD3ζ in which two of the three ITAM motifs (the second and third ITAM motifs) have been altered to be non-functional. More specifically, both tyrosine (Y) phosphorylation sites in the second and third ITAMs may be substituted by phenylalanine, thereby rendering those sites incapable of being phosphorylated. This altered CD3ζ signaling domain is described in Feucht et al (Nat Med. 201925: 82-88). In any embodiment, the car may have signaling from CD3ζ, CD28 and 4-1BB. The term CAR is not limited specifically to CAR molecules but also includes CAR variants. CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero- dimerization of the two portions of the split CAR is pharmacologically controlled (e.g., as described in PCT publication no. WO 2014 / 127261 A1 and US Patent Application No. 2015 / 0368342 A1, the disclosures of which are incorporated herein by reference in their entirety). CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013) ;5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98;Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Useful CARs also include the anti-CD19—4-1BB—CD3ζ CAR expressed by lentivirus loaded CTL019 (Tisagenlecleucel-T) CAR-T cells as commercialized by Novartis (Basel, Switzerland). The terms “T cell receptor” and “TCR” are used interchangeably and will generally refer to a molecule found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR complex is a disulfide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (α) and beta (β) chains expressed as part of a complex with CD3 chain molecules. Many native TCRs exist in heterodimeric αβ or γδ forms. The complete endogenous TCR complex in heterodimeric αβ form includes eight chains, namely an alpha chain (referred to herein as TCRα or TCR alpha), beta chain (referred to herein as TCRβ or TCR beta), delta chain, gamma chain, two epsilon chains and two zeta chains. In some instances, a TCR is generally referred to by reference to only the TCRα and TCRβ chains, however, as the assembled TCR complex may associate with endogenous delta, gamma, epsilon and / or zeta chains an ordinary skilled artisan will readily understand that reference to a TCR as present in a cell membrane may include reference to the fully or partially assembled TCR complex as appropriate. Recombinant or engineered individual TCR chains and TCR complexes have been developed. References to the use of a TCR in a therapeutic context may refer to individual recombinant TCR chains. As such, engineered TCRs may include individual modified TCRα or modified TCRβ chains as well as single chain TCRs that include modified and / or unmodified TCRα and TCRβ chains that are joined into a single polypeptide by way of a linking polypeptide Any engineered TCR having immune cell activation function can be induced using a method of the present disclosure. Such TCRs include, e.g., antigen-specific TCRs, Monoclonal TCRs (MTCRs), Single chain MTCRs, High Affinity CDR2 Mutant TCRs, CD1-binding MTCRs, High Affinity NY-ESO TCRs, VYG HLA-A24 Telomerase TCRs, including e.g., those described in PCT Pub Nos. WO 2003 / 020763, WO 2004 / 033685, WO 2004 / 044004, WO 2005 / 114215, WO 2006 / 000830, WO 2008 / 038002, WO 2008 / 039818, WO 2004 / 074322, WO 2005 / 113595, WO 2006 / 125962; Strommes et al. Immunol Rev. 2014; 257(1):145-64; SchmittAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT et al. Blood. 2013; 122(3):348-56; Chapuls et al. Sci Transl Med. 2013; 5(174):174ra27; Thaxton et al. Hum Vaccin Immunother. 2014; 10(11):3313-21 (PMID:25483644); Gschweng et al. Immunol Rev. 2014; 257(1):237-49 (PMID:24329801); Hinrichs et al. Immunol Rev. 2014; 257(1):56-71 (PMID:24329789); Zoete et al. Front Immunol. 2013; 4:268 (PMID:24062738); Marr et al. Clin Exp Immunol. 2012; 167(2):216-25 (PMID:22235997); Zhang et al. Adv Drug Deliv Rev. 2012; 64(8):756-62 (PMID:22178904); Chhabra et al. Scientific World Journal. 2011; 11:121-9 (PMID:21218269); Boulter et al. Clin Exp Immunol. 2005; 142(3):454-60 (PMID:16297157); Sami et al. Protein Eng Des Sel. 2007; 20(8):397-403; Boulter et al. Protein Eng. 2003; 16(9):707-11; Ashfield et al. IDrugs. 2006; 9(8):554-9; Li et al. Nat Biotechnol. 2005; 23(3):349-54; Dunn et al. Protein Sci. 2006; 15(4):710-21; Liddy et al. Mol Biotechnol. 2010; 45(2); Liddy et al. Nat Med. 2012; 18(6):980-7; Oates, et al. Oncoimmunology. 2013; 2(2):e22891; McCormack, et al. Cancer Immunol Immunother. 2013 Apr;62(4):773-85; Bossi et al. Cancer Immunol Immunother. 2014; 63(5):437-48 and Oates, et al. Mol Immunol. 2015 Oct;67(2 Pt A):67-74; the disclosures of which are incorporated herein by reference in their entirety. HLA-Independent TCRs (Eyquem et al. 2022 Feb;28(2):345-352) are also included in this definition. Pro-inflammatory cytokines Pro-inflammatory cytokines are secreted from the cell. In these embodiments, the circuit may comprise a nucleic acid containing a promoter that is activated by the released transcriptional activator, and a coding sequence encoding a pro-inflammatory protein. In this disclosure, the term “pro-inflammatory cytokine” is intended to encompass any cytokine that have a pro-inflammatory activity (e.g., IL-2 , CCL-21, IL-12, IL-7, IL-15 and IL-21, etc.), as well as non-natural or “engineered” cytokines that have pro-inflammatory activity such as super IL-2 (see, e.g., Levin et al Nature 2012484: 529–533, which has the following amino acid substitutions L80F, R81D, L85V, I86V, and I92F relative to wild type), mini-TGF-Beta (which blocks TGF-Beta signaling) and DR-18 (an IL-18 variant), etc.). Engineered cytokines include superkines, which often have up to 10 amino acid substitutes relative to a natural cytokine, as well as natural cytokines that have been truncated, and dominant variants. Cytokines of interest include selected from IL-2, IL-12, IL-15, IL-7, CD40L, or a non-natural variant of IL-2, IL-12, IL-15, IL-7, CD40L that has pro-inflammatory activity. CytokinesAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT include "ortho" cytokines that can be paired with a receptor in the immune cell (see, e.g., Sockolosky et al. 2018). Sources for exemplary pro-inflammatory proteins are listed below. Payload Exemplary sources of payload sequences IL2 NCBI Gene ID: 3558 2.As would be appreciated, pro-inflammatory proteins are secreted from the cell and their coding sequence will encode a secretion signal. In some embodiments the immune cell may additionally express a recombinant receptor for the pro-inflammatory protein, which may enhances the immune cell’s response. For example, if the pro-inflammatory protein is an “ortho2”, then the immune cell may additionally express a receptor for that pro-inflammatory protein. Immune checkpoint inhibitors Immune checkpoint inhibitors are also secreted from the cell. These molecules block interactions with PD1, CTLA4, BTLA, CD160, KRLG-1, 2B4, Lag-3, Tim-3 and other immune checkpoints. See, e.g., Odorizzi and Wherry (2012) J. Immunol. 188:2957; and Baitsch et al. (2012) PLoSOne 7: e30852. Exemplary immune checkpoint inhibitors include antibodies to CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. Many examples of such antibodies are known in the art. For example, anti-PD1 antibodies include Pembrolizumab, Nivolumab, Cemiplimab, Dostarlimab, JTX-4014, Spartalizumab,Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT Camrelizumab, Sintilimab, Tislelizumab, Toripalimab, INCMGA00012, AMP-224, and AMP- 514. Anti-PDL1 antibodies include Atezolizumab, Avelumab, Durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189. Multi-specific antibodies In some embodiments, activation of the BTTS may induce expression of a multi-specific antibody. While such an antibody may perform other roles in killing cancer cells, in some embodiments, the antibody may be a monoclonal antibody that stimulates ADCC, or an NK cell engager (NKCE), such as a BiKE (bispecific killer cell engager) or TriKE (trispecific killer cell engager). BiKEs and TriKEs are reviewed in Felices et al (Methods Mol Biol. 2016; 1441: 333– 346). Such molecules tether NK cells to a tumor cell and induce their activation at that site. BiKEs and TriKEs are molecules that contain a single variable portion of an antibody linked to one (BiKE) or two (TriKE) variable portions from other antibodies of different specificity. See, e.g., Shanshal et al (Cancers (Basel). 202315: 2824) Multispecific antibodies can be in a variety of different formats, including, but not limited to IgG-like antibody formats (including an Fc domain) and non-IgG-like antibody formats (without an Fc domain). Multispecific antibodies with IgG-like antibody formats can be in a variety of different formats, including, but not limited to knob-into-hole (KIH), TrioMab, Duobody, κλ body, CrossMab, common light chain, strand exchange engineered domain bodies (SEEDBodies), Azymetric heterodimeric Fc, dual action Fab (DAF), dual-variable-domain immunoglobulin (DVD-Ig), IgG-scFv, Fab-Fab-Fc, DutaMab, and DutaFab. Non-IgG-like antibody formats may lack an Fc region entirely. For example, Fab, Fv and VHH antibody regions may be genetically engineered and combined in various orientations and pairings. Multispecific antibodies in non-IgG-like formats can be in a variety of different formats, including, but not limited to bivalent dual-affinity re-targeting protein (DART), tetravalent DART, half-life extended bispecific T-cell engager (HLE-BiTE), bispecific T-cell engager (BiTE), immune mobilizing monoclonal T-cell receptor (ImmTAC), tandem diabody (TandAb), bispecific killer cell engager (BiKE), trispecific killer cell engager (TRiKE), multispecific scFV single-chain variable fragment, trispecific T-cell activation construct (TriTAC), bispecific nanobody, and cross-over dual variable region (CODV). Cytokine sinksAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT In some embodiments, the payload may be an immunosuppressive protein, e.g., a “sink” for a pro-inflammatory cytokine or, alternatively, an anti-inflammatory cytokine, Cytokine sinks include a protein that binds to a pro-inflammatory cytokine (e.g., INF-γ, IL-1, IL-2, IL-6, IL-8, IL-10, IL-18, TNF-α, MCP-1, GM-CSF) and prevents it from interacting with its cognate receptor on other cells. A pro-inflammatory cytokine sink may be based on the natural receptor for the pro-inflammatory cytokine (e.g., the receptors for INF-γ, IL-1, IL-2, IL-6, IL-8, IL-10, IL-18, TNF-α, MCP-1, GM-CSF). In these embodiments, the ligand binding domain of the receptor, e.g., the extracellular domain (in some embodiments without the intracellular signaling domain) may be expressed on the surface of the cell. In these embodiments, the cell itself binds to the pro-inflammatory cytokine and prevents it from binding to its receptor on other cells. In other embodiments, the cell may secrete a soluble form of the receptor, which binds to the pro- inflammatory cytokine in solution. For example, in one embodiment the sink may contain at least the extracellular domain of CD25 (which is the receptor for IL-2), although others could be used too. In alternative embodiments, an antibody (e.g., a scFv) that binds to the pro-inflammatory cytokine may be used. In these embodiments, the antibody may be tethered to the cell, e.g., via a transmembrane domain, or secreted. Anti-inflammatory cytokine In some embodiments, the payload induced by the BTTS may be an anti-inflammatory cytokine, which will be secreted from the cell. In these embodiments, the circuit may comprise a nucleic acid containing a promoter that is activated by the released transcriptional activator, and a coding sequence encoding an anti-inflammatory cytokine. In this disclosure, the term “anti- inflammatory cytokine” is intended to encompass natural cytokines that have anti-inflammatory activity (e.g., Il-1ra, IL-4, IL-10, IL-11, IL-13, TGFβ, etc.), as well as non-natural or “engineered” cytokines that have anti-inflammatory activity. As would be appreciated, cytokines are secreted from the cell and therefore require a secretion signal. Growth factors In alternative embodiments, the payload may be growth factor, i.e., a molecule that is capable of stimulating cell proliferation and / or wound healing. Growth factors of interest include KGF, HGF, BMP4, WNT, PDGF, VEGF, EGF family (such as EGF and TGF-α), IGF family (such as IGF), FGF family (such as bFGF and KGF), TGF-β family (e.g., any of TGF-Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT β1‒3) and GM-CSF, many of which have been implicated in tissue repair and / or regeneration (see, e.g., Desai (Respir Res. 20023: 2) and Chu (Frontiers in Medicine 202014: 262–272) among many others. In any embodiments, the payload may be a protease inhibitor, enzyme (which may be intracellular or secreted) or a transcription factor (which may or may not alter cell fate). In any embodiment, the BTTS may activate expression of more than one payloads via an divergent promoter or IRES, for example. In any embodiment, the therapeutic protein may be a chimeric antigen receptor (CAR). In these embodiments, the CAR may be a tandem chimeric antigen receptor (CAR) that has a first binding domain that recognizes to Ephrin type-A receptor 2 (EphA2) and a second binding domain that recognizes Interleukin-13 receptor subunit alpha-2 (IL13RA2). In any embodiment, the therapeutic protein may be an antigen-specific therapeutic that recognizes a cancer-specific antigen in the brain. Such antigens include, but are not limited to: EGFRvIII, Ephrin type-A receptor 2 (EphA2), Ephrin type-A receptor 3 (EphA3), Interleukin- 13 receptor subunit alpha-1 (IL13RA1), Interleukin-13 receptor subunit alpha-2 (IL13RA2), Epidermal growth factor receptor (EGFR), erb-b2 receptor tyrosine kinase 2 (ERBB2), and B7- H3, etc. Other antigens may be targeted, depending on the nature of the tumor (e.g., whether it has metastacized, etc.) For example, if the therapeutic protein is a CAR, then the CAR may recognize a cancer-selective antigen. Host cells The host cell may be an immune cell or stem cell. For example, the cell may be a CD8+ (cytotoxic) T cell, a regulatory T cell (T reg), CD4+ T cell, a macrophage, an NK cell or a somatic cell such as a stem cell, or a mesenchymal stromal cell. However, they can be used in certain circumstances. The present cells include cells that are genetically modified to produce the components of the present disclosure or to which a nucleic acid, as described above, has been otherwise introduced. In some instances, the subject cells have been transduced with one or more nucleic acids and / or expression vectors to express one or more components of a circuit of the present disclosure. Suitable mammalian immune cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodentAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT (e.g., mouse, rat) cell lines, and the like. In some instances, the cell is not an immortalized cell line, but is instead a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell, immune cell progenitor or immune stem cell obtained from an individual. As an example, the cell is a lymphoid cell, e.g., a lymphocyte, or progenitor thereof, obtained from an individual. Cells encoding a circuit of the present disclosure may be generated by any convenient method. Nucleic acids encoding one or more components of a subject circuit may be stably or transiently introduced into the subject immune cell, including where the subject nucleic acids are present only temporarily, maintained extrachromosomally, or integrated into the host genome. Introduction of the subject nucleic acids and / or genetic modification of the subject immune cell can be carried out in vivo, in vitro, or ex vivo. In some cases, the introduction of the subject nucleic acids and / or genetic modification may be carried out ex vivo. For example, a primary T lymphocyte may be obtained from an individual; and the cell obtained from the individual is modified to express components of a circuit of the present disclosure. Other embodiments may use “off the shelf”, allogeneic cells. If the payload recognizes a cancer cell and the cell is cytotoxic, the cell may be a cytotoxic T cell, for example. If the transmembrane signaling protein itself is a CAR or TCR, then the cell may be CAR-Treg and TCR-Treg, where the cell dampens immune responses locally by secreting immune suppressive cytokines such as TGF-β, IL-10 or IL-35 and / or by interacting with other cells such as dendritic cells. Such cells may by generated by transduction of polyclonal Tregs with CAR construct or cotransduction of T cells with CAR construct and forkhead box P3 (FoxP3) gene, for example. Populations of cells A population of the cells is also provided. In some embodiments, these cells may be present in vitro and may be progenitors of primary cells that have been genetically modified to contain the present circuit, as described above. As noted above, some cells may be genetically modified to be allogeneic in a human host. In these embodiments, the cells may be frozen. The population may comprise any number of the cells (e.g. 100,000-1 Bn cells). However, in some embodiments, the population may contain 1M-500M of the cells, or less. In some embodiments, the harvested cells may be cryopreserved, where the term “cryopreserved” refers to cells that have been preserved or maintained by cooling to low sub-Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT zero temperatures, such as 77 K or -196 deg. C. (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. Useful methods of cryopreservation and thawing cryopreserved cells, as well as processes and reagents related thereto, include but are not limited to e.g., those described in U.S. Patent Nos. 10370638; 10159244; 9078430; 7604929; 6136525; and 5795711, the disclosures of which are incorporated herein by reference in their entirety. In contrast, the term “fresh”, as used herein with reference to cells, may refer to cells that have not been cryopreserved and, e.g., may have been directly obtained and / or used (e.g., transplanted, cultured, etc.) following collection from a subject or organ thereof. Harvested therapeutic cell populations produced by the methods as described herein and therapeutic or pharmaceutical compositions thereof may be present in any suitable container (e.g., a culture vessel, tube, flask, vial, cryovial, cryo-bag, etc.) and may be employed (e.g., administered to a subject) using any suitable delivery method and / or device. Such populations of cells and pharmaceutical compositions may be prepared and / or used fresh or may be cryopreserved. In some instances, populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a “ready-to-use” format, including e.g., where the therapeutic cells are present in a suitable diluent and / or at a desired delivery concentration (e.g., in unit dosage form) or a concentration that can be readily diluted to a desired delivery concentration (e.g., with a suitable diluent or media). Populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a delivery device or a device compatible with a desired delivery mechanism or the desired route of delivery, such as but not limited to e.g., a syringe, an infusion bag, or the like. In some instances, the present disclosure provides one or a plurality of cell therapy doses, e.g., each contained in suitable container. Cell therapy doses may be generated through a variety of methods. Aliquoting expanded populations of therapeutic cells into cell therapy doses may be performed by a variety of means. In certain embodiments, the compositions may include the therapeutic cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCl, MgCl2, KCl, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acidAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N- tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions. A population may include a therapeutically effective amount of the cells. By “therapeutically effective amount” it is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to affect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease or disorder associated, e.g., with the target cell or a population thereof, as compared to a control. An effective amount can be administered in one or more administrations. A “therapeutically effective amount” of such cells may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the cells to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In certain embodiments, a therapeutically effective amount of T cells may be 100,000-50M of the T cells. However, in other embodiments, a therapeutically effective amount of T cells may be 50M- 500M cells. The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated for administration by combination with appropriate excipients, diluents and / or the like. Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration. An aqueous formulation of the cells may be prepared in a pH-buffered solution, e.g., at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation. A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM. In some embodiments, a composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m- cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v). Methods of treatment Also provided is a method comprising administering a cell as described above to a subject. In these embodiments, the subject disease or disorder of the brain, e.g., has brain cancer or a metastasis to the brain, or a non-cancerous brain disease or disorder such as an inflammatory disorder, a brain injury or brain degeneration. This method may be a method of treating the subject for the disease or disorder.Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT In some embodiments, the method may comprise administering an effective amount the cells to a patient in need thereof where, in some embodiments, an effective amount may be in the range of 10,000-1 Bn cells. In any embodiment, the cells may be autologous / autogeneic (“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells obtained from the subject to whom the therapeutic cells are later administered. “Allogeneic” as used herein refers to cells obtained from a donor other than the subject to whom the therapeutic cells are administered. In some embodiments, the cells (e.g., T cells) are cells obtained from a mammalian subject. In certain embodiments, the mammalian subject is a primate. In some embodiments, the cells are obtained from a human. Cells are typically injected into the patient, although routes of administration can be used. In any embodiment, the patient may be a patient where the treatment may result in at least an amelioration of one or more symptoms associated with the condition of the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition. Routes for administration include intravenous injection, although any other suitable route may be used.

[0071] In some instances, the subject circuit may be delivered by administering to the subject a cell expressing the circuit. In some instances, the subject circuit may be delivered by administering to the subject a nucleic acid comprising one or more nucleotide sequences encoding the circuit. Administering to a subject a nucleic acid encoding the circuit may include administering to the subject a cell containing the nucleic acid where the nucleic acid may or may not yet be expressed. In some instances, administering to a subject a nucleic acid encoding the circuit may include administering to the subject a vector designed to deliver the nucleic acid to a cell.

[0072] Accordingly, in the subject methods of treatment, nucleic acids encoding a circuit or components thereof may be administered in vitro, ex vivo or in vivo. In some instances, cells may be collected from a subject and transfected with nucleic acid and the transfected cells mayAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT be administered to the subject, with or without further manipulation including but not limited to e.g., in vitro expansion. In some instances, the nucleic acid, e.g., with or without a delivery vector, may be administered directly to the subject.

[0073] In some embodiments, the subject may have a brain cancer selected from glioblastoma, a child brain tumor (e.g., medulloblastoma, diffuse midline glioma (previously called DIPG), ependymoma, craniopharyngiom, embryonal tumor (previously known as PNET), pineoblastoma, brainstem glioma, choroid plexus carcinoma or germ cell tumor, or one or more adult brain tumors, e.g., pituitary adenoma, acoustic neuroma (also known as vestibular schwannoma), meningioma, oligodendroglioma, haemangioblastoma, CNS lymphoma, non- GBM (or low grade) astrocytoma, or a tumor with unknown cells (i.e., an unspecified glioma). Cancer-specific antigens associated with many of such tumors are known or may become known.

[0074] In some embodiments, the disease may be a cancer from a non-brain or CNS tissue that has metastasized to the brain. Brain metastases can develop from any type of cancer. The most common types of cancer that spread to the brain are breast cancer, lung cancer, kidney cancer, melanoma, colon cancer, and thyroid cancer.

[0075] In some embodiments, the subject may have Alzheimer's disease, stroke, brain and spinal cord injury, brain cancer, HIV infection in the brain, ataxia-producing disorders, amyotrophic lateral sclerosis (ALS), Huntington disease, childhood inborn genetic errors affecting the brain, Parkinson's disease, swelling of the brain, autoimmune encephalomyelitis, or multiple sclerosis, for example.

[0076] In some embodiments, the subject may have brain a brain injury of damage, which may have been caused by a physical insult or a disease. EXAMPLES

[0077] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. UnlessAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.

[0078] Programmable brain-sensing T cells were engineered to locally deliver therapeutic payloads customized for cancer or neuroinflammation. First, a set of CNS-specific extracellular ligands was identified. Next, antibodies against the identified ligands were screened for and used to generate CNS-activated synthetic Notch (synNotch) receptors (engineered receptors that sense an extracellular antigen and respond by inducing a transcriptional response). This platform was harnessed to locally produce a set of genetically-encoded payloads directed towards different CNS diseases. CNS-triggered expression of CARs can be used to clear primary and secondary brain cancers, including glioblastoma and breast cancer metastases. Conversely CNS- induced expression of the immunosuppressive cytokine, IL-10, can ameliorate neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). This tissue-targeted cell strategy provides dual-level targeting specificity: production of the therapeutic payload is anatomically restricted only to the tissue of interest and the payload itself also has its own intrinsic molecular targeting specificity within the target tissue. Such a local delivery strategy thereby avoids potential toxic systemic cross- reactions of the molecular payload in other non-disease tissues.

[0079] Example 1: Engineering synNotch sensors for CNS-specific extracellular molecules. To construct brain-sensing cells, synNotch receptors that detect the candidate antigens were designed and tested. These synNotch sensors could then be used to conditionally induce the transcription of desired genetically-encoded therapeutic payloads. Briefly, synNotch receptors consist of a variable extracellular recognition domain (e.g. a single chain antibody), a cleavable Notch-based transmembrane domain, and a transcriptional intracellular domain. Upon antigen binding, the intramembrane receptor is cleaved, releasing the transcriptional domain that can enter the nucleus to activate the expression of a transgene of choice from the synNotchAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT responsive promoter. Therapeutic payloads could be chosen depending on the nature of the target disease.

[0080] For this study, a list of 59 candidate antigens was narrowed down to 7 antigens to build cognate synNotch receptors including: three CNS ECM molecules - CSPG5, BCAN, and PTPRZ1; three neural surface molecules - CDH10 (cadherin 10), NrCAM (neural cell adhesion molecule), and GRM3 (metabotropic glutamate receptor - mGluR); and one oligodendrocyte surface molecule - MOG. A phage antibody library was panned against the human versions of these proteins, and 1-10 antibody recognition domains were successfully identified against each specific target. MOG binder sequences were identified from literature. Based on the binding properties of these antibodies, the list was narrowed down to design and build a total of 40 new synNotch receptors, using scFvs designed from the antibody sequences (40 receptors including the different antigen targets, antibodies, and heavy and light chain scFv orientations). These receptors were first screened for surface expression, and then screened for antigen-inducible synNotch activity, with low basal background. Because it was desired to evaluate the in vivo targeting function of these receptors in living mice, synNotch receptors that were also cross- reactive against the mouse cognate antigen were screened for. To functionally test synNotch receptor cross-reactivity, receptors were expressed in CD4+T cells with an inducible GFP reporter. The engineered T cells were co-cultured with either primary mouse brain cell cultures or K562 cells expressing the cognate mouse antigen. Constructs showing antigen-induced expression of GFP, without significant basal leakage, were selected for further study. This process resulted in a set of synNotch receptors that can be used to sense six candidate CNS- specific antigens. The induction profile of the anti-BCAN, CSPG5, PTPRZ1 and NrCAM synNotch receptors was investigated.

[0081] Illustrative data for selected anti-BCAN, anti-PTPRZ1 anti-CDH10 and anti-NrCAM synNotch receptors are shown in Fig. 1. The scFvs for these synNotch receptors are identified in the following table. scFv TargetAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT

[0082] The anti-BCAN synNotch receptor was analyzed in further detail below.

[0083] synNotch receptors targeting the ECM were first developed since the ECM is prevalent throughout the brain (~20% of brain volume), and because they could function as an ideal disease-agnostic CNS target. BCAN, in particular, is one of the most highly prevalent molecules in the brain ECM. Astrocytes synthesize high levels of BCAN. Therefore, to test the activity of the α-BCAN synNotch receptor, primary T cells expressing this receptor were co-cultured with primary mouse astrocytes in vitro. It was observed that T cells contact the surface of astrocytes and subsequently turn green from induction of the GFP synNotch activation reporter. Thus, cultured primary mouse astrocytes produce enough BCAN to drive activation of this synNotch receptor. Reconstituted BCAN-containing hydrogels were shown to activate cells with the anti- BCAN synNotch receptor (data not shown), indicating this induction can occur in a cell-free manner. Overall, these findings highlight that cells can be engineered to sense specific non- cellular microenvironmental features such as the ECM as a means to recognize a particular tissue such as the brain.

[0084] Example 2: Using a BCAN sensor to anatomically target primary brain tumors in vivo. A concrete application of CNS-sensing synNotch receptors is to deploy them to direct CAR T cell killing of brain tumors, such as glioblastoma (GBM). As with most solid tumors, there is no perfect single antigen to target on GBM that is both absolutely tumor specific and homogeneously expressed on tumor cells. Nonetheless, there are several glioma-associated antigens, like ephrin type A receptor 2 (EphA2) and interleukin 13 receptor α2 (IL13Rα2), that are homogeneously expressed on the surface of most of GBM cells at high levels. However, these antigens are also expressed in normal tissues, and are only specific to GBM within the confines of the brain (both EphA2 and IL13Ra2 are not normally expressed in brain). Thus, if T cells could be engineered to only induce expression of an anti-EphA2 / IL13Ra2 CAR when in the brain, effective GBM killing could potentially be achieved while also preventing CAR killing of cross-reactive off-tumor tissues outside of the brain. From RNAseq data in the Cancer Genome Atlas Program (TCGA), it was also found that BCAN was the most strongly expressed of the brain-specific antigens within GBM tumor samples. Thus, T cells containing the following synNotch circuit were tested: α-BCAN synNotch ^ α-EphA2 / IL13Ra2 CAR (a tandem CAR that kills cells expressing either antigen) (Fig. 2A).Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT

[0085] The ability of CD8+T cells engineered with this BCAN-induced CAR circuit to kill GBM6 cells (a GBM PDX tumor cell line) was then tested. A strong killing of GBM6 cells was observed in vitro, but only in the presence of K562 cells engineered to express BCAN (only low levels of GBM6 killing was observed when co-cultured with BCAN- K562 cells, Fig. 2B). Thus, although GBM6 cells do express some level of BCAN themselves, this level of BCAN does not appear to be sufficient for maximal synNotch activation. SynNotch priming by neighboring BCAN+K562 cells appears to strongly induce CAR expression and the subsequent killing of GBM6 cells (Fig. 2B), without causing toxicity to the trans-priming BCAN+cells (not shown).

[0086] This circuit was then tested in vivo using mice bearing GBM xenografts. GBM6 tumors were implanted in the brains of immunodeficient NCG mice and the mice were infused intravenously with either T cells bearing the α-BCAN synNotch–α-EphA2 / IL13Rα2 CAR circuit or untransduced control T cells (Fig. 2C). All the mice receiving the untransduced T cells (n = 5) died of tumor progression by day 51 after tumor implantation. Notably, all the mice treated with the synNotch-CAR T cells showed complete and long-term remission of the GBM6 tumors (P = 0.018, mixed-effects analysis), reflected in their significantly increased survival (P = 0.003, log-rank Mantel-cox test). Notably, the BCAN driven T cells were far more effective than similar MOG primed T cells, as they cleared GBM6 tumors far more rapidly and with near perfect consistency, suggesting that this brain ECM target is a more prevalent and effective brain-priming antigen.

[0087] To determine with higher resolution where these BCAN induced T cells are active, confocal imaging was performed on mouse brain samples after 10 days of treatment (Fig. 2D). synNotch induced T cells were observed throughout the brain slices (induced expression of CAR-GFP), but only killing / apoptosis (active caspase 3 staining) was observed within the tumor. Adjacent neurons (stained for NeuN) did not show apoptosis. Thus, these T cells did not kill the normal brain, but only specifically killed the EphA2 / IL13Rα2 expressing tumor cells.

[0088] To further examine if BCAN synNotch induction was restricted to the brain, flow cytometry analysis of α-BCAN synNotch^α-EphA2 / IL13Rα2 CAR T cells isolated from the blood, spleen and brain of GBM6 tumor-bearing mice at day 6 after T cell injection was performed. Induced CAR expression was found only in T cells harvested from the brain, consistent with brain restricted synNotch activation (Fig. 2E). The T cells isolated from the brain also show induced expression of CD69 and CD103, which are markers of T cell activationAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT and retention (not shown). CAR expression was not observed in the T cells isolated from the blood and spleen. All together, these data are consistent with a brain-restricted anti-tumor response. Importantly, the α-BCAN synNotch–α-EphA2 / IL13Rα2 CAR T cells persisted in the brains even after tumor regression, consistent with the durable anti-tumor response (not shown). The brain-only localization of synNotch-activated T cells is consistent with prior studies that show that synNotch-induced CAR expression decays with a half-life of a few hours upon removal of the priming antigen.

[0089] To confirm that the endogenous brain BCAN is sufficient to induce CAR activity, CRISPR-Cas9 was used to completely knockout BCAN expression in the GBM6 tumor cells (they still express the target killing CAR antigens, EphA2 and IL13Rα2). First, it was confirmed in vitro that the GBM6 BCAN KO cells could only be killed by α-BCAN synNotch^ CAR T cells when co-cultured with BCAN expressing K562s cells (but not with parental K562 cells) (not shown). Next, the GBM6 BCAN KO cells were implanted intracranially in mice and treated with α-BCAN synNotch^ CAR circuit T cells, and tumor size was monitored. Even with no BCAN being expressed by the GBM6 cells, the α-BCAN synNotch^ CAR circuit T cells could clear the brain implanted tumors, presumably through priming by the endogenous brain BCAN (not shown). Because BCAN was not required on the tumor cells, it is possible that other GBM PDX cells lacking BCAN might also be efficiently cleared, making this approach more generalizable. A different glioblastoma PDX line, GBM39, was identified as being BCAN negative and it was confirmed that GBM39 cells could not be killed by BCAN synNotch^ CAR circuit T cells in vitro (not shown). Consistent with previous findings, the α- BCAN synNotch^ CAR circuit T cells could efficiently clear GBM39 tumors in vivo when they were implanted within the mouse brain (not shown). Thus, BCAN in normal brain tissue is sufficient to prime killing of neighboring tumor cells lacking any BCAN expression.

[0090] To confirm that induced tumor-killing CAR activity was restricted to the brain milieu, dual tumor mouse studies were performed, in which GBM6 tumors were implanted both in the brain and in the flank, within the same animal. To eliminate possible priming from BCAN expressed in the GBM6 flank tumors, the GBM6 BCAN KO tumor cells described above were used (which is cleared when implanted in brain). Thus, the flank implanted tumor lacks both intrinsic and environmental BCAN expression, but still expresses the target CAR antigens, EphA2 and IL13Rα2. The dual implanted tumor mice were then treated with intravenouslyAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT infused T cells expressing the α-BCAN synNotch^ CAR circuit, and the size of both tumors were monitored. These results showed that only the brain-implanted tumor was cleared, while the flank tumor grew and failed to regress (Fig. 2F). Thus, CAR killing activity is not observed outside of the brain, even for potential target cells that express the CAR antigens. This lack of extra-brain killing even occurs while the T cells are actively killing the tumors within the brain. This high degree of specific anatomical targeting is consistent with the induced expression of T cell retention molecules (CD69 and CD103), as well as previous measurements showing that synNotch induced CAR expression rapidly decays with a half-life of hours after loss of synNotch stimulation.

[0091] Example 3: Using BCAN synNotch sensor to anatomically target secondary brain tumors. Equipped with a reliable in vivo CNS-sensing synNotch platform, it was desired to test its versatility by expanding its use to other brain tumors such as secondary, metastatic brain tumors. Breast cancer (BC) is the most common cancer worldwide. While it is curable in ~70– 80% of early-stage patients with non-metastatic disease, it remains lethal in advanced metastatic stages, resulting in more than half a million deaths annually worldwide. A major unmet need is the treatment of the lethal brain metastases (BM) (extracerebral disease can be controlled in 50% of HER2+breast cancer patients). For HER2+breast cancers, an α-HER2 CAR is predicted to exhibit off-tumor toxicity since HER2 is expressed on several healthy tissues. However, because HER2 expression is minimal in the normal brain, restricting the killing action of a HER2 CAR to the CNS could allow for effective and safe clearance of breast cancer metastases (Fig. 3A). It was found that, in vitro, CD8+T cells engineered with the α-BCAN synNotch^α- HER2 CAR circuit could effectively kill BT-474 tumor cells, a HER2+breast cancer model cell line, but only in the presence of BCAN+K562 cells to induce priming (BT-474 cells do not to express BCAN) (Fig. 3B).

[0092] BT-474 HER2+breast cancer tumors were implanted in the brains of NSG mice to emulate metastases, and then infused with T cells bearing the α-BCAN synNotch^α-HER2 CAR circuit. The BCAN primed T cells efficiently cleared the BT-474 brain implanted tumors and increased mouse survival, compared to mice treated with untransduced T cells (Fig. 3C).

[0093] Using a similar strategy, triple negative (negative for estrogen, progesterone, and HER2 receptors) breast cancers (TNBC) that show an even lower patient survival rate compared to HER2+tumors were examined. The surface antigen TROP2 has recently received muchAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT attention, as it is overexpressed in TNBCs and can be effectively targeted with an antibody-drug conjugate therapy. TROP2 is also expressed in other normal tissues, but its levels of expression in the normal brain are minimal (not shown). Thus, TROP2 could also be a good CAR target for brain metastases, when combined with brain / CNS restricted induction. It was found that CD8+T cells engineered with the α-BCAN synNotch^α-TROP2 CAR circuit could effectively kill BT- 20 tumor cells, a TNBC TROP2+cell line, in vitro, but only in the presence of BCAN+K562s to induce priming (BT-20 cells do not to express BCAN) (not shown). BT-20 tumors were then implanted in the brains of immunodeficient NSG mice to emulate breast cancer brain metastases, and treated with T cells bearing the α-BCAN synNotch^α-TROP2 CAR circuit. The BCAN-primed T cells efficiently cleared the BT-20 tumors and increased mouse survival relative to control mice treated with untransduced T cells (Fig. 3C).

[0094] In summary, the α-BCAN brain sensing synNotch showed the ability to induce clearance of breast cancer tumors implanted within the brain and, thus, offers a potential strategy to target both HER2+and triple negative breast cancer metastases to the brain, which are often far more resistant to standard therapies than the primary tumor.

[0095] Example 4: Engineering brain-targeted immune suppressor cells. Neuroinflammatory diseases, such as MS, represent an extremely difficult therapeutic challenge, as it is challenging to develop therapies that effectively suppress inflammation in the CNS, while not causing systemic immune suppression. While B cell depleting therapies have shown great improvement in relapsing forms of the MS, they do not resolve progressive MS, including chronic active lesions which are histologically comprised of T cells and activated macrophages / microglia foci, suggesting that broader approaches are needed.

[0096] Numerous immunomodulatory molecules (cytokines, chemokines, antibodies) have shown potential to address neuroinflammation, but their clinical efficacy has proven disappointing, due to insufficient penetration of the molecule into the CNS (poor crossing of the BBB). One example is the cytokine interleukin-10 (IL-10), a potent anti-inflammatory molecule. Expression of IL-10 directly in the brain via adenovirus injection successfully ameliorated disease in mouse models of neuroinflammation. Nonetheless, intravenous infusion of IL-10 in mouse models and in human clinical trials did not show efficacy, likely due to IL- 10’s short half-life (∼3 h in both humans and mice) and inability to cross the BBB. Thus, this data suggests that IL-10 could be effective at ameliorating neuroinflammation, if it could beAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT effectively delivered to the brain. Most efforts have focused on engineering variants of cytokines that can alter their lifetime, BBB permeability, and receptor specificity. The results presented here, however, suggest an alternative orthogonal strategy in which brain-sensing T cells could be used as a vehicle to produce IL-10 selectively in the brain. Thus, IL-10 is an ideal proof-of-principle payload with which to test the broader capabilities of the brain-sensing T cell platform (Fig. 4A).

[0097] CD4+T cells were engineered with an α-BCAN synNotch^IL10 circuit and it was demonstrated that these cells could effectively produce IL-10 in vitro, but only in the presence of BCAN+K562 cells (Fig. 4B). To evaluate their anti-inflammatory potential, these cells were tested for their ability to inhibit the inflammatory activation of both CNS autoreactive T cells and microglia cells in vitro. CNS autoreactive T cells from genetically engineered α-MOG- specific TCR (2D2) transgenic mice were tested (Fig. 4C), and their activation (determined by CD25 staining and IFN-γ secretion) was found to be significantly inhibited by α-BCAN synNotch^IL10 CD4+T cells, in the presence of BCAN+K562 cells (Fig. 4C). Analogously, mouse microglia cells activated with LPS and IFN-γ were examined (Fig. 4C). In the presence of BCAN+K562 inducer cells, the α-BCAN synNotch^IL10 CD4+T cells significantly reduced secretion of proinflammatory cytokines (IL-6 and TNF-α) by activated microglia cells (Fig. 4C). Thus, the α-BCAN synNotch^IL10 CD4+T cells, when induced, could exert immunosuppressive activity in vitro.

[0098] Example 5: Brain-targeted suppressor cells ameliorate disease in mouse model of neuroinflammation. To determine which brain-priming antigens might be best to direct cell therapies against neuroinflammation, published RNA-seq analysis of chronically active MS lesions (compared to healthy CNS tissue of patients) were examined. These data showed maintenance of expression of both BCAN and CDH10 in MS lesions, but a marked decrease in MOG expression (likely caused by demyelination in the disease). Thus, both BCAN and CDH10 are good candidates for priming brain-targeted responses in neuroinflammatory diseases like MS. An experimental autoimmune encephalomyelitis (EAE) mouse model of MS was used to test the efficacy of these brain-targeted suppressor cells in vivo. An established adoptive transfer disease model was used (Fig. 5A), in which pathogenic polyclonal MOG autoreactive Th17 CD4+T cells were generated by direct immunization with MOG peptide. These pathogenic cells were then adoptively transferred into a recipient immunocompromised RAG-1-Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT / - mouse, inducing severe and usually fatal neurological disease. In this model, the ability of infused engineered T cells to reduce disease severity was tested. Neurological disease severity and progression was tracked using the EAE clinical scoring system, which scores increasing levels of paralysis and neurological disfunction (Fig. 5A).

[0099] Adoptive transfer of α-MOG autoreactive T cells results in severe EAE disease in the recipient RAG-1- / -mice, yielding scores close to the maximum of 5. To test the suppressor cells, the mice were injected i.v. every 4 days with the therapeutic suppressor T cells (or control T cells expressing BFP), starting seven days after adoptive transfer of the disease causing α-MOG autoreactive T cells. The α-BCAN synNotch ^ IL-10 CD4+T cells significantly improved disease outcome: less severe EAE scores were observed (P = 0.003, mixed analysis), cumulative EAE scores were lowered, and mouse survival increased, relative to mice treated with control T cells (Fig. 5B). Amelioration of EAE disease by the therapeutic cells was supported by the overall increased mobility in the mice treated with α-BCAN synNotch ^ IL-10 CD4+T cells (control treated mice show increased paralysis). Similarly, treatment with the α-CDH10 synNotch^IL-10 CD4+T cells, also resulted in significantly lower EAE severity scores and increased survival (Fig. 5C). Different dosing regimens of therapeutic T cells provided similar protection (not shown). Thus, IL-10 produced by brain-sensing T cells (using either BCAN or CDH10 as a priming antigen) are able to improve EAE symptoms. These findings validate the concept that brain-sensing cells can serve as a vehicle to deliver anti-inflammatory payloads to the brain.

[0100] Here, a general cell platform for delivering diverse therapeutic payloads to the brain is developed. Several classes of CNS-specific antigens that can be used for CNS targeting of therapeutic cell activity are identified (Fig. 1). These target antigens include components of the brain ECM, which offer an abundant and ubiquitous targetable substrate, as the ECM has a highly unique molecular composition and represents about 20% of the CNS volume. synNotch receptors that detect the brain ECM component BCAN can effectively sense the native CNS in an in vivo context. This brain-induced platform could be used effectively to locally deliver different model payloads directed against two distinct types of CNS diseases: cancer and autoimmunity. This highlights the broad spectrum of CNS diseases that this approach can be used to target.Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT

[0101] In the case of brain cancer (and cancer in general), it is almost impossible to find a target antigen for CAR T cell therapy that is both homogeneously expressed on all the cancer cells yet not expressed in any healthy tissues. As shown here, this conundrum can in some cases be resolved by restricting killing action to a specific anatomical compartment, such as the brain. This opens broad new flexibility to safely attack cancers using target antigens that are absent in the brain (or other target tissue), even if they are expressed on healthy tissues elsewhere.

[0102] Therapeutic cells that can deliver immunomodulatory cytokines or other biologics to a target tissue, such as the CNS, can not only make delivery more effective but they can also reduce the risk of systemic toxicity. Many of these biologics have pleiotropic effects on multiple tissues, which can lead to major toxicities outside of the target tissue. For example, in the case of chronic inflammation, such as MS, systemic treatment with anti-inflammatory drugs can result in increased risk of infections and other pathologies.

[0103] Here, a model anti-inflammatory molecule, IL-10, is tested and it is validated that delivery of IL-10 by a brain-sensing cell improves outcomes in an animal model of neuroinflammation. This study opens the door to a much wider examination of diverse cell- delivered payloads. It may be possible to develop more effective combinatorial payloads or to use further optimized versions of key biologics to further improve therapeutic outcomes. Further improving pharmacodynamic and specificity features of the payload cytokine will undoubtedly also lead to more optimized therapies. There are also many additional ways that can be explored to improve the specificity and efficiency of brain or CNS targeting by the cells. This could include ways to improve cell trafficking and residence in the brain, as well as to improve cell migration through the BBB. Other important issues to address will be ways to both amplify and tune the level of payload production and to increase durability / survival of the therapeutic cells. The current results show that brain-sensing cells can be used as a general platform to treat a broad set of CNS diseases, including brain tumors, brain metastases, neuroinflammation, or even neurodegeneration (Fig. 5D).

[0104] More generally, the power of subcontracting a key part of therapeutic targeting function to engineered immune cells is demonstrated, rather than depending only on the physical targeting properties of an isolated molecular therapeutic itself. In this case, the molecular-scale specificity of the therapeutic payload is layered on top of the anatomical-scale specificity of theAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT cell, yielding much higher combinatorial therapeutic specificity compared to systemic payload administration. This concept represents the first step towards a broader therapeutic “tissue GPS” cell targeting platform which could allow application of this modular approach to diseases targeting a larger set of tissues. Tissue-targeted cell delivery provides a novel general strategy to make therapies more specific and effective, and to reduce systemic toxicity. Materials and Methods.

[0105] Construct Design. SynNotch receptors were built by fusing the various scFv sequences to mouse Notch1 (NM_008714) minimal regulatory region (res.1427-1752) and Gal4 DBD VP64. All synNotch receptors contain N-terminal CD8a signal peptide (MALPVTALLLPLALLL HAARP) for membrane targeting and α-myc-tag (EQKLISEEDL) for detecting surface expression with α-myc A647 (cell-signaling #2233); see Morsut et al. Cell (2016) 164:780-791 for synNotch sequence. Receptors were cloned into a modified pHR’SIN:CSW vector containing a PGK or SFFV promoter. The pHR’SIN:CSW vector was also used to make response element plasmids with five copies of the Gal4 DNA binding domain target sequence (GGAGCACTGTCCTCC GAACG) upstream from a minimal CMV promoter. Response element plasmids also contain a PGK promoter that constitutively drives blue fluorescent protein (BFP) expression to easily identify transduced T cells. CARs were built by fusing IL13 Mutein [E13K,K105R]-G4Sx4-EphA2 scFv (3), α-Her2 (4D5) or α-TROP2 (hRS7) to the hinge region of the human CD8α chain and transmembrane and cytoplasmic regions of the human 4-1BB, and CD3z signaling domains. Inducible CAR constructs or cytokines were cloned into a site 3' to the Gal4 response elements. CARs were tagged c-terminally with GFP or RFP, or n-terminally with myc tag, or flag tag to verify surface expression.

[0106] Primary Human T Cell Isolation and Culture. Primary CD4+and CD8+T cells were isolated from donor blood after apheresis by negative selection (STEMCELL Technologies #15062 and #15063). Blood was obtained from Blood Centers of the Pacific, as approved by the University of California San Francisco (UCSF) Institutional Review Board. T cells were cryopreserved in RPMI-1640 (UCSF cell culture core) with 20% human AB serum (Valley Biomedical, #HP1022) and 10% dimethyl sulfoxide. After thawing, T cells were cultured in human T cell medium consisting of X-VIVO 15 (Lonza #04-418Q), 5% Human AB serum, and 10 mM neutralized N-acetyl L-Cysteine (Sigma-Aldrich #A9165) supplemented with 30Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT units / mL interleukin (IL)-2 (NCI BRB Preclinical Repository) for all experiments except for the IncuCyte experiments. IncuCyte experiments were cultured in RPMI-1640 with 5% human AB serum supplemented with 30 units / mL IL-2.

[0107] Lentiviral Transduction of Human T Cells. Pantropic vesicular stomatitis virus G (VSV- G) pseudotyped lentivirus was produced via transfection of Lenti-X 293T cells (Clontech #11131D) with a pHR’SIN:CSWvector and the viral packaging plasmids pCMVdR8.91 and pMD2.G using Fugene HD (Promega #E2312). Primary T cells were thawed the same day and, after 24 hours in culture, were stimulated with Human T-Activator CD3 / CD28 Dynabeads (Life Technologies #11131D) at a 1:3 cell:bead ratio. At 48 hours, viral supernatant was harvested and, in some assays, concentrated using Lenti-X concentrator (Clontech #631231). Primary T cells were exposed to the lentivirus for 24 hours. At day 5 after T cell stimulation, Dynabeads were removed, and sorted with a Beckton Dickinson (BD) FACS ARIA Fusion or Sony SH800S Cell Sorter. AND-gate T cells exhibiting basal CAR expression were gated out during sorting. T cells were expanded until rested and could be used in assays.

[0108] Human T Cell Phenotyping. T cells phenotypes were assessed using the following antibodies: PE anti-CD25 (clone BC96, 302606, BioLegend) for T cell activation; PE-Cy7 anti- PD-1 (clone EH12.1, 561272, BD Biosciences), BV785 anti-TIM-3 (clone F38-2E2, 345031 BioLegend) and AF700 anti-LAG-3 (clone 3DS223H, 56-2239-42, Thermo Fisher) for T cell exhaustion; BV605 anti-CD45RA (clone HI100, 304133, BioLegend) and APC-Cy7 anti- CD62L (clone DREG-56, 304813, BioLegend) T cell differentiation state. Briefly, post- transfected T cells were sorted, expanded and rested for about 10 days, then analyzed by flow cytometry. To assess influence of antigen-dependent activation on T cell phenotype, T cells were cocultured E:T 1:1 (50,000:50,000) for 24 hours with target GBM6 cells pre-stained with CellTrace far red (C34564, Thermo Fisher) per manufacturer’s instructions to distinguish them from T cells. A 1:100 antibody dilution was used. A total volume of 50 µL per staining reaction was used in staining buffer (phosphate-buffered saline with 2% fetal bovine serum). Samples were incubated at 4 °C for 15 minutes and washed with staining buffer. T cells were analyzed by flow cytometry.

[0109] Cell Lines. Cell lines used were K562 myelogenous leukemia cells (ATCC #CCL-243), GBM6, GBM39 PDX cells (gift of Frank Furnari, Ludwig Institute and UCSD), BT-474 (ATCC # #HTB-20) and BT-20 (ATCC #HTB-19). Cells were lentivirally transduced to stably expressAtty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT GFP or mCherry, and enhanced firefly luciferase under control of the spleen focus-forming virus (SFFV) promoter and sorted. For transgene expression, K562s were transduced with the lentiviral vector CD510-B1 (System Biosciences) and puromycin selected.

[0110] GBM6 and GBM39 cells were cultured in DMEM / F12 media, with supplements of epidermal growth factor (EGF, 20 µg / mL), fibroblast growth factors (FGF, 20 µg / mL), and heparin (5 µg / mL). K562 and BT-20 cells were cultured in DMEM 10% FBS. BT-474 cells were cultured in RPMI 10% FBS.

[0111] In Vitro Stimulation of SynNotch T cells. For in vitro synNotch induction, the engineered T cell and tumor / target cells were co-cultured at 1:1 ratio, 1 x 105cells each in a flat bottom 96-well tissue culture plate for 48h. Cells were analyzed by flow cytometry using BD Fortessa; analysis was performed by FlowJo software (TreeStar). When cytokine release assays were conducted, the supernatant was collected and processed by ELISA.

[0112] In Vitro Mixed Neuronal Cultures. Cortexes were dissected from P0 mice of both sexes, dissociated in 0.25% trypsin, washed three times with Hank’s Balanced Salt Solution (HBSS) containing 10 mM HEPES and 20 mM glucose, triturated and plated on poly-L-lysine coated coverslips at 350 cells / mm2. Neurons were plated in Minimal Essential Medium containing B27 (Gibco, 17504044), 2 mM glutaMAX (ThermoFisher, 35050061), 5% FBS, 21 mM glucose (Sigma, G8769) and 1x penicillin / streptomycin (ThermoFisher, 15070063). After one day in vitro (DIV1), 3 / 4 of the medium was changed to Neurobasal (ThermoFisher, 21103049) with B27, glutaMAX and penicillin / streptomycin.

[0113] In Vitro Astrocyte Culture. Astrocytes were isolated from the adult NCG mouse brain tissue using Adult Brain Disassociation kit (Cat no: 130-107-677, Miltenyi Biotec). Briefly, the extracellular matrix is enzymatically digested along with mechanical dissociation on gentleMACS™ Dissociator with heat. Following disassociation, myelin and cell debris is removed using Debris Removal Solution. The anti-ASCA-2 microbead kit (Cat. No: 130-097- 678, Miltenyi Biotec) was used to isolate astrocytes from the single cell suspension. The astrocytes were cultured in pre-coated glass-bottom 6- well plate in AstroMACS medium (Cat no: 130-117-031, Miltenyi Biotec) for 7 days.

[0114] Assessment of Astrocyte-synNotch CAR T cell interaction. The cultured astrocytes, labeled with MemGlow™ 590 (20nM, Cat no: MG03-02, Cytoskeleton, Inc.), were co- incubated with α-BCAN synNotch CD8+T cells and loaded on to a pre-warmed stage (370C,Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT 5% C02) of Zeiss Spinning Disk Confocal. The live cell imaging (20X magnification) was done for 8 hours with image acquisition at every 5-minute interval.

[0115] Assessment of SynNotch-CAR T Cell Cytotoxicity. CD8+synNotch-CAR T cells were stimulated for up to 72 hours with target cells expressing the killing antigens and when needed priming K562 cells (1:1:1, 5 × 105). The degree of specific lysis of target cells was determined by comparing the fraction of target cells alive in the culture compared to treatment with non- transduced T cell controls, unless stated otherwise. Cell death was monitored by shift of target cells out of the side scatter and forward scatter region normally populated by the target cells. Alternatively, cell viability was analyzed using the IncuCyte Zoom system (Essen Bioscience). Tumor cells were plated into a 96-well plate at a density of 2.5 × 104cells per well in triplicate overnight. 5 × 104T cells (and K562 cells when specified) were added into each well next day. Target cells and T cells were co-cultured as described above. At least two fields of view were taken per well every 2-3 hours. Mean florescence intensity (MFI) was calculated using IncuCyte Zoom software (Essen BioScience) to determine target cell survival. Data were summarized as mean ± error.

[0116] Assessment of autoreactive T cell activation. CD4+control or synNotch-IL10 T cells were cultured with K562s expressing BCAN, CD4+, CD25+MOG TCR (isolated from C57BL / 6-Tg(Tcra2D2,Tcrb2D2)1Kuch / J mice, Jackson lab), APC (splenic cells that are CD4-, isolated from C57BL / 6-Tg(Tcra2D2,Tcrb2D2)1Kuch / J mice, Jackson lab) in presence of MOG P35-55 (50μg / mL) at 1:1:1:1 ratio (5 × 104each) for 4 days. Cells and supernatant were further processed as described in figures.

[0117] Assessment of microglial inflammation inhibition. CD4+control or synNotch-IL10 T cells were cultured with K562s expressing BCAN, BV2 microglia cells, 1 × 105, 1 × 105and 1 × 104respectively for 24 hours in media containing LPS (100 ng / mL) and murine IFN-γ (0.5 ng / mL). Cells and supernatant were further processed as described in figures.

[0118] In Vivo Mouse Experiments. All mouse experiments were conducted according to Institutional Animal Care and Use Committee (IACUC)–approved protocols. For orthotopic model with GBM6, and GBM395.0 × 104GBM6-luc-mCherry or GBM39-luc-mCherry cells were implanted intracranially into 6- to 8-week-old female NCG mice (Charles River). For orthotopic model with BT-474 and BT-20, 1.0 × 105luc-GFP expressing cells were implanted intracranially into 8- to 12-week-old female NSG mice.Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT

[0119] Following anesthesia with 1.5% isofluorane, stereotactic surgery for tumor cell implantation (injection volume: 2µl) was performed with the coordination of the injection site at 2 mm right and 1 mm anterior to the bregma and 3 mm into the brain. Before and for three days after surgery, mice were treated with an analgesic and monitored for adverse symptoms in accordance with the IACUC.

[0120] In the subcutaneous model, NCG mice were injected with 1.2 × 105GBM6-luc-mcherry cells subcutaneously in 100 μl of HBSS on day 0.

[0121] Tumor progression was evaluated by luminescence emission on Xenogen IVIS Spectrum after intraperitoneal injection of 1.5mg D-luciferin (GoldBio, injection volume 100 μl). Prior to treatment, mice were randomized such that initial tumor burden in control and treatment groups were equivalent. Mice were treated with engineered or non-transduced T cells at indicated doses intravenously via tail vein in 100 μl of phosphate buffered saline (PBS). Survival was evaluated over time until predetermined IACUC-approved endpoint (hunching, neurological impairments such as circling, ataxia, paralysis, limping, head tilt, balance problems, seizures, body-weight loss) was reached.

[0122] In the EAE experiments, an adoptive transfer model was used. Briefly, naive 8-14 week-old C57BL / 6 female mice were injected subcutaneously with 50 µg / mouse of MOG peptide in 0.1 ml emulsion of CFA containing 4 mg / ml Mycobacterium tuberculosis H37Ra (DIFCO Laboratories) and PBS (1:1). Ten days later, draining lymph nodes were collected, single-cell suspensions were prepared and cells were stimulated at 5 × 106cells / ml with 25 µg / ml of relevant MOG peptide in the presence of recombinant murine IL-12 (25 ng / ml) and anti-murine IFN-γ mAb (BD). After three days of culture, cells were harvested, washed, and 20- 25 × 106cells were injected i.p. into each in naive recipient RAG1- / -mice.

[0123] Immunofluorescence. Mice were euthanized before being perfused transcardially with cold PBS. Brains were then removed and fixed overnight in 4% paraformaldehyde–PBS before being transferred to 30% sucrose and were allowed to sink (1-2 days). Subsequently, brains were embedded in O.C.T. Compound (Tissue-Tek; 4583; Sakura Finetek). Serial 10-mm coronal sections were then cut on freezing microtome and stored at -80 °C. Sections were later thawed, fixed with 10% formalin for 10 mins followed by incubation in blocking buffer (PBS- 5% normal donkey serum) for 40 minutes and stained with primary antibodies overnight at 4 °C. Primary antibodies used were: CD45 (D9M8I) XP Rabbit mAb (Cell Signaling Technologies,Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT 1:100), Cleaved Caspase 3 (Asp175) Rabbit mAb (Cell Signaling Technologies, 1:250), and NeuN mouse mAb (Milipore, clone A60, 1:500).

[0124] Conjugated secondary antibodies were used at 4 °C for two hours to detect primary labeling. Sections were stained with DAPI (Thermo Fisher). Images were acquired using either a Zeiss Axio Imager 2 microscope (×20 magnification) with TissueFAXS scanning software (TissueGnostics) or a Zeiss LSM 780 microscope (x20 magnification) with Zeiss Zen imaging software. Exposure times and thresholds were kept consistent across samples within imaging sessions. When needed to improve the visibility of an image, linear adjustment of contrast and brightness was applied to the entire image in accordance with Science guidelines (see Fig. 2D).

[0125] Assessment of Engineered T cells In Vivo. For all experiments involving phenotyping of adoptively transferred engineered T cells, brain and spleen were harvested following perfusion with cold PBS. Brains were mechanically minced and treated at 37 °C for 30 minutes with digestion mix consisting of Collagenase D (30mg / ml) and DNAse (10mg / ml) and Soybean trypsin Inhibitor (20mg / ml). The resulting brain homogenate was resuspended in 30% Percoll (GE Healthcare), underlaid with 70% Percoll, and then centrifuged for 30 minutes at 650 x g. Enriched brain infiltrating T cells were recovered at the 70-30% interface and stained with fluorescently conjugated antibodies against CD3 (5 µl, Cat no: 555342, BD Biosciences), CD45 (5 µl, Cat no: 564357, BD Biosciences), CD69 (5ul, Cat no: 567066 BD Biosciences), and CD103 (5ul, Cat no: 25-1038-42, Thermo Fisher Scientific) for one hour at 4 °C. Prior to staining with antibodies, cells were stained with BD Horizon Fixability Viability Stain 780 (BD Biosciences) to discriminate live from dead cells. Data was collected on an Attune NxT Flow Cytometer and the analysis was performed in FlowJo software (TreeStar).

[0126] Assessment of Mouse Motility. Mice movements were recorded using an iPhone after the experimenter introduced their hand in the cage, gave a gentle push or by moving the mice to the center of the cage. Individual traces of the mice were obtained using the Fiji plugin Manual Tracking for the next 240 frames (8s). To account for camera movements, the corners of the cage were also tracked and the mouse displacements were adjusted accordingly.

[0127] Statistical Analysis. All statistical analyses were performed with Prism software version 9.0 (GraphPad) as described in the figures and legends.Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT

[0128] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT CLAIMS What is claimed is:

1. A transmembrane signaling protein comprising: (a) an extracellular binding domain comprising a scFv having the CDRs of an antibody selected from Table 1; (b) a transmembrane domain; and (c) an intracellular signaling domain.

2. The transmembrane signaling protein of claim 1, wherein the scFv comprises: a heavy chain variable domain that is at least 90% identical to the heavy chain variable domain of the selected antibody; and a light chain variable domain that is at least 90% identical to the light chain variable domain of the selected antibody.

3. The transmembrane signaling protein of claim 1 or 2, wherein the scFv comprises: an amino acid sequence that is at least 90% identical to any of SEQ ID NOs. 41-45.

4. The transmembrane signaling protein of any prior claim, wherein the transmembrane signaling protein is: a binding-triggered transcriptional switch (BTTS); an engineered immune receptor; or a synCAM.

5. The transmembrane signaling protein of any prior claim, wherein the transmembrane signaling protein is a BTTS, and the BTTS comprises: the extracellular binding domain; the transmembrane domain; an intracellular signaling domain that comprises a transcriptional activator, and one or more protease cleavage domains;Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT wherein binding of the extracellular domain to an antigen in the brain results in cleavage of the BTTS at the one or more protease cleavage domains to release the transcriptional activator.

6. A nucleic acid encoding the transmembrane signaling protein of any prior claim.

7. A cell comprising a nucleic acid of claim 6.

8. The cell of claim 7, wherein the transmembrane signaling protein is a BTTS and the cell further comprises: a coding sequence for a therapeutic protein, and a regulatory sequence operably linked to the coding sequence, wherein a transcriptional activator released by the BTTS binds to the regulatory sequence and induces expression of the therapeutic protein.

9. The cell of claim 8, wherein the therapeutic protein is secreted.

10. The cell of claim 8 or 9, wherein the therapeutic protein is a secreted peptide, enzyme or antibody.

11. The cell of claim 8, wherein the therapeutic protein is intracellular.

12. The cell of claim 8, wherein the therapeutic protein is a cell surface protein.

13. The cell of claim 8, wherein the therapeutic protein is a receptor.

14. The cell of claim 12 or 13, wherein the therapeutic protein is a chimeric antigen receptor (CAR).Atty. Dkt: UCSF-798WO Client Ref.: SF-2024-199-2-PCT 15. The cell of claim 14, wherein the CAR is a tandem chimeric antigen receptor (CAR) that has a first binding domain that recognizes to Ephrin type-A receptor 2 (EphA2) and a second binding domain that recognizes Interleukin-13 receptor subunit alpha-2 (IL13RA2).

16. The cell of any of claims 7-15, wherein the cell is an immune cell or stem cell.

17. The cell of any of claims 7-16, wherein the cell is a T cell.

18. A method comprising administering a cell of any of claims 7-17 to a subject.

19. The method of claim 18, wherein the subject has a disease or disorder of the brain.

20. The method of claim 18 or 19, wherein the subject has brain cancer or a metastasis to the brain.

21. The method of claim 18 or 19, wherein the subject has a non-cancerous brain disease or disorder.

22. The method of any of claims 18-21, wherein the method is a method of treatment.

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