Synthetic protein switch for regulating t-cell signaling and method of manufacture and use thereof

Abstract

A synthetic protein switch is provided that has an active state and an inactive state and includes a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11 and a sensing domain, and wherein the synthetic protein switch reversibly converts from the inactive state to the active state upon interaction with an inducer and reversibly converts from the active state to the inactive state upon removal of the inducer. Also provided are methods for reversible modulation of T-cell signaling, treating a cytokine release syndrome (CRS), and treating a hematological malignancy using such synthetic protein switch. Further provided is a method of producing such synthetic protein switch, the method including transferring to a host cell or transfecting the host cell with a viral vector having a nucleic acid encoding the synthetic protein switch.

Description

Attorney Docket No. 0073605-001129 SYNTHETIC PROTEIN SWITCH FOR REGULATING T-CELL SIGNALING AND METHOD OF MANUFACTURE AND USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application Serial No. 63 / 759,672, filed on February 18, 2025, and entitled “APPARATUS, MATERIAL AND PROCESS FOR REGULATION OF T-CELL SIGNALING,” the entirety of which is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] Not Applicable.FIELD OF THE INVENTION

[0003] The present invention generally relates to the field of immunotherapy and T-cell therapy. In particular, the present invention is directed to a synthetic protein switch for regulating T-cell signaling and method of manufacture and use thereof.REFERENCE TO SEQUENCE LISTING

[0004] This specification includes a sequence listing submitted herewith, which includes the file entitled 0073605-001129. xml having the following size: 100,889 bytes which was created February 12, 2026, the contents of which are incorporated by reference herein.BACKGROUND

[0005] T cell activation is governed by a finely balanced interplay between protein tyrosine kinases and phosphatases, where modest perturbations in proximal signaling can lead to substantial downstream consequences. An example of these regulators includes C-terminal Src kinase (CSK), which, together with Src-family kinases (SFKs), form a conserved regulatory module that constrains tyrosine kinase signaling across eukaryotic evolution. CSK plays a central role by maintaining basal inhibition of Src-family kinases, particularly LCK, through phosphorylation of the an inhibitory C-terminal tyrosine residue; unlike most kinases, CSK functions as a negative regulator that suppresses downstream signaling cascades. While the biochemical role of CSK in T cell signaling has been well established, approaches to externally regulate CSK activity in a reversible and modular manner have remained relatively underexplored. Despite its central role in immune signaling, tools to reversibly and inducibly activate CSK in living cells have remained limited.

[0006] Chimeric antigen receptor (CAR) T cell immunotherapy has transformed the treatment of hematologic malignancies and is now being actively explored for solid tumors by harnessing the immune system to eliminate malignant cells. To date, the U.S. Food and Drug Administration has approved seven CAR-T cell therapies, including five targeting CD 19 in B-cell leukemias andAttorney Docket No. 0073605-001129 lymphomas and two targeting B-cell maturation antigen (BCMA) in multiple myeloma. In parallel, the recent approval of a transgenic T-cell receptor (TCR) therapy targeting MAGE-A4 highlights the broader emergence of engineered T-cell medicines in clinical care. Despite these advances, engineered T-cell therapies remain associated with substantial toxicities, most notably cytokine release syndrome (CRS), which arises from excessive immune activation during tumor engagement. CRS is common rather than rare, with reported incidence rates exceeding 70% in many clinical cohorts, and can progress rapidly to life-threatening organ dysfunction.

[0007] Current clinical management of CRS relies primarily on pharmacological interventions, each of which presents important limitations. High-dose corticosteroids, often used as first-line therapy, broadly suppress T-cell receptor signaling and have been associated with reduced CAR-T cell persistence and diminished therapeutic efficacy. Alternatively, IL-6 blockade with tocilizumab effectively alleviates systemic inflammatory symptoms but does not cross the blood-brain barrier, limiting its utility in CAR-T-cell-related encephalopathy syndrome (CRES). To overcome these pharmacological constraints, genetic “safety switches” have been developed to embed control mechanisms directly within therapeutic cells. These safety switches include suicide gene systems such as inducible caspase-9 (iCasp9) and herpes simplex virus thymidine kinase (HSV-TK), as well as antibody-mediated elimination markers such as truncated EGFR (huEGFRt). However, a fundamental limitation of these approaches is their binary and irreversible nature: they permanently eliminate the therapeutic cells rather than modulating their activity. In addition, strategies relying on viral proteins introduce non-human epitopes that may promote immunogenicity, while many existing safety switches require redesign for each CAR architecture or target antigen, limiting their generalizability.

[0008] Recent advances in synthetic biology have introduced more nuanced regulatory strategies, including “rheostat” systems such as CRASH-IT switches and lenalidomide-gated CARs. These platforms enable reversible and tunable control over T-cell function through small-molecule-induced protein degradation or recruitment of inhibitory signaling domains, representing a significant improvement over irreversible suicide mechanisms. Nevertheless, these approaches remain fundamentally open-loop systems that depend on physician recognition of toxicity, drug administration, and pharmacokinetic distribution. In the setting of hyper-acute CRS, where clinical deterioration can occur rapidly, this inherent diagnostic and interventional latency may limit their ability to prevent severe toxicity.

[0009] To address this limitation, there is growing interest in autonomous, closed-loop regulatory systems capable of self-modulating cellular activity in real time. Such concepts have beenAttorney Docket No. 0073605-001129 validated in other contexts, including glucose-responsive insulin circuits that function as beta-cell mimetics to restore metabolic homeostasis. More recently, landmark work demonstrated the feasibility of engineering complex protein logic gates and feedback loops in mammalian cells.However, these systems have largely been characterized in model cell lines that lack the specialized signaling networks and cytotoxic machinery of functional T cells. As a result, a translational gap remains in adapting closed-loop synthetic circuits to operate reliably within the highly dynamic and inflammatory environment of therapeutic T cells.SUMMARY OF THE DISCLOSURE

[0010] An aspect of the present disclosure is a synthetic protein switch having an active state and an inactive state, the synthetic protein switch including: (I) a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11; and (II) a sensing domain. The synthetic protein switch (i) reversibly converts from the inactive state to the active state upon interaction with an inducer; and (ii) reversibly converts from the active state to the inactive state upon removal of the inducer.

[0011] In some embodiments, the sensing domain of the synthetic protein switch includes a FKBP12F36Vsensing domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 9.

[0012] In some embodiments, the CSK domain of the synthetic protein switch contains a Y304 autophosphorylation site.

[0013] In some embodiments, when the synthetic protein switch is under the active state, the sensing domain is in a dimeric state; and when the synthetic protein switch is under the inactive state, the sensing domain is in a monomeric state.

[0014] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch catalyzes phosphorylation of lymphocyte-specific protein tyrosine kinase (LCK), thereby attenuating or suppressing T-cell signaling.

[0015] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch enhances phosphorylation at Y505 position of the LCK and suppresses phosphorylation at Y394 position of the LCK, thereby stabilizing the LCK in an inactive conformation.

[0016] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch suppresses production or secretion of an inflammatory cytokine. In some embodiments, the inflammatory cytokine includes an extracellular inflammatory cytokine. In some embodiments, the inflammatory cytokine includes an intracellular cytokine. InAttorney Docket No. 0073605-001129 some embodiments, the inflammatory cytokine includes one or more of interleukin-2 (IL-2), interferon-y (IFNy), and tumor necrosis factor-a (TNFa).

[0017] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch suppresses CD3^ phosphorylation.

[0018] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch suppresses cytotoxic killing of a target cell.

[0019] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch suppresses antigen-dependent CAR-T activation without causing nonspecific cellular dysfunction.

[0020] In some embodiments, the inducer that activates the synthetic protein switch includes a dimerizer that converts the sensing domain from the monomeric state to the dimeric state. In some embodiments, the dimerizer includes (lR,rR)-(((((2-((dimethylamino)methyl)propane-l,3-diy 1 )b i s(azanediyl))bi s(2-oxoethane-2, 1 -di y 1 ))b i s(oxy))bi s(3 , 1 -phenylene))bis(3 -(3,4-dimethoxyphenyl)propane- 1,1 -diyl) (2S,2’S)-bis(l-((S)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate), which is also known as AP20187 or a B / B homodimerizer (molecular formula: C82H107N5O20).

[0021] In some embodiments, the inducer that activates the synthetic protein switch includes a cytokine. In some embodiments, the cytokine includes interleukin-6 (IL-6). In some embodiments, the CSK domain of the synthetic protein switch includes an interleukin-6 (IL-6) receptor.

[0022] In some embodiments, the synthetic protein switch has a transmembrane configuration.

[0023] In some embodiments, the CSK domain of the synthetic protein switch is to be positioned at an inner leaflet of a plasma membrane.

[0024] In some embodiments, the synthetic protein switch further includes a CD8 hinge region that is at least 70% identical to, or a functional variant of, SEQ ID NO: 26. In some embodiments, the synthetic protein switch further includes a CD28 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 25.

[0025] In some embodiments, the sensing domain of the synthetic protein switch includes an hIL6Ra extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 13.

[0026] In some embodiments, the sensing domain of the synthetic protein switch includes an hIL6Ra transmembrane domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 14.Attorney Docket No. 0073605-001129

[0027] In some embodiments, the sensing domain of the synthetic protein switch includes a gpl30 extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 16.

[0028] In some embodiments, the sensing domain of the synthetic protein switch includes a gpl30 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 22.

[0029] In some embodiments, the sensing domain of the synthetic protein switch includes a GP130 domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 17.

[0030] In some embodiments, the sensing domain of the synthetic protein switch includes an IL6Ra domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 19

[0031] In some embodiments, the sensing domain of the synthetic protein switch includes an IL7TM domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 21.

[0032] In some embodiments, the synthetic protein switch further includes an N-terminal myristoylation signal having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 8.

[0033] In some embodiments, the synthetic protein switch further includes a signal peptide having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 15.

[0034] In some embodiments, the synthetic protein switch further includes a TEL linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 10. In some embodiments, the synthetic protein switch further includes a (GsS)4 linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 12.

[0035] In some embodiments, the T-cell signaling is mediated by a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0036] In some embodiments, the synthetic protein switch has a molar mass of from approximately 70kDa to proximately 100 kDa, preferably approximately 80 kDa, as determined by, e.g., immunoblot analysis.

[0037] In some embodiments, the CSK domain of the synthetic protein switch contains one or more point mutations and one or more of S186A, Y188A, and W134A positions.Attorney Docket No. 0073605-001129

[0038] In some embodiments, the synthetic protein switch has an amino acid sequence that is at least 70% identical to, or a functional variant of, any one of SEQ ID NOs: 1-7.

[0039] Another aspect of the present disclosure is a method for reversible modulation or regulation of T-cell signaling in a subject in need thereof, the method including providing or administering to the subject a therapeutically effective amount of a synthetic protein switch having an active state and an inactive state. The synthetic protein switch includes (I) a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11; and (II) a sensing domain, wherein an interaction between the synthetic protein switch and an inducer reversibly converts the synthetic protein switch from the inactive state to the active state.

[0040] Another aspect of the present disclosure is a method of treating a cytokine release syndrome (CRS) in a subject in need thereof, the method including providing or administering to the subject a therapeutically effective amount of a synthetic protein switch having an active state and an inactive state. The synthetic protein switch includes (I) a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11; and (II) a sensing domain, wherein an interaction between the synthetic protein switch and an inducer reversibly converts the synthetic protein switch from the inactive state to the active state.

[0041] Another aspect of the present disclosure is a method of treating a medical condition, such as a hematological malignancy, the method including providing or administering to the subject (A) a therapeutically effective amount of T cells; and (B) a therapeutically effective amount of a synthetic protein switch having an active state and an inactive state. The synthetic protein switch includes a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11; and (II) a sensing domain, wherein an interaction between the synthetic protein switch and an inducer reversibly converts the synthetic protein switch from the inactive state to the active state, thereby modulating or regulating (e.g., suppressing, attenuating, or downregulating) an activity of the T cells.

[0042] In some embodiments of the methods, an attenuation or removal of the inducer reversibly converts the synthetic protein switch from the active state to the inactive state.

[0043] In some embodiments of the methods, the sensing domain of the synthetic protein switch includes a FKBP12F36Vsensing domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 9.

[0044] In some embodiments of the methods, the CSK domain of the synthetic protein switch contains a Y304 autophosphorylation site.Attorney Docket No. 0073605-001129

[0045] In some embodiments of the method, when the synthetic protein switch is under the active state, the sensing domain is in a dimeric state; and when the synthetic protein switch is under the inactive state, the sensing domain is in a monomeric state.

[0046] In some embodiments, the method further includes catalyzing phosphorylation of lymphocyte-specific protein tyrosine kinase (LCK), using the synthetic protein switch under its active state, thereby attenuating or suppressing T-cell signaling.

[0047] In some embodiments, the method further includes enhancing phosphorylation at Y505 position of the LCK and suppressing phosphorylation at Y394 position of the LCK, using the synthetic protein switch under its active state, thereby stabilizing the LCK in an inactive conformation.

[0048] In some embodiments, the method further includes suppressing production or secretion of an inflammatory cytokine using the synthetic protein switch under its active state. In some embodiments of the method, the inflammatory cytokine includes an extracellular inflammatory cytokine. In some embodiments of the method, the inflammatory cytokine includes an intracellular cytokine. In some embodiments of the method, the inflammatory cytokine includes one or more of interleukin-2 (IL -2), interferon-y (IFNy), and tumor necrosis factor-a (TNFa).

[0049] In some embodiments, the method further includes suppressing CD3^ phosphorylation using the synthetic protein switch under its active state.

[0050] In some embodiments, the method further includes suppressing cytotoxic killing of a target cell using the synthetic protein switch under its active state.

[0051] In some embodiments, the method further includes suppressing antigen-dependent CAR-T activation without causing nonspecific cellular dysfunction using the synthetic protein switch under its active state.

[0052] In some embodiments, the method further includes converting the sensing domain of the synthetic protein switch from the monomeric state to the dimeric state using a dimerizer. In some embodiments of the method, the dimerizer includes AP20187, in accordance with details described throughout the present disclosure.

[0053] In some embodiments of the method, the inducer that activates the synthetic protein switch includes a cytokine. In some embodiments of the method, the cytokine includes interleukin-6 (IL-6). In some embodiments of the method, the CSK domain of the synthetic protein switch includes an interleukin-6 (IL-6) receptor.

[0054] In some embodiments of the method, the synthetic protein switch has a transmembrane configuration.Attorney Docket No. 0073605-001129

[0055] In some embodiments of the method, the CSK domain of the synthetic protein switch is to be positioned at an inner leaflet of a plasma membrane.

[0056] In some embodiments of the method, the synthetic protein switch further includes a CD8 hinge region that is at least 70% identical to, or a functional variant of, SEQ ID NO: 26.

[0057] In some embodiments of the method, the synthetic protein switch further includes a CD28 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 25.

[0058] In some embodiments of the method, the sensing domain of the synthetic protein switch includes an hIL6Ra extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 13.

[0059] In some embodiments of the method, the sensing domain of the synthetic protein switch includes an hIL6Ra transmembrane domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 14.

[0060] In some embodiments of the method, the sensing domain of the synthetic protein switch includes a gpl30 extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 16.

[0061] In some embodiments of the method, the sensing domain of the synthetic protein switch includes a gpl30 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 22.

[0062] In some embodiments of the method, the sensing domain of the synthetic protein switch includes a GP130 domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 17.

[0063] In some embodiments of the method, the sensing domain of the synthetic protein switch includes an IL6Ra domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 19.

[0064] In some embodiments of the method, the sensing domain of the synthetic protein switch includes an IL7TM domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 21.

[0065] In some embodiments of the method, the synthetic protein switch further includes an N-terminal myristoylation signal having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 8.Attorney Docket No. 0073605-001129

[0066] In some embodiments of the method, the synthetic protein switch further includes a signal peptide having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ IDNO: 15.

[0067] In some embodiments of the method, the synthetic protein switch further includes a TEL linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 10. In some embodiments of the method, the synthetic protein switch further includes a (G3S)4linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ IDNO: 12.

[0068] In some embodiments of the method, the T-cell signaling is mediated by a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0069] In some embodiments of the method, the synthetic protein switch has a molar mass of from approximately 70kDato proximately 100 kDa, preferably approximately 80 kDa, as determined by, e.g., immunoblot analysis.

[0070] In some embodiments of the method, the CSK domain of the synthetic protein switch contains one or more point mutations and one or more of S186A, Y188A, and W134A positions.

[0071] In some embodiments of the method, the synthetic protein switch has an amino acid sequence that is at least 70% identical to, or a functional variant of, any one of SEQ ID NOs: 1-7.

[0072] Another aspect of the present disclosure is the use of the synthetic protein switch described herein in the manufacture of a medicament for reversible modulation or regulation of T-cell signaling in a subject in need thereof.

[0073] Another aspect of the present disclosure is the use of the synthetic protein switch described herein in the manufacture of a medicament for treating cytokine release syndrome (CRS) in a subject in need thereof.

[0074] Another aspect of the present disclosure is the use of the synthetic protein switch described herein in combination with one or more T cell therapies in the manufacture of a medicament for treating a hematological malignancy in a subject in need thereof.

[0075] In some embodiments, the T cell therapy to be used in combination with the synthetic protein switch described herein includes a chimeric antigen receptor (CAR) T cell therapy or a transgenic T-cell receptor (TCR) therapy.

[0076] Another aspect of the present disclosure is a method of producing the synthetic protein switch described herein, the method including transferring to a host cell or transfecting the host cell with a viral vector having a nucleic acid (e.g., a plasmid) encoding the synthetic protein switch. In some embodiments, the viral vector includes a lentiviral vector. In some embodiments, the methodAttorney Docket No. 0073605-001129 further includes culturing the transfected host cell under suitable conditions to produce the synthetic protein switch.

[0077] These and other aspects and features of nonlimiting embodiments of the present invention will become apparent to those skilled in the art upon review of the following description of specific nonlimiting embodiments of the invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] For the purpose of illustrating the invention, the drawings show aspects of one or more embodiments of the invention. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities shown in the drawings.

[0079] FIG. 1A-1E depict exemplary schematics and experimental data showing that chemical induction of CSK dimerization activates CSKdim phosphorylation at Y304. FIG. 1A is an exemplary schematic of proximal T cell receptor (TCR) signaling in activated T cells. Engagement of the TCR complex leads to autophosphorylation of the Src-family kinase LCK at the activating residue Y394, initiating downstream signaling. In resting or inhibited states, C-terminal Src kinase (CSK) suppresses TCR signaling by phosphorylating LCK at the inhibitory residue Y505, maintaining LCK in an inactive conformation. FIG. IB depicts an exemplary schematic showing the design and mechanism of the chemically inducible CSK dimerization construct (CSKdim). The construct is expressed under the EFla promoter and includes an N-terminal myristoylation signal for plasma membrane localization, an FKBP12(F36V) sensing domain, a truncated ETV6 (TEL)-derived linker (amino acids 125-335), and the CSK kinase domain (amino acids 195-449). Addition of the smallmolecule dimerizer AP20187 induces FKBP-mediated homodimerization at the membrane, enforcing proximity -driven activation of CSK and autophosphorylation at tyrosine 304 (Y304). FIG.1C-1D depict an exemplary flow cytometry analysis of CSK activation in lurkat T cells. Cells expressing either shCSK alone or shCSK complemented with CSKdim were treated with DMSO or AP20187 and stained with a phospho-specific antibody against pCSK Y304. Representative contour plots and histograms show a marked increase in CSK Y304 phosphorylation upon dimerizer treatment in CSKdim-expressing cells but not in shCSK controls. Quantification of mean fluorescence intensity (MFI) demonstrates a significant induction of pCSK Y304 following chemical dimerization (***P < 0.001). FIG. IE depicts exemplary results showing an immunoblot validation of chemically induced CSK activation. Jurkat T cells expressing CSKdim, with or without endogenous CSK knockdown (shCSK), were treated with AP20187 as indicated. Western blot analysis reveals a robust increase in phosphorylation of CSK at Y304 upon dimerizer treatment, while total CSKdim expression remains unchanged. GAPDH is shown as a loading control.Attorney Docket No. 0073605-001129

[0080] FIG. 2A-2H depict exemplary schematics and experimental data showing that an inducible CSKdim described herein suppresses T cell receptor signaling by increasing inhibitory LCK Y505 phosphorylation and reducing activating LCK Y394 phosphorylation. FIG. 2A depicts an exemplary conceptual model of LCK regulation by CSKdim. In resting T cells, LCK cycles between open and closed conformations. Activation of CSKdim promotes phosphorylation of LCK at the inhibitory residue Y505, stabilizing LCK in a closed, inactive conformation and preventing autophosphorylation at the activating residue Y394. This shift can suppress proximal T cell receptor (TCR) signaling. FIG. 2B-2C depict an exemplary flow cytometry analysis of inhibitory LCK Y505 phosphorylation in Jurkat T cells expressing CSKdim or control CSK. Cells were stimulated with anti-CD3 antibodies in the presence of DMSO or the dimerizer AP20187 and stained with a phospho-specific antibody against pLCK Y505. Representative contour plots and histograms demonstrate a robust increase in Y505 phosphorylation upon CSKdim activation, whereas control CSK-expressing cells show minimal change. Quantification of mean fluorescence intensity (MFI) reveals a significant induction of pLCK Y505 following chemical activation of CSKdim (***P < 0.001). FIG 2D depicts exemplary results of an immunoblot validation of LCK Y505 phosphorylation. Jurkat-CSKdim cells were treated with AP20187 as indicated, and whole-cell lysates were analyzed by Western blot. Dimerizer treatment increases phosphorylation of LCK at Y505 without altering total LCK levels. GAPDH is shown as a loading control. FIG. 2E-2G depict an exemplary flow cytometry analysis of activating LCK Y394 phosphorylation following TCR stimulation. Jurkat T cells expressing CSKdim or control CSK were left unstimulated or stimulated with CD3 / CD28 antibodies in the presence of DMSO or AP20187. Representative contour plots and histograms show that TCR stimulation induces strong Y394 phosphorylation, which is markedly suppressed upon CSKdim activation. In contrast, dimerizer treatment does not significantly affect Y394 phosphorylation in control CSK-expressing cells. Quantification of pLCK Y394 levels by flow cytometry. Mean fluorescence intensity (MFI) demonstrates that chemical activation of CSKdim significantly reduces TCR-induced LCK Y394 phosphorylation, whereas control CSK cells maintain high Y394 phosphorylation upon stimulation (***p < 0.001). FIG. 2H depicts an exemplary immunoblot analysis of LCK Y394 phosphorylation in wild-type and Jurkat-CSKdim cells. Cells were stimulated with CD3 / CD28 antibodies in the presence or absence of AP20187. Consistent with flow cytometry data, activation of CSKdim suppresses phosphorylation of LCK at Y394 without affecting total LCK expression. GAPDH is shown as a loading control.

[0081] FIG. 3A-3G depict exemplary schematics and experimental data showing that a CSKdim described herein reversibly inhibits inflammatory cytokine secretion in activated T cells. FIG. 3A-3BAttorney Docket No. 0073605-001129 depict an exemplary conceptual schematic illustrating CSKdim-mediated regulation of T cell activation. In activated T cells, TCR engagement induces LCK autophosphorylation at Y394, driving cytokine secretion and effector function. Chemical activation of CSKdim promotes inhibitory phosphorylation of LCK at Y505, suppressing downstream signaling and cytokine production, thereby returning cells to a resting-like state. FIG. 3C-3E depict an exemplary schematic of experimental timeline and exemplary results from an ELISA analysis of cytokine secretion in T cells expressing CSKdim. Cells were stimulated with CD3 / CD28 Dynabeads for 16 hours, treated with 10 nM AP20187 to activate CSKdim, and re-stimulated following dimerizer removal. IFNy and IL-2 ELISA results show that CSKdim activation significantly reduces cytokine secretion, which is restored upon dimerizer withdrawal, demonstrating reversible control. FIG. 3F depicts exemplary data showing dose-dependent regulation of IL-2 secretion by CSKdim in Jurkat T cells. IL-2 levels were measured following treatment with increasing concentrations of AP20187. CSKdim-expressing cells exhibit tunable suppression of IL-2 secretion, whereas wild-type and shLCK control cells show minimal response. FIG. 3G depicts exemplary results of NF AT- and IL-2-luciferase reporter assays in Jurkat T cells. Cells were stimulated with CD3 / CD28 antibodies in the presence of DMSO or 10 nM AP20187. Activation of CSKdim significantly reduces NF AT- and IL-2-dependent luciferase activity compared to controls, confirming suppression of TCR-driven transcriptional programs.

[0082] FIG. 4A-4C depict exemplary schematics and data showing that activation of CSKdim inhibits T cell-mediated cytotoxic killing. FIG. 4A depicts an exemplary schematic illustration of T cell-mediated killing in the presence and absence of CSKdim activation. Blinatumomab (anti-CD3 x anti-CD19 BiTE) redirects cytotoxic T cells to CD19+cancer cells, inducing LCK Y394 phosphorylation, CD3(^ signaling, and target cell killing when CSKdim is OFF. Chemical activation of CSKdim by the dimerizer AP20187 promotes inhibitory LCK Y505 phosphorylation, suppressing TCR signaling, and cytotoxic activity. FIG. 4B-4C depict an exemplary flow cytometry analysis of T cell-mediated killing of CD19+BV173 cells. CSKdim-expressing T cells were co-cultured with BV173 cells in the presence of Blinatumomab and treated with DMSO or AP20187. Representative flow plots and quantification show that activation of CSKdim significantly reduces the percentage of CD19+target cells killed. FIG. 4C depicts an exemplary flow cytometry analysis of T cell-mediated killing of CD19+RAJI cells. Similar to BV173 cells, activation of CSKdim significantly inhibits cytotoxic killing of RAJI targets and reduces CD3+T cell expansion. Quantification confirms consistent suppression of T cell effector function upon CSKdim activation.

[0083] FIG. 5A-5F depict exemplary schematics and experimental results showing that CSKdim inhibits extracellular and intracellular cytokine production in CAR-T cells. FIG. 5A depicts anAttorney Docket No. 0073605-001129 exemplary conceptual schematic illustrating CAR-T activation and inhibition by CSKdim. In active CAR-T cells, antigen engagement induces LCK phosphorylation at the activating residue Y394, leading to secretion of inflammatory cytokines. Chemical activation of CSKdim by AP20187 promotes inhibitory phosphorylation of LCK at Y505, suppressing downstream CAR signaling and cytokine production. FIG. 5B-5D depict an exemplary schematic of experimental timeline and exemplary results from an ELISA analysis of extracellular cytokine secretion in third-generation FMC63 anti-CD19 CAR-T cells. CAR-T cells were co-cultured with CD19+BV173 cells, treated with 10 nM AP20187 to activate CSKdim, and re-assessed following dimerizer removal. ELISA measurements of IL-2 and IFNy show significant suppression during CSKdim activation with recovery upon dimerizer withdrawal. FIG. 5E-5F depict an exemplary intracellular cytokine analysis in CAR-T cells. FMC63 CAR-T cells expressing CSKdim were co-cultured with CD19+target cells (BV173, K562-CD19+, RAJI-CD19+) or CD19’ K562 cells in the presence of DMSO or AP20187. Flow cytometry quantification shows that activation of CSKdim significantly reduces the proportion of CD3+CAR-T cells producing IL-2, IFNy, and TNFa in response to CD19~ targets, while responses to CD 19" cells remain low.

[0084] FIG. 5G-5J depict exemplary results showing that inhibition of inflammatory cytokine secretion was observed in all generations of the CAR-T.

[0085] FIG. 6A-6D depict exemplary schematics and experimental data showing that activation of the CSKdim switch inhibits cytotoxic killing by second-generation (4-1BB) CAR-T cells. FIG. 6A depicts an exemplary schematic illustration of CAR-T cell-mediated killing in the presence and absence of CSKdim activation. When CSKdim is OFF, engagement of CD19 by second-generation (4-1BB) CAR-T cells induces LCK phosphorylation at Y394, leading to CD3^ signaling, cytokine release, and tumor cell killing. Chemical activation of CSKdim by AP20187 promotes inhibitory LCK Y505 phosphorylation, suppressing CAR signaling and cytotoxic activity. FIG. 6B depicts exemplary results from a CAR-T killing assay across multiple effector-to-target (E:T) ratios. FMC63 anti-CD194-1BB CAR-T cells expressing CSKdim were co-cultured with BV173-CD19+target cells in the presence of DMSO or 10 nM AP20187. Activation of CSKdim significantly reduces the percentage of CD19+target cells killed across all tested E:T ratios (***P < 0.001). FIG. 6C-6D depict exemplary results showing antigen-specific inhibition of CAR-T expansion and cytotoxicity by CSKdim. FMC 63 -CAR-T -CSKdim cells were co-cultured with CD19+target cell lines (BV173-CD19+, RAJI-CD19+, K562-CD19+) or CD19’ K562 cells. Activation of CSKdim significantly reduces CAR-T expansion (fold increase) and CD19+target cell killing, while minimal killing of CD 19“ cells is observed under all conditions.Attorney Docket No. 0073605-001129

[0086] FIG. 7A-7C depict exemplary data showing that CSKdim-mediated inhibition of CAR-T killing is reversible. FIG. 7A-7B depict exemplary data showing the reversibility of CAR-T cytotoxicity against CD19+leukemia targets. FMC63 (4-1BB) CAR-T cells expressing CSKdim were co-cultured with BV173-CD19+or RAJI-CD19+target cells in the presence of 10 nM AP20187 or DMSO. Quantification of CD3 CAR-T expansion (fold increase) and percentage of CD19+target cells killed shows that CSKdim activation suppresses CAR-T killing, while removal of the dimerizer restores both CAR-T expansion and cytotoxic function. FIG. 7C depicts exemplary results from a temporal washout experiment demonstrating reversibility of CAR-T killing in HEK 293-hCD19+cells. FMC63 -CAR-T -CSKdim cells were co-cultured with HEK 293-hCD19+targets and treated with AP20187 for two days, followed by ligand washout and continued co-culture. Percent CD19+target cell killing was quantified after ligand removal. CSKdim activation suppresses CAR-T cytotoxicity, whereas ligand washout restores killing capacity. Minimal killing is observed against CD19 HEK 293 controls. (***P < 0.001).

[0087] FIG. 8A-8C depict exemplary schematics and experimental results pertaining to reengineering of CSKdim from a cytoplasmic to an extracellularly configurable transmembrane switch. FIG. 8A depicts exemplary schematic representations of CSKdim architectures. The original intracellular CSKdim (inCSKdim) includes an N-terminal myristoylation signal, tandem FKBP12(F36V) sensing domains, a TEL-derived linker, and the CSK kinase domain. Extracellular CSKdim variants replace the myristoylation signal with a signal peptide (SP), CD8 hinge region, and CD28 transmembrane domain, positioning CSK intracellularly while enabling extracellular control. Two linker configurations were tested: a flexible (G4S)a linker (exCSKdim-G4S) and a TEL-derived linker (exCSKdim-TEL). FIG. 8B depicts exemplary results from IL-2 ELISA in Jurkat T cells expressing intracellular or extracellular CSKdim constructs. Cells were left unstimulated or stimulated with CD3 / CD28 antibodies in the presence or absence of 10 nM B / B homodimerizer. Activation of intracellular and extracellular CSKdim significantly suppresses IL-2 secretion compared to stimulated controls. shLCKl serves as a control for LCK-dependent signaling. FIG. 8C depicts an exemplary flow cytometry analysis of LCK activation. Representative histograms show phosphorylation of LCK at Y394 following CD3 / CD28 stimulation in Jurkat cells expressing intracellular or extracellular CSKdim constructs. Activation of CSKdim reduces pLCK-Y394 levels across both cytoplasmic and transmembrane designs. Bar graph summarizes suppression of LCK activation upon CSKdim activation, confirming preserved inhibitory function following cytoplasmic-to-extracellular reprogramming.Attorney Docket No. 0073605-001129

[0088] FIG. 9A-9E depict exemplary schematics and experimental results showing that engineering an IL-6-responsive CSK switch enables closed-loop inhibition of TCR signaling. FIG.9A depicts an exemplary conceptual schematic illustrating the stepwise re-engineering of CSKdim. An intracellular, chemically inducible CSK dimerization system is first established using FKBP12(F36V) homodimerization. This system is then converted into a transmembrane, dimerization-dependent switch using a CD8 hinge and CD28 transmembrane domain. Finally, FKBP-based dimerization is replaced with IL-6-dependent heterodimerization of IL-6Ra and gpl30 extracellular domains, coupling IL-6 sensing to CSK activation and inhibition of Src-family kinases (LCK and FYN). FIG. 9B depicts exemplary results showing cytokine suppression by IL-6-dependent CSK activation. Jurkat T cells expressing IL6Ra-TEL-CSK and gpl30-TEL-CSK were stimulated with CD3 / CD28 antibodies in the presence of increasing concentrations of IL-6. IFNy and IL-2 secretion was quantified and shows dose-dependent suppression compared to wild-type controls. FIG. 9C depicts an exemplary immunoblot analysis of inhibitory LCK phosphorylation. Cells expressing IL6Ra / gpl30-CSK constructs were treated with increasing concentrations of IL-6. Western blotting demonstrates increased phosphorylation of LCK at Y5O5 in response to IL-6, while total protein levels remain unchanged. P-actin serves as a loading control. FIG. 9D depicts an exemplary flow cytometry analysis of pLCK-Y505. Representative histograms show increased inhibitory phosphorylation of LCK following IL-6 stimulation in IL6Ra / gpl30-CSK-expressing cells compared to controls. FIG. 9E depicts an exemplary flow cytometry analysis of activating pLCK-Y394. IL-6 treatment suppresses CD3 / CD28-induced phosphorylation of LCK at Y394 in cells expressing IL6Ra / gpl30-CSK, confirming inhibition of proximal TCR signaling.

[0089] FIG. 10A-10D depict exemplary results showing that the IL-6-responsive split inhibitory receptors modulate LCK phosphorylation at CRS-relevant IL-6 concentrations. FIG. 10A depicts a summary of reported serum IL-6 concentrations associated with different clinical grades of cytokine release syndrome (CRS), ranging from healthy individuals to severe disease. FIG. 10B depicts a schematic illustration of the IL-6-responsive split inhibitory receptor (IL6-SIR) design. The extracellular domains of IL-6Ra and gp!30 are fused to transmembrane domains and intracellular CSK kinase domains via a TEL-derived linker. IL-6 binding promotes receptor association, bringing CSK kinase domains into proximity. Domain organization of IL6-SIR constructs showing extracellular receptor domains, transmembrane regions, TEL linker, and CSK kinase domain containing the Y304 autophosphorylation site. FIG. 10C depicts an exemplary immunoblot analysis of inhibitory LCK phosphorylation. Cells expressing IL6-SIRs were treated with increasing concentrations of IL-6. Phosphorylation of LCK at Y505 and total LCK levels are shown, withAttorney Docket No. 0073605-001129 GAPDH as a loading control. Quantification of pLCK-Y505 normalized to total LCK. Bar graph summarizes changes in inhibitory LCK phosphorylation across IL-6 treatment conditions (ns, not significant; ***P < 0.001 as indicated). FIG. 10D depicts an exemplary immunoblot analysis of activating LCK phosphorylation during CD3 / CD28 stimulation in the presence of increasing IL-6 concentrations. Quantification of pLCK-Y394 normalized to total LCK. Bar graph shows reduced activating LCK phosphorylation in the presence of IL-6 during TCR stimulation.

[0090] FIG. 11 A-l ID depict exemplary schematics and experimental results showing that mutational analysis of IL-6 split inhibitory receptors reveals residue-specific effects on LCK Y505 phosphorylation. FIG. 11A depicts an exemplary schematic of IL-6 split inhibitory receptor (IL6-SIR) constructs. The extracellular domains of IL-6Ra or gpl30 are fused to their respective transmembrane domains and intracellular CSK kinase domains via a TEL-derived linker. Point mutations (W134A, S186A, Y188A) were introduced into the IL-6R01 extracellular domain. FIG. 11B depicts an exemplary immunoblot analysis of inhibitory LCK phosphorylation (pLCK-Y505) in cells expressing IL6-SIR constructs with wild-type or mutant IL-6Ra extracellular domains under no-IL-6 and IL-6-treated conditions. Total LCK is shown as a loading reference. FIG. 11C-1 ID depict an exemplary immunoblot analysis of pLCK-Y505 following exposure to elevated IL-6 concentration relevant to severe inflammatory conditions. Differences in pLCK-Y505 levels are observed across IL-6Ra mutants relative to the unmutated receptor. Quantification of pLCK-Y505 normalized to total LCK. Bar graph summarizes relative inhibitory LCK phosphorylation across IL-6Ra variants. Statistical annotations are shown as indicated (ns, not significant; ***P < 0.001).

[0091] FIG. 12A-12C depict exemplary schematics and experimental data showing that a single-chain IL-6 inhibitory receptor influences activating and inhibitory LCK phosphorylation. FIG.12A depicts an exemplary schematic of the single-chain IL-6 inhibitory receptor (IL6 scIR). The extracellular domains of gp!30 and IL-6Ra are fused in cis and linked to a transmembrane domain, followed by a TEL-derived linker and the CSK kinase domain containing the Y304 autophosphorylation site. This design is inspired by prior CAR-T chimeric cytokine receptor architectures using IL-6Ra and gp!30 extracellular domains. FIG. 12B depicts an exemplary immunoblot analysis of activating LCK phosphorylation (pLCK-Y394). Cells expressing the IL6 scIR were stimulated with CD3 / CD28 antibodies in the presence of increasing IL-6 concentrations. Total LCK and GAPDH are shown as controls. Quantification of pLCK-Y394 normalized to total LCK. Bar graph summarizes relative levels of LCK activation across conditions. Statistical annotations are shown as indicated. FIG. 12C depicts an exemplary immunoblot analysis of inhibitory LCK phosphorylation (pLCK-Y505) in scIR-expressing cells following IL-6 treatment.Attorney Docket No. 0073605-001129 Total LCK and GAPDH serve as loading controls. Quantification of pLCK-Y505 normalized to total LCK. Bar graph summarizes relative inhibitory phosphorylation levels across IL-6 conditions (ns, not significant; ***P < 0.001 as indicated).

[0092] In certain instances, details that are not necessary for an understanding of the embodiments or that render other details difficult to perceive may have been omitted.DETAILED DESCRIPTION

[0093] The present disclosure demonstrates a reversible (bio)chemical regulation of C-terminal Src kinase (CSK) activity through engineered proximity. Drawing inspiration from receptor tyrosine kinase activation and oncogenic kinase fusions, the present disclosure in particular provides a chemically inducible CSK construct (CSKdim) that combines membrane localization with forced dimerization. Induced CSK proximity is associated with increased inhibitory phosphorylation of LCK and attenuation of proximal T cell receptor (TCR) signaling in T cells. Because LCK and FYN are important signaling nodes for both endogenous TCRs and chimeric antigen receptors (CARs), this strategy provides a suitable means to reversibly modulate engineered T cell activity.

[0094] Building on an initial open-loop design and moving towards autonomous regulation, the present disclosure demonstrates that CSK activation can be coupled to inflammatory cues relevant to cytokine release syndrome (CRS) and that CSKdim can accordingly be reconfigured into a cytokine-responsive system. This approach demonstrates that endogenous inflammatory cues can be harnessed to provide reversible, intrinsic feedback control over engineered T-cell activity.Specifically, by reconfiguring CSKdim into IL-6-responsive receptor architectures, IL-6 sensing can be linked to CSK-mediated inhibitory signaling, and the resulting IL-6-responsive CSK constructs can accordingly influence proximal TCR signaling, consistent with the role of CSK as a negative regulator of Src-family kinases.

[0095] Together, the present disclosure demonstrates the feasibility of repurposing a kinase regulatory motif into both chemically controlled and cytokine-responsive formats. While further optimization and validation may be required, these results provide a framework for integrating reversible, inhibitory feedback into engineered T cell systems without permanently disabling cellular function.

[0096] To facilitate the understanding of this invention, a number of terms are defined below and throughout the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. The terminology herein is used to describe specific embodiments of the invention, but theirAttorney Docket No. 0073605-001129 usage does not limit the invention, except as outlined in the claims. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0097] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0098] As used herein, unless stated otherwise, the terms “about” or “approximately” refer to a value that is within 10% above or below the value being described.

[0099] As used herein, a “subject” includes, but is not limited to, humans and non-human vertebrates such as wild, domestic, and farm animals. The terms “subject” and “patient” may be used interchangeably throughout this disclosure. As a nonlimiting example, a subject or patient can include a human of any age, sex, gender, race, ethnicity, health record, etc., as deemed relevant and / or suitable by a person of ordinary skill in the art, upon reviewing the entirety of this disclosure. Additional nonlimiting examples of subjects or patients can include mammals or birds, such as without limitation, non-human primates, cats, dogs, cows, horses, rodents, pigs, sheep, goats, and poultry. The term subject can refer to any individual in need of treatment. While the present invention primarily describes treatment of human subjects, it should be noted that the scope of the present invention is not limited to human subjects.

[0100] As used herein, the terms “administration” or “administering” refer to a method of giving a dosage of a compound or pharmaceutical composition to a subject. A composition described herein may be administered to a subject by any one of a variety of manners or a combination of varieties of manners. For example, a composition may be administered orally, nasally, intraperitoneally, or parenterally, by intravenous, intramuscular, topical, or subcutaneous routes, or by injection into tissue.

[0101] As used herein, an “effective amount” or “therapeutically effective amount” is the amount of a composition of this disclosure which, when administered to a subject, is sufficient to effect treatment of a disease or condition in the subject. The amount of a composition of this disclosure which constitutes a “therapeutically effective amount” may vary depending on theAttorney Docket No. 0073605-001129 composition, the condition and its severity, the manner of administration, and the age of the subject to be treated.

[0102] As used herein, the terms “treat”, “treating”, or “treatment” refer to administration of a compound or pharmaceutical composition for a therapeutic purpose. To “treat a disorder” or use for “therapeutic treatment” refers to administering treatment to a patient already suffering from a disease to ameliorate the disease or one or more symptoms thereof to improve the patient’s condition (e.g., by reducing one or more symptoms of a neurological disorder). The term “therapeutic” includes the effect of mitigating deleterious clinical effects of certain processes (i.e., consequences of the process, rather than the symptoms of processes). As nonlimiting examples, a treatment may include (i) preventing a disease or condition from occurring in a subject, in particular, when such subject is predisposed to the condition but has not yet been diagnosed as having it; (ii) inhibiting a disease or condition, i.e., arresting its development; (iii) relieving a disease or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from a disease or condition, i.e., relieving pain without addressing the underlying disease or condition.

[0103] It will be appreciated that variations to the embodiments of the invention can be made while still falling within the scope of the invention. Alternative features serving the same, equivalent, or similar purpose can replace features disclosed in the specification, unless stated otherwise. Thus, unless stated otherwise, each feature disclosed represents one example of a generic series of equivalent or similar features.Synthetic Protein Switch

[0104] An objective of the present disclosure is to provide a synthetic protein switch that reversibly modulates or regulates T-cell signaling. As used herein, a “synthetic protein switch” is a synthetic chemical species that (i) includes one or more amino acid sequences or peptide sequences; (ii) has both an active state and an inactive state; by switching between its active and inactive states, the protein switch initiates, terminates, increases, suppresses, and / or otherwise regulates the biological activity of another chemical species.

[0105] Specifically, the synthetic protein switch includes (I) a C-terminal Src kinase (CSK) domain. As used herein, a “C-terminal Src kinase domain,” “CSK domain,” or “CSK kinase domain” is a portion or fragment of the synthetic protein switch that effectively functions as a C-terminal Src kinase. As used herein, “C-terminal Src kinase” or “CSK” is a tyrosine kinase that acts as a negative regulator of a Src family kinase by phosphorylating a tyrosine residue on its C-terminal and converting it into an inactive, closed conformation.Attorney Docket No. 0073605-001129

[0106] In some embodiments, the CSK domain of the synthetic protein switch contains a Y304 autophosphorylation site.

[0107] In some embodiments, the CSK domain of the synthetic protein switch contains one or more point mutations and one or more of S186A, Y188A, and W134A positions.

[0108] The synthetic protein switch further includes (II) a sensing domain. As used herein, a “sensing domain” is a portion or fragment of the synthetic protein switch that, upon interacting with a chemical species of interest, such as an inducer described herein, initiates or triggers a structural or conformational change of at least part of the synthetic protein switch, thereby regulating the biological activity of the protein switch.

[0109] In some embodiments, the interconversion between the active and inactive states of synthetic protein switch is driven by the proximity between the constituents of the sensing domain.

[0110] In some embodiments, the sensing domain includes a dimerization domain that interconverts between a monomeric state and a dimeric state. In some embodiments, when the synthetic protein switch is under the active state, the sensing domain is in a dimeric state; and when the synthetic protein switch is under the inactive state, the sensing domain is in a monomeric state. In some embodiments, the sensing domain includes or forms a homodimer when under its active state. In some embodiments, the sensing domain includes or forms a heterodimer when under its active state. Accordingly, the synthetic protein switch described herein can be referred to as CSKdim throughout the entirety of the present disclosure.

[0111] As used herein, a “monomeric state” is a state in which a molecule, such as a protein, does not bind to, or interact or form a quaternary structure with, another molecule or protein through noncovalent interactions. A molecule, when under such monomeric state, can be referred to as a “monomer.”

[0112] As used herein, a “dimeric state” is a state in which two discrete molecules, such as such as two proteins or protein units, aggregate or assemble through noncovalent interactions. Such aggregate or assembly can accordingly be referred to as a “dimer.” Depending on whether the two constituent molecules are the same or different, a dimer can be referred to as a “homodimer” or “heterodimer,” respectively.

[0113] In some embodiments, when the synthetic protein switch is under the active state, the sensing domain is in a folded or crosslinked state; and when the synthetic protein switch is under the inactive state, the sensing domain is in unfolded or un-crosslinked state. Such folding or crosslinking can be a result of the synthetic protein switch interacting with an inducer, in accordance with details described throughout the present disclosure.Attorney Docket No. 0073605-001129

[0114] The regulatory strategy used in embodiments of the synthetic protein switch described herein mimics the endogenous mechanisms that govern kinase activity in T cells. C-terminal Src kinase (CSK) functions as a central negative regulator of Src-family kinases, including LCK and FYN. Although CSK is typically described as a cytosolic monomer with limited basal activity, structural and biochemical studies have shown that its catalytic activity can be enhanced through scaffold-mediated dimerization, including interactions with the pseudokinase Pragmin. In parallel, oncogenic fusion proteins involving tyrosine kinases and oligomerization domains from TEL (ETV6) demonstrate that enforced proximity is sufficient to drive kinase activation. Accordingly, CSKdim combines membrane localization with inducible dimerization to modulate CSK activity. The enforced proximity of CSK at the membrane enhances inhibitory phosphorylation of LCK and attenuates T-cell signaling, in accordance with details described throughout the present disclosure.

[0115] The synthetic protein switch, when under its active state, effectively functions as an enzyme. As used herein, an “enzyme” is a biological catalyst, often a protein, having a three-dimensional structure specifically tailored for fitting a substrate or reactant and catalyzing a chemical reaction therefrom. Enzymes are often characterized by their high catalytic activity, high specificity toward substrates, and sensitivity to environmental factors such as temperature and pH. A substrate may interact with an enzyme (and its active / binding site) via a lock-and-key mechanism or an induced fit. As used herein, a “catalyst” is a chemical capable of accelerating a chemical reaction by lowering at least an activation barrier along a reaction coordinate and increasing at least a rate constant associated with the at least an activation barrier. In some embodiments, to perform a catalytic function, a catalyst may first be consumed by one or more reactants to form one or more intermediates, then be regenerated as the one or more intermediates are converted to one or more products. In some embodiments, one or more reactants may bind to a catalyst, participate in a chemical reaction, then dissociate from the catalyst as one or more products. The catalytic function of a catalyst or enzyme may be described using mathematical tools such as Arrhenius equation, Eyring equation, Michaelis-Menten equation, Lineweaver-Burk equation, among others, as deemed suitable by a person of ordinary skill in the art upon reviewing the entirety of this disclosure.

[0116] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch catalyzes phosphorylation of lymphocyte-specific protein tyrosine kinase (LCK), thereby attenuating or suppressing T-cell signaling, in accordance with details described throughout the present disclosure.

[0117] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch enhances phosphorylation at Y505 position of the LCKAttorney Docket No. 0073605-001129 and suppresses phosphorylation at Y394 position of the LCK, thereby stabilizing the LCK in an inactive conformation. This process can subsequently suppress downstream signaling and / or cytokine production, thereby returning a cell to a resting-like state, in accordance with details described throughout the present disclosure.

[0118] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch suppresses CD3(^ phosphorylation.

[0119] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch suppresses cytotoxic killing of a target cell.

[0120] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch suppresses antigen-dependent CAR-T activation without causing nonspecific cellular dysfunction.

[0121] The CSK domain of the synthetic protein switch has an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11.

[0122] It should be noted that, when a protein or amino acid sequence described in the present disclosure is referred to as having an amino acid sequence that is “at least 70% identical” to a sequence ID number, the amino acid sequence can be, for example and without limitation, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to that sequence ID number. As nonlimiting examples, the CSK domain of the synthetic protein switch can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 11.

[0123] Sequence identity between two proteins or amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid or base, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percentAttorney Docket No. 0073605-001129 identity involves the production of an optimal alignment, taking into consideration gap penalties. A person of ordinary skill in the art, upon reviewing the entirety of the present disclosure, would be able to identify suitable means to compare sequence identity between two proteins or amino acid sequences.

[0124] As used herein, a “functional variant” of a base protein is a protein that differs from the base protein by one or more amino acids while maintaining at least 70% of the sequence identity and substantially of the functional characteristics of the base peptide. As a nonlimiting example, a functional variant of an enzyme, when applied to the same substrate, can catalyze the conversion of the substrate in a manner similar to that of the enzyme itself. A functional variant can be created via amino acid substitutions, additions, or deletions, but none of such changes should significantly alter the tertiary structure of the base protein from which the functional variant is derived.

[0125] In some embodiments, a functional variant can be created via one or more conservative or non-conservative amino acid substitutions. As used herein, a “conservative amino acid substitution” is a change in amino acid sequence where an amino acid is replaced with a different amino acid with broadly similar properties. As used herein, a “non-conservative amino acid substitution” is a change in amino acid sequence where one amino acid is replaced with another amino acid of a different type. As nonlimiting examples, common amino acids can be categorized as follows: nonpolar amino acids include Ala, Vai, Leu, He, Pro, Met, Phe, and Trp; uncharged polar amino acids include Gly, Ser, Thr, Cys, Tyr, Asn, and Gin; acidic amino acids include Asp and Glu; and basic amino acids include Lys, Arg, and His. Since side chains of amino acids in the same category have similar polarities and are capable of establishing or participating in similar electrostatic interaction(s), hydrogen bond(s), and / or van der Waals contact(s), altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide. Non-conservative substitutions are also possible provided that these substitutions do not disrupt the tertiary structure of an epitope within the peptide. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide. Suitably, functional variants can be at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., with respect to the base protein while retaining the biological function of the protein.

[0126] In some embodiments, the sensing domain of the synthetic protein switch includes a FKBP12F36Vsensing domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 9. As nonlimiting examples, the FKBP12F36Vsensing domain canAttorney Docket No. 0073605-001129 have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 9.

[0127] In some embodiments, the sensing domain of the synthetic protein switch includes an hIL6Ra extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 13. As nonlimiting examples, the hIL6Ra extracellular domain can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 13.

[0128] In some embodiments, the sensing domain of the synthetic protein switch includes an hIL6Ra transmembrane domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 14. As nonlimiting examples, the hIL6Ra transmembrane domain can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 14.

[0129] In some embodiments, the sensing domain of the synthetic protein switch includes a gpl30 extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 16. As nonlimiting examples, the gpl30 extracellular domain can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 16.

[0130] In some embodiments, the sensing domain of the synthetic protein switch includes a gpl30 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 22. As nonlimiting examples, the gpl30 transmembrane domain can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 22.

[0131] In some embodiments, the sensing domain of the synthetic protein switch includes a GP130 domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 17. As nonlimiting examples, the GP130 domain can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 17.Attorney Docket No. 0073605-001129

[0132] In some embodiments, the sensing domain of the synthetic protein switch includes an IL6Ra domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 19. As nonlimiting examples, the IL6Ra domain can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 19.

[0133] In some embodiments, the sensing domain of the synthetic protein switch includes an IL7TM domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 21. As nonlimiting examples, the IL7TM domain can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 21.

[0134] In some embodiments, the synthetic protein switch has a molar mass of from approximately 70kDa to proximately 100 kDa, preferably approximately 80 kDa, as determined by, e.g., immunoblot analysis.Reversible Induction

[0135] The synthetic protein switch described herein (i) reversibly converts from the inactive state to the active state upon interaction with an inducer; and (ii) reversibly converts from the active state to the inactive state upon removal of the inducer. In some embodiments, removal of the inducer reversibly restores the expansion and / or cytotoxic function of the T cell. This reversibility distinguishes CSKdim of the present disclosure from other irreversible suicide switches and supports its utility as a dynamic safety and control module for engineered T cell therapies.

[0136] As used herein, an “inducer” is a chemical or biochemical species that reacts or interacts with a target chemical species, such as the synthetic protein switch described herein, thereby initiating the biological function of the target chemical species.

[0137] In some embodiments, the inducer that activates the synthetic protein switch includes a dimerizer that converts the sensing domain from the monomeric state to the dimeric state. In some embodiments, the dimerizer includes (lR,l’R)-(((((2-((dimethylamino)methyl)propane-l,3-diy 1 )b i s(azanediyl))bi s(2-oxoethane-2, 1 -di y 1 ))b i s(oxy))bi s(3 , 1 -phenylene))bi s(3 -(3,4-dimethoxyphenyl)propane- 1,1 -diyl) (2S,2’S)-bis(l-((S)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate), which is also known as AP20187 or a B / B homodimerizer (molecular formula: C82H107N5O20). Specifically, addition of AP20187 can induceAttorney Docket No. 0073605-001129 FKBP-mediated homodimerization at the cell or plasma membrane, enforcing proximity-driven activation of CSK and autophosphorylation at tyrosine 304 (Y304).

[0138] In some embodiments, the inducer that activates the synthetic protein switch includes a cytokine. In some embodiments, the cytokine includes interleukin-6 (IL-6). IL-6 is a cytokine that plays a central role in the inflammatory cascade underlying systemic toxicity and neuroinflammation For example, severe CRS is typically characterized by rapid elevations in circulating IL-6. Rather than treating IL-6 solely as a biomarker, the present disclosure describes direct coupling of IL-6 sensing to inhibitory signaling within engineered T cells. Such IL-6-responsive transmembrane kinase design can enable closed-loop suppression of TCR signaling and dynamically suppress TCR signaling in response to inflammatory cues.

[0139] Accordingly, in some embodiments, the CSK domain of the synthetic protein switch includes an interleukin-6 (IL-6) receptor. Specifically, IL-6 engagement promotes receptor association, enabling signal propagation across the membrane to influence intracellular CSK activity and thereby attenuate LCK- and FYN-dependent signaling. By incorporating into, or replacing at least a portion of, the chemical sensing domains with one or more extracellular IL-6 receptor components, inflammatory cytokine detection is effectively converted into proximity-driven CSK activation. For example, FKBP-based dimerization module can be replaced with the extracellular domains of IL-6 receptor a (IL-6Ra) and gpl30, thereby coupling CSK activation directly to IL-6-induced receptor complex formation.Impact on Downstream Inflammatory Cytokine Production

[0140] In some embodiments, when the synthetic protein switch is under the active state, the CSK domain of the synthetic protein switch suppresses production or secretion of an inflammatory cytokine. In some embodiments, the inflammatory cytokine includes an extracellular inflammatory cytokine. In some embodiments, the inflammatory cytokine includes an intracellular cytokine. In some embodiments, the inflammatory cytokine includes one or more of interleukin-2 (IL-2), interferon-y (IFNy), and tumor necrosis factor-a (TNFa). In some embodiments, the production or secretion of the inflammatory cytokine can be suppressed as a function of the concentration of the inducer, in a dose-dependent manner.

[0141] In some embodiments, the T-cell signaling is mediated by a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In active CAR-T cells, antigen engagement induces LCK phosphorylation at the activating residue Y394, leading to secretion of inflammatory cytokines. Transmembrane Configuration

[0142] In some embodiments, the synthetic protein switch has a transmembrane configuration.Attorney Docket No. 0073605-001129

[0143] In some embodiments, the synthetic protein switch further includes a CD8 hinge region that is at least 70% identical to, or a functional variant of, SEQ ID NO: 26. In some embodiments, the synthetic protein switch further includes a CD28 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 25.

[0144] In some embodiments, the CSK domain of the synthetic protein switch is to be positioned at an inner leaflet of a plasma membrane while regulatory elements are exposed extracellularly.Split Receptor Design vs. Single-Chain Inhibitory Receptor (scIR) Architecture

[0145] In some embodiments, the synthetic protein switch has a split receptor design, in accordance with details described throughout the present disclosure. In some embodiments, the synthetic protein switch has a single-chain inhibitory receptor (scIR) architecture, in accordance with details described throughout the present disclosure.

[0146] While split receptor designs offer conceptual advantages in terms of tunability and geometric control, single-chain architectures can be used in CAR-T contexts to reduce complexity. As a nonlimiting example, an IL-6Ra / gpl30 single-chain configuration can be adapted to couple IL-6 sensing to CSK-mediated inhibitory signaling rather than cytokine-driven activation, in accordance with details described throughout the present disclosure.Signal Peptide

[0147] In some embodiments, the synthetic protein switch further includes anN-terminal myristoylation signal. Such N-terminal myristoylation signal promotes plasma membrane localization of the synthetic protein switch. In some embodiments, the N-terminal myristoylation signal has an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 8. As nonlimiting examples, the myristoylation signal can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 8.

[0148] In some embodiments, the synthetic protein switch further includes a signal peptide having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 15. As nonlimiting examples, the signal peptide can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 15.LinkerAttorney Docket No. 0073605-001129

[0149] In some embodiments, the synthetic protein switch further includes a TEL linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 10. As nonlimiting examples, the TEL linker can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 10. The activation mechanism described herein can be independent of TEL’s transcriptional function and instead reflect its role as a structural linker that increases the effective local concentration of kinase domains. As a nonlimiting example, the TEL PNT oligomerization domain can be deliberately removed to prevent constitutive, ligandindependent clustering and basal CSK activation. Instead, the TEL segment provides appropriate spatial separation and conformational flexibility, allowing FKBP-mediated dimerization to mimic the proximity-driven activation mechanism observed in TEL-SFK oncogenic fusions, but under precise chemical control.

[0150] In some embodiments, the synthetic protein switch further includes a (638)4 linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 12. As nonlimiting examples, the (638)4 linker can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to SEQ ID NO: 12.

[0151] In some embodiments, CSKdim incorporates a myristoylation signal for membrane recruitment, a TEL-derived structural linker, and an FKBP12F36Vsensing domain, enabling reversible activation through chemical dimerization. As a nonlimiting example, CSKdim can incorporate four modular elements: (i) an N-terminal myristoylation signal to target the construct to the plasma membrane and ensure proximity to endogenous Lek; (ii) tandem FKBP12F36V(DmrB) domains that undergo rapid, dose-dependent homodimerization upon addition of the small-molecule dimerizer AP20187; (iii) a truncated TEL-derived linker included as a flexible spacer between the dimerization modules and the kinase domain; and (iv) the CSK domain as the catalytic effector.

[0152] In some embodiments, the synthetic protein switch, or one or more subunits thereof, has an amino acid sequence that is at least 70% identical to, or a functional variant of, any one of SEQ ID NOs: 1-7. As nonlimiting examples, the synthetic protein switch can have an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, etc., to any one of SEQ ID NOs: 1-7.Attorney Docket No. 0073605-001129 Method of Using the Synthetic Protein Switch

[0153] Another objective of the present disclosure is to provide a method for reversible modulation or regulation of T-cell signaling in a subject in need thereof, the method including providing or administering to the subject a therapeutically effective amount of a synthetic protein switch having an active state and an inactive state. The synthetic protein switch includes (I) a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11; and (II) a sensing domain, wherein an interaction between the synthetic protein switch and an inducer reversibly converts the synthetic protein switch from the inactive state to the active state. It should be noted that the method described herein can be used to regulate the signaling activity of any suitable type of T cells, and / or in any suitable type of T-cell therapy, that is deemed suitable or applicable by a person of ordinary skill in the art, upon reviewing the entirety of the present disclosure. As nonlimiting examples, the methods described herein can be used to modulate or regulate the signaling activity of second-generation and third-generation CAR T cells, including FMC63 anti-CD19 second-generation or third-generation (4-1BB) CAR-T cells.

[0154] Another objective of the present disclosure is to provide a method of treating a cytokine release syndrome (CRS) in a subject in need thereof, the method including providing or administering to the subject a therapeutically effective amount of a synthetic protein switch having an active state and an inactive state. The synthetic protein switch includes (I) a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11; and (II) a sensing domain, wherein an interaction between the synthetic protein switch and an inducer reversibly converts the synthetic protein switch from the inactive state to the active state.

[0155] As used herein, “cytokine release syndrome” or “CRS” is a systemic inflammatory response caused by a rapid, massive release of cytokines from immune cells, leading to symptoms including without limitation fever, fatigue, nausea, low blood pressure, headache, and rash. CRS is commonly triggered by certain immunotherapies, such as CAR-T cell therapy.

[0156] Similarly, in some embodiments, the method described herein can used to treat CAR-T-cell-related encephalopathy syndrome (CRES), also known as ICANS.

[0157] Another objective of the present disclosure is to provide a method of treating a hematological malignancy, the method including providing or administering to the subject (A) a therapeutically effective amount of T cells; and (B) a therapeutically effective amount of a synthetic protein switch having an active state and an inactive state. The synthetic protein switch includes a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, orAttorney Docket No. 0073605-001129 a functional variant of, SEQ ID NO: 11; and (II) a sensing domain, wherein an interaction between the synthetic protein switch and an inducer reversibly converts the synthetic protein switch from the inactive state to the active state, thereby modulating or regulating (e.g., suppressing, attenuating, or downregulating) an activity of the T cells. Nonlimiting examples of hematological malignancy that can be treated using the method described herein include relapsed or refractory blood cancers, including various types of lymphomas, leukemia, and the like, such as leukemia B-cell acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, high-grade B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma, and multiple myeloma, among others.

[0158] In some embodiments of the methods, an attenuation or removal of the inducer reversibly converts the synthetic protein switch from the active state to the inactive state, in accordance with details described throughout the present disclosure.

[0159] In some embodiments of the methods, the sensing domain of the synthetic protein switch includes a FKBP12F36sensing domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 9, in accordance with details described throughout the present disclosure.

[0160] In some embodiments of the methods, the CSK domain of the synthetic protein switch contains a Y304 autophosphorylation site, in accordance with details described throughout the present disclosure.

[0161] In some embodiments of the method, when the synthetic protein switch is under the active state, the sensing domain is in a dimeric state; and when the synthetic protein switch is under the inactive state, the sensing domain is in a monomeric state, in accordance with details described throughout the present disclosure.

[0162] In some embodiments, the method further includes catalyzing phosphorylation of lymphocyte-specific protein tyrosine kinase (LCK), using the synthetic protein switch under its active state, thereby attenuating or suppressing T-cell signaling, in accordance with details described throughout the present disclosure.

[0163] In some embodiments, the method further includes enhancing phosphorylation at Y505 position of the LCK and suppressing phosphorylation at Y394 position of the LCK, using the synthetic protein switch under its active state, thereby stabilizing the LCK in an inactive conformation, in accordance with details described throughout the present disclosure.Attorney Docket No. 0073605-001129

[0164] In some embodiments, the method further includes suppressing production or secretion of an inflammatory cytokine using the synthetic protein switch under its active state. In some embodiments of the method, the inflammatory cytokine includes an extracellular inflammatory cytokine. In some embodiments of the method, the inflammatory cytokine includes an intracellular cytokine. In some embodiments of the method, the inflammatory cytokine includes one or more of interleukin-2 (IL-2), interferon-y (IFNy), and tumor necrosis factor-a (TNFa) , in accordance with details described throughout the present disclosure.

[0165] In some embodiments, the method further includes suppressing CD3(^ phosphorylation using the synthetic protein switch under its active state, in accordance with details described throughout the present disclosure.

[0166] In some embodiments, the method further includes suppressing cytotoxic killing of a target cell using the synthetic protein switch under its active state, in accordance with details described throughout the present disclosure.

[0167] In some embodiments, the method further includes suppressing antigen-dependent CAR-T activation without causing nonspecific cellular dysfunction using the synthetic protein switch under its active state, in accordance with details described throughout the present disclosure.

[0168] In some embodiments, the method further includes converting the sensing domain of the synthetic protein switch from the monomeric state to the dimeric state using a dimerizer. In some embodiments of the method, the dimerizer includes AP20187, in accordance with details described throughout the present disclosure, in accordance with details described throughout the present disclosure.

[0169] In some embodiments of the method, the inducer that activates the synthetic protein switch includes a cytokine. In some embodiments of the method, the cytokine includes interleukin-6 (IL-6). In some embodiments of the method, the CSK domain of the synthetic protein switch includes an interleukin-6 (IL-6) receptor, in accordance with details described throughout the present disclosure.

[0170] In some embodiments of the method, the synthetic protein switch has a transmembrane configuration, in accordance with details described throughout the present disclosure.

[0171] In some embodiments of the method, the synthetic protein switch further includes a CD8 hinge region that is at least 70% identical to, or a functional variant of, SEQ ID NO: 26, in accordance with details described throughout the present disclosure. In some embodiments of the method, the synthetic protein switch further includes a CD28 transmembrane domain that is at leastAttorney Docket No. 0073605-001129 70% identical to, or a functional variant of, SEQ ID NO: 25, in accordance with details described throughout the present disclosure.

[0172] In some embodiments of the method, the CSK domain of the synthetic protein switch is to be positioned at an inner leaflet of a plasma membrane, while regulatory elements are exposed extracellularly, in accordance with details described throughout the present disclosure.

[0173] In some embodiments of the method, the sensing domain of the synthetic protein switch includes an hIL6Ra extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 13, in accordance with details described throughout the present disclosure.

[0174] In some embodiments of the method, the sensing domain of the synthetic protein switch includes an hIL6Ra transmembrane domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 14, in accordance with details described throughout the present disclosure.

[0175] In some embodiments of the method, the sensing domain of the synthetic protein switch includes a gpl30 extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 16, in accordance with details described throughout the present disclosure.

[0176] In some embodiments of the method, the sensing domain of the synthetic protein switch includes a gpl30 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 22, in accordance with details described throughout the present disclosure.

[0177] In some embodiments of the method, the sensing domain of the synthetic protein switch includes a GP130 domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 17, in accordance with details described throughout the present disclosure.

[0178] In some embodiments of the method, the sensing domain of the synthetic protein switch includes an IL6Ra domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 19, in accordance with details described throughout the present disclosure.

[0179] In some embodiments of the method, the sensing domain of the synthetic protein switch includes an IL7TM domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 21, in accordance with details described throughout the present disclosure.Attorney Docket No. 0073605-001129

[0180] In some embodiments of the method, the synthetic protein switch further includes an N-terminal myristoylation signal having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 8, in accordance with details described throughout the present disclosure.

[0181] In some embodiments of the method, the synthetic protein switch further includes a signal peptide having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 15, in accordance with details described throughout the present disclosure.

[0182] In some embodiments of the method, the synthetic protein switch further includes a TEL linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 10, in accordance with details described throughout the present disclosure.

[0183] In some embodiments of the method, the synthetic protein switch further includes a (G3S)4linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 12, in accordance with details described throughout the present disclosure.

[0184] In some embodiments of the method, the T-cell signaling is mediated by a T cell receptor (TCR) or a chimeric antigen receptor (CAR), in accordance with details described throughout the present disclosure.

[0185] In some embodiments of the method, the synthetic protein switch has a molar mass of from approximately 70kDato proximately 100 kDa, preferably approximately 80 kDa, as determined by, e.g., immunoblot analysis, in accordance with details described throughout the present disclosure.

[0186] In some embodiments of the method, the CSK domain of the synthetic protein switch contains one or more point mutations and one or more of S186A, Y188A, and W134A positions, in accordance with details described throughout the present disclosure.

[0187] In some embodiments of the method, the synthetic protein switch has an amino acid sequence that is at least 70% identical to, or a functional variant of, any one of SEQ ID NOs: 1-7, in accordance with details described throughout the present disclosure.Use of the Synthetic Protein Switch

[0188] Another objective of the present disclosure is to demonstrate the use of the synthetic protein switch described herein in the manufacture of a medicament for reversible modulation or regulation of T-cell signaling in a subject in need thereof, in accordance with details described throughout the present disclosure.

[0189] Another objective of the present disclosure is to demonstrate the use of the synthetic protein switch described herein in the manufacture of a medicament for treating cytokine releaseAttorney Docket No. 0073605-001129 syndrome (CRS) in a subject in need thereof, in accordance with details described throughout the present disclosure.

[0190] Another objective of the present disclosure is to demonstrate the use of the synthetic protein switch described herein in combination with one or more T cell therapies in the manufacture of a medicament for treating a hematological malignancy in a subject in need thereof, in accordance with details described throughout the present disclosure.

[0191] In some embodiments, the T cell therapy to be used in combination with the synthetic protein switch described herein includes a chimeric antigen receptor (CAR) T cell therapy or a transgenic T-cell receptor (TCR) therapy, in accordance with details described throughout the present disclosure.Method of Producing the Synthetic Protein Switch

[0192] Another objective of the present disclosure is to provide a method of producing the synthetic protein switch described herein, the method including transferring to a host cell or transfecting the host cell with a viral vector having a nucleic acid (e.g., a plasmid) encoding the synthetic protein switch. In some embodiments, the viral vector includes a lentiviral vector. In some embodiments, the method further includes culturing the transfected host cell under suitable conditions to produce the synthetic protein switch. In some embodiments, the viral vector can be used as part of a pharmaceutical composition for treating a subject in need thereof (i.e., by producing the synthetic protein switch in vivo), in accordance with details described throughout the present disclosure.

[0193] In some embodiments, the viral vector is produced using a plasmid. As used herein, a “plasmid” is a circular, double-stranded DNA molecule. Plasmids are distinct from a cell’s chromosomal DNA and are capable of autonomous replication. Plasmids can be used for insertion, expression, and propagation of foreign genes within a cell or host organism. Plasmids can include specific sequences for an origin of replication, selectable markers, and cloning sites, enabling manipulation and study of genetic material for applications. As nonlimiting examples, the plasmid used for producing such viral vector can include a packaging plasmid such as psPAX2 (Addgene plasmid #12260) and an envelope plasmid such as pMD2.G (Addgene plasmid #12259).

[0194] The type of host cells or cell cultures that can be used for producing the synthetic protein switch described herein is not particularly limited and can include any type of cell or cell culture deemed suitable by a person of ordinary skill in the art, upon reviewing the entirety of the present disclosure. In some embodiments, the host cell or cell culture includes one or more immortalized cell lines, such as without limitation human cervical cancer cell lines including HeLa, CaSki, and SiHa;Attorney Docket No. 0073605-001129 Human Embryonic Kidney 293 cells (HEK 293); Chinese hamster ovary cells (CHO); human breast cancer cells including MCF-7; rat pheochromocytoma cells including PC-12; etc. In some embodiments, the host cell or cell culture includes one or more types of stem cells, such as without limitation one or more of embryonic stem cells (ES), induced pluripotent stem cells (iPS), and mesenchymal stem cells (MSCs). In some embodiments, the cell culture includes a bacterial culture, such as without limitation, Escherichia coli (E coll), Saccharomyces cerevisiae (5. cerevisiae or Baker’s Yeast), Bacillus subtilis (B. subtilis), Agrobacterium tumefaciens (A. tumefaciens), Lactobacillus Geobacter, and / or Shewanella. In some embodiments, the host cell or cell culture includes a fungal culture, such as without limitation, Penicillium, Aspergillus, etc. In some embodiments, the host cell or cell culture includes a microalgal culture.EXAMPLE

[0195] The present example demonstrates that CSK can be reprogrammed to inducibly inhibit SFK function as a chemically dimerizable integral membrane fusion protein. This reversible CSK-based switch modulates TCR and CAR-T cell activity with a fully humanized design. This innovative synthetic protein switch offers significant translational potential, including without limitation:

[0196] 1 -Universal Applicability: the synthetic protein switch can be seamlessly introduced alongside any CAR-T or transgenic TCR therapy, making it a versatile tool in various treatment protocols.

[0197] 2-Reversible Control: instead of employing a kill switch that permanently eliminates the cell therapy, our design allows for temporary inhibition of an expensive therapy that is essentially working too well, preserving the therapeutic cells for continued use.

[0198] 3 -Reduced Immunogenicity: by avoiding viral sequences and utilizing a fully humanized design, the switch is less likely to trigger an immune response, enhancing patient safety and therapy compatibility.

[0199] The present example addresses a critical issue in CAR-T therapy: the inability to precisely control T cell activity, which can lead to life-threatening side effects such as cytokine release syndrome (CRS) and tumor lysis syndrome (TLS). Current methods to mitigate these side effects, like "kill switches" or steroids, often compromise the efficacy of the therapy by dampening the CAR-T cell response. The lack of a reversible and fine-tuned control mechanism poses significant risks for patients undergoing CAR-T therapy.Attorney Docket No. 0073605-001129

[0200] Unlike existing technologies that permanently deactivate CAR-T cells or require broad immunosuppressive drugs, our CSK-based switch provides a reversible, immune-compatible method for modulating T cell activity. By controlling LCK activation through the addition or removal of a clinical -grade small molecule (AP20178), this technology allows for the precise tuning of CAR-T cell function without permanently altering or compromising their therapeutic potential. Its fully humanized design further enhances compatibility with clinical use, minimizing the risk of immune rejection.

[0201] The technology described herein can be developed into a therapeutic product integrated into CAR-T cell therapies. The product might take the form of engineered CAR-T cells equipped with the CSKdim switch, along with a corresponding small-molecule activator (AP20178) that clinicians could administer as needed to regulate T cell activity during treatment. This synthetic protein switch can be used as part of a next-generation CAR-T therapy package, offering enhanced safety.

[0202] Overall, the approach described in the present example provides a safer, more adaptable method for controlling engineered T-cell functions in clinical settings.Results

[0203] Referring now to FIG. 1A-1E, the present example demonstrates that a chemically inducible CSK dimerization switch can be engineered to suppress TCR signaling.

[0204] T cell receptor (TCR) signaling is governed by a finely balanced phosphorylation network in which modest perturbations in kinase or phosphatase activity can produce disproportionately large effects on downstream activation. A central node in this regulatory system is C-terminal Src kinase (CSK), a unique cytoplasmic tyrosine kinase whose catalytic activity suppresses Src-family kinases (SFKs), most notably Lek, by phosphorylating the conserved inhibitory residue Y505. Phosphorylation at Y505 stabilizes Lek in a closed, inactive conformation, thereby preventing autophosphorylation at Y394 and blocking initiation of proximal TCR signaling. Because CSK functions as a negative regulator rather than a signal amplifier, strategies that enhance CSK activity represent a powerful means to attenuate T cell activation. Although CSK provides basal inhibition in resting T cells, multiple studies have demonstrated that its kinase activity is strongly regulated by induced proximity mechanisms, including membrane recruitment, scaffolding interactions, and self-association. Structural and biochemical analysis of the pseudokinase Pragmin revealed that enforced dimerization of a non-catalytic scaffold potently activates CSK by increasing the local effective concentration of CSK kinase domains, thereby promoting transautophosphorylation at tyrosine 304 (Y304) within the CSK activation loop. Phosphorylation atAttorney Docket No. 0073605-001129 Y304 stabilizes CSK in an open, catalytically competent conformation and serves as a robust biochemical marker of CSK activation. Complementary studies have further shown that CSK activity is sensitive to oligomeric state and SH3 -mediated interactions, reinforcing the concept that CSK functions as a proximity-regulated kinase rather than a constitutively active enzyme.

[0205] In parallel, oncogenic kinase fusion studies have established that forced oligomerization of kinase domains is sufficient to drive ligand-independent activation. In particular, the ETS-family transcriptional repressor TEL (ETV6) has been shown to act as a potent oligomerization scaffold in multiple fusion proteins involving Src-family kinases and ABL. The seminal PNAS study by Melnick et al. demonstrated that TEL-mediated self-association converts otherwise tightly regulated cytoplasmic kinases into constitutively active signaling enzymes by enforcing proximity of kinase domains, thereby promoting trans-autophosphorylation and stabilization of the active conformation. Importantly, this activation mechanism is independent of TEL’s transcriptional function and instead reflects its role as a structural linker that increases the effective local concentration of kinase domains.

[0206] Guided by these principles, we engineered a synthetic, chemically inducible CSK construct, termed CSKdim, designed to recapitulate native proximity -dependent CSK activation in a reversible and tunable format (FIG. IB). The CSKdim architecture incorporates four modular elements: (i) an N-terminal myristoylation signal to target the construct to the plasma membrane and ensure proximity to endogenous Lek; (ii) tandem FKBP12F36V(DmrB) domains that undergo rapid, dose-dependent homodimerization upon addition of the small-molecule dimerizer AP20187; (iii) a truncated TEL-derived linker included as a flexible spacer between the dimerization modules and the kinase domain; and (iv) the CSK kinase domain as the catalytic effector. Notably, the TEL PNT oligomerization domain was deliberately removed to prevent constitutive, ligand-independent clustering and basal CSK activation. Instead, the TEL segment provides appropriate spatial separation and conformational flexibility, allowing FKBP-mediated dimerization to mimic the proximity-driven activation mechanism observed in TEL-SFK oncogenic fusions, but under precise chemical control.

[0207] We first assessed whether chemically induced dimerization of CSKdim was sufficient to activate CSK in T cells. Jurkat T cells expressing CSKdim were treated with AP20187 and analyzed by flow cytometry using a phospho-specific antibody against CSK pY304. Dimerizer treatment resulted in a marked increase in Y304 phosphorylation compared to untreated cells (FIG. 1C-1D), indicating that DmrB-mediated clustering successfully drove CSK into an active, autophosphorylated state. This result is consistent with prior structural models in which increasedAttorney Docket No. 0073605-001129 local kinase concentration promotes trans-autophosphorylation and stabilization of the active CSK conformation. CSKdim activation was further validated by immunoblot analysis (FIG. IE). The exogenous CSKdim fusion protein migrated as a discrete band at approximately 80 kDa, consistent with the predicted molecular weight of the kinase domain fused to the synthetic regulatory components. In the absence of the dimerizer, CSKdim exhibited minimal basal Y304 autophosphorylation. In contrast, treatment with AP20187 resulted in a robust increase in exogenous pCSK Y304 levels, demonstrating efficient activation of the engineered kinase.

[0208] Collectively, these data establish that CSKdim reconstitutes a native regulatory mechanism in which CSK activity is controlled by induced proximity. By combining membrane targeting, chemically inducible dimerization, and a TEL -derived structural linker, CSKdim enables rapid activation of a potent negative regulator of TCR signaling, providing a synthetic platform to modulate early T cell activation with high precision.

[0209] Referring now to FIG. 2A-2H, the present example further shows that inducible activation of CSKdim suppresses proximal TCR signaling by reciprocally regulating LCK phosphorylation.

[0210] Having established that chemical dimerization robustly activates CSKdim through autophosphorylation at Y304 (FIG. 1A-1E), we next asked whether CSKdim activation functionally suppresses proximal T cell receptor (TCR) signaling by modulating the phosphorylation state of its primary substrate, LCK. LCK activity is tightly controlled by phosphorylation at two conserved tyrosine residues: phosphorylation at Y394 promotes kinase activation, whereas phosphorylation at Y505 by CSK stabilizes LCK in a closed, inactive conformation. Thus, a shift toward increased Y505 phosphorylation and reduced Y394 phosphorylation represents a definitive biochemical signature of CSK-mediated inhibition of TCR signaling.

[0211] We first examined whether activation of CSKdim increases inhibitory phosphorylation of LCK at Y505. Serum-starved Jurkat T cells expressing CSKdim and wild type were briefly stimulated through the TCR using anti-CD3 / CD28 antibodies and treated with either DMSO or the FKBP dimerizer AP20187 for 10 minutes. Flow cytometric analysis using a phospho-specific antibody against pLCK Y505 revealed a marked increase in Y505 phosphorylation upon dimerizer treatment in CSKdim-expressing cells, whereas control CSK-expressing cells showed minimal change (FIG. 2B-2C). Quantification of mean fluorescence intensity confirmed a significant induction of inhibitory LCK phosphorylation following chemical activation of CSKdim. This result was further validated by immunoblot analysis, which demonstrated a robust increase in pLCK Y505 levels upon dimerizer treatment without changes in total LCK expression (FIG. 2D).Attorney Docket No. 0073605-001129

[0212] We next assessed whether CSKdim activation concomitantly suppresses activating phosphorylation of LCK at Y394. Jurkat T cells expressing CSKdim were left unstimulated or stimulated with CD3 / CD28 antibodies in the presence of DMSO or AP20187. As expected, TCR stimulation induced strong phosphorylation of LCK at Y394. However, chemical activation of CSKdim markedly reduced Y394 phosphorylation in stimulated cells, as measured by flow cytometry (FIG. 2E). Consistent with the flow cytometry data, immunoblot analysis of whole-cell lysates confirmed that activation of CSKdim suppresses TCR-induced phosphorylation of LCK at Y394 without altering total LCK protein levels (FIG. 2F). Together, these data demonstrate that chemical activation of CSKdim enforces a coordinated shift in LCK phosphorylation state — enhancing inhibitory Y5O5 phosphorylation while suppressing activating Y394 phosphorylation — thereby effectively downregulating proximal TCR signaling. Collectively, these findings establish that inducible CSKdim activation functionally reprograms early TCR signaling by directly modulating LCK activity, validating CSKdim as a potent and tunable negative regulator of T cell activation.

[0213] Referring now to FIG. 3 A-3G, the present example further shows that the CSKdim described herein can reversibly suppress inflammatory cytokine secretion downstream of TCR activation

[0214] Having shown that inducible activation of CSKdim suppresses proximal T cell receptor (TCR) signaling by enforcing inhibitory LCK phosphorylation (FIGS. 1-2), we next investigated whether this signaling inhibition translates into functional suppression of T cell effector responses. Upon TCR engagement, activated T cells secrete inflammatory cytokines such as interleukin-2 (IL-2), interferon-y (IFNy), and tumor necrosis factor-a (TNFa), which are critical for T cell proliferation, survival, and effector differentiation. We therefore assessed whether chemical activation of CSKdim inhibits cytokine secretion and whether this effect is reversible.

[0215] To conceptually frame this regulatory mechanism, we modeled T cell activation and inhibition states (FIG. 3A-3B). In the absence of CSKdim activation, TCR stimulation induces LCK autophosphorylation at Y394, triggering downstream signaling cascades that drive cytokine production. In contrast, chemical dimerization of CSKdim promotes phosphorylation of LCK at the inhibitory residue Y505, stabilizing LCK in an inactive conformation and suppressing cytokine secretion, thereby returning T cells to a resting-like state.

[0216] We first examined cytokine secretion in primary T cells expressing CSKdim following CD3 / CD28 stimulation. T cells were stimulated with CD3 / CD28 Dynabeads for 16 hours, treated with the dimerizer AP20187 to activate CSKdim, and then re-stimulated following dimerizer removal (FIG. 3C-3E). Enzyme-linked immunosorbent assays (ELISA) revealed that activation of CSKdimAttorney Docket No. 0073605-001129 significantly reduced secretion of both IFNy and IL-2 compared to stimulated controls. Importantly, removal of the dimerizer restored cytokine secretion to levels comparable to untreated, stimulated cells, demonstrating that CSKaim-mediated inhibition is reversible and does not permanently impair T cell cytokine-producing capacity.

[0217] We next assessed the dose dependence of CSKaim-mediated cytokine suppression in Jurkat T cells. IL-2 secretion was measured across increasing concentrations of the dimerizer. In CSKaim-expressing cells, IL-2 production decreased in a dose-dependent manner following dimerizer addition, whereas wild-type and shLCK control cells did not exhibit comparable suppression (FIG.3F). These results confirm that cytokine inhibition is specifically mediated by CSKaim activation and is tunable based on dimerizer concentration.

[0218] Finally, we evaluated downstream transcriptional outputs of TCR signaling using NF AT- and IL-2-luciferase reporter Jurkat cells. Upon CD3 / CD28 stimulation, robust luciferase activity was observed in control cells. In contrast, activation of CSKaim by AP20187 significantly reduced both NF AT- and IL-2-driven luciferase activity (FIG. 3G), consistent with upstream inhibition of LCK signaling and suppression of TCR-dependent transcriptional programs.

[0219] Together, these data demonstrate that inducible activation of CSKaim functionally suppresses inflammatory cytokine secretion downstream of TCR activation in a reversible, dosedependent manner. This result establishes CSKaim as a tunable molecular switch capable of dynamically regulating T cell effector function without permanently compromising T cell responsiveness.

[0220] Referring now to FIG. 4A-4C, the present example further demonstrates that activation of CSKaim suppresses T cell-mediated cytotoxic killing.

[0221] After establishing that CSKaim activation suppresses proximal TCR signaling and downstream cytokine production in a reversible manner (FIGS. 1-3), we next evaluated whether this molecular inhibition translates into functional suppression of T cell-mediated cytotoxicity. Effective T cell killing requires sustained TCR signaling, LCK activation, and CD3(^ phosphorylation, all of which are negatively regulated by CSK. We therefore examined whether inducible activation of CSKdim inhibits T cell-mediated killing of CD19+target cells.

[0222] To model antigen-dependent cytotoxicity, we used the bispecific T cell engager Blinatumomab (anti-CD3 x anti -CD 19 bispecific T cell engager), which redirects T cells to kill CD19+leukemia cells independently of CAR expression (FIG. 4A). In the absence of CSKdim activation, Blinatumomab engagement induces robust T cell activation, LCK Y394 phosphorylation,Attorney Docket No. 0073605-001129 CD3(^ signaling, and target cell killing. In contrast, chemical activation of CSKdim by AP20187 enforces inhibitory LCK Y505 phosphorylation, suppressing TCR signaling and cytotoxic function.

[0223] Consistent with this model, flow cytometric analysis revealed that activation of CSKdim significantly reduced T cell-mediated killing of CD19+BV173 cells. In the presence of Blinatumomab, CSKdim-expressing T cells treated with DMSO efficiently eliminated BV173 targets, whereas addition of 10 nM AP20187 markedly decreased the percentage of CD 19“ target cells killed (FIG. 4B-4C). Quantification across biological replicates confirmed a significant reduction in cytotoxic activity upon CSKdim activation.

[0224] We next evaluated whether CSKdim-mediated inhibition of killing was generalizable across distinct CD19+target cell lines. Similar experiments performed using RAJI cells yielded comparable results, with chemical activation of CSKdim significantly suppressing T cell-mediated cytotoxicity (FIG. 4B-4C). In parallel, activation of CSKdim reduced CD3+T cell expansion, consistent with attenuated activation and effector function following inhibition of proximal TCR signaling.

[0225] Together, these data demonstrate that inducible activation of CSKdim functionally suppresses T cell-mediated killing of CD19+target cells. This establishes CSKdim as an effective molecular switch capable of dynamically controlling not only TCR signaling and cytokine secretion but also the ultimate effector function of cytotoxic T cells.

[0226] Referring now to FIG. 5A-5J, the present example further demonstrates that CSKdim activation suppresses both extracellular and intracellular cytokine production in CAR-T cells.

[0227] Having demonstrated that CSKdim activation suppresses proximal signaling, cytokine secretion, and cytotoxic function in TCR-driven T cells (FIG. 1A-1E, 2A-2H, and 3A-3G), we next examined whether this regulatory strategy extends to chimeric antigen receptor (CAR) T cells. CAR-T activation relies on the same Src-family kinases, LCK and FYN, to transmit signals downstream of CD3^ and co-stimulatory domains across different CAR generations. We therefore hypothesized that inducible activation of CSKdim would suppress CAR-T-mediated cytokine production by enforcing inhibitory phosphorylation of LCK. Conceptually, in the absence of CSKdim activation, engagement of CD19 by CAR-T cells induces phosphorylation of LCK at Y394, driving transcriptional and effector programs that result in secretion of inflammatory cytokines such as IL-2, IFNy, and TNFa (FIG. 5A, top). In contrast, chemical activation of CSKdim via the FKBP homodimerizer AP20187 promotes inhibitory phosphorylation of LCK at Y505, suppressing CAR signaling and shifting CAR-T cells toward a resting-like state (FIG. 5A, bottom).Attorney Docket No. 0073605-001129

[0228] We first assessed extracellular cytokine secretion in third-generation FMC63 anti-CD19 CAR-T cells expressing CSKdim. CAR-T cells were co-cultured with CD19+BV173 target cells for 10 hours, followed by addition of 10 nM AP20187 to activate CSKdim. Supernatants were collected either during CSKdim activation or after dimerizer removal, and cytokine levels were quantified by ELISA (FIG. 5B-5D). Activation of CSKdim significantly reduced secretion of both IL-2 and IFNy compared to stimulated CAR-T controls. Importantly, removal of AP20187 restored cytokine secretion toward baseline stimulated levels, indicating that CSKdim-mediated suppression of CAR-T cytokine output is reversible.

[0229] We next examined whether CSKdim activation also suppresses intracellular cytokine production during antigen recognition. FMC63 CAR-T cells expressing CSKdim were co-cultured with CD19+target cell lines (BV173, K562-CD19+, and RAJI) or CD 19' K562 controls in the presence of DMSO or AP20187. Intracellular staining revealed robust induction of IL-2, IFNy, and TNFa in CAR-T cells upon antigen engagement. However, activation of CSKdim significantly reduced the frequency of cytokine-producing CD3+CAR-T cells across all CD19+targets, while responses to CD19" targets remained minimal (FIG. 5E-5F). These results demonstrate that CSKdim selectively suppresses antigen-dependent CAR-T activation rather than causing nonspecific cellular dysfunction.

[0230] Together, these data establish that inducible activation of CSKdim suppresses both extracellular cytokine secretion and intracellular cytokine production in CAR-T cells in a reversible and antigen-dependent manner. These findings extend the CSKdim regulatory paradigm from TCR signaling to CAR signaling and demonstrate its capacity to dynamically modulate inflammatory outputs of engineered T cell therapies.

[0231] Referring now to FIG. 6A-6D, the present example further shows that activation of the CSKdim switch suppresses cytotoxic killing by second-generation (4-1BB) CAR-T cells

[0232] Having shown that CSKdim activation suppresses CAR-T cytokine production across multiple readouts (FIG. 5A-5J), we next evaluated whether this inhibition extends to the cytotoxic function of clinically relevant second-generation CAR-T cells. Second-generation CARs incorporating the 4-1BB (CD137) co-stimulatory domain are widely used in approved anti-CD19 therapies and rely on sustained Src-family kinase signaling downstream of CD3(^ to mediate target cell killing. We therefore evaluated whether inducible activation of CSKdim could dynamically suppress killing by 4-1BB CAR-T cells. Conceptually, in the absence of CSKdim activation, engagement of CD19 by CAR-T cells induces LCK phosphorylation at Y394, leading to CD3(^ signaling, cytokine release, and efficient tumor cell killing (FIG. 6A, left). In contrast, chemicalAttorney Docket No. 0073605-001129 activation of CSKdim via the FKBP homodimerizer AP20187 enforces inhibitory phosphorylation of LCK at Y505, suppressing proximal CAR signaling and attenuating cytotoxic activity (FIG. 6A, right).

[0233] We first assessed CAR-T-mediated killing across a range of effector-to-target (E:T) ratios using FMC63 anti-CD19 second-generation (4-1BB) CAR-T cells expressing CSKdim and CD19+BV173 target cells. In the absence of dimerizer, CSKdim-expressing CAR-T cells efficiently killed BV173 targets in a dose-dependent manner as E:T ratios increased. However, activation of CSKdim with 10 nM AP20187 significantly reduced killing across all tested ratios (FIG. 6B), demonstrating that CSKdim activation robustly suppresses CAR-T cytotoxicity even under conditions of high effector pressure.

[0234] We next evaluated whether this inhibitory effect was consistent across multiple CD19+target cell lines. FMC63-CAR-T-CSKdim cells were co-cultured with BV173, RAH, or K562-CD19+targets in the presence of DMSO or AP20187. Flow cytometric analysis revealed that CSKdim activation significantly reduced both CAR-T expansion (fold increase) and the percentage of CD19+target cells killed across all CD19+targets (FIG. 6C-6D). In contrast, minimal killing was observed against CD I 9 K562 cells under all conditions, confirming antigen specificity and indicating that CSKdim activation does not induce nonspecific cytotoxic dysfunction. Together, these data demonstrate that inducible activation of CSKdim effectively suppresses cytotoxic killing by second-generation (4- IBB) CAR-T cells in an antigen-dependent and tunable manner. These findings establish CSKdim as a powerful molecular switch capable of dynamically controlling the effector phase of CAR-T cell therapy.

[0235] The present example further demonstrates that CSKdim-mediated inhibition of CAR-T cytotoxicity is fully reversible across multiple target cell contexts.

[0236] Referring now to FIG. 7A-7C, the present example demonstrates that CSKdim-mediated inhibition of CAR-T cytotoxicity is fully reversible across multiple target cell contexts.

[0237] A key limitation of existing CAR-T safety switches is their irreversible nature, which permanently ablates therapeutic cells once activated. Having demonstrated that CSKdim activation suppresses CAR-T cytotoxicity (FIG. 6A-6D), we next evaluated whether this inhibition is functionally reversible, allowing CAR-T cells to regain killing capacity upon removal of the dimerizer. Demonstration of reversibility is an important factor to consider for enabling transient control of CAR-T activity without compromising long-term therapeutic efficacy.

[0238] We first evaluated reversibility of killing using second-generation FMC63 (4-1BB) CAR-T cells expressing CSKdim co-cultured with CD19+leukemia targets. CAR-T cells wereAttorney Docket No. 0073605-001129 incubated with BV173-CD19+or RAJI-CD19+cells in the presence of either 10 nM AP20187 or DMSO, followed by assessment of CAR-T expansion and target cell killing. As expected, activation of CSKdim significantly reduced both CD3+CAR-T expansion and the percentage of CD19+target cells killed. In contrast, removal of the dimerizer restored CAR-T expansion and cytotoxicity to levels comparable to untreated controls (FIG. 7A-7B). These data demonstrate that CSKdim-mediated suppression of CAR-T killing is reversible and does not permanently impair effector function.

[0239] To further validate reversibility in a prolonged and clinically relevant killing context, we next employed a temporal washout experiment using HEK 293 cells engineered to express CD 19. CAR-T-CSKdim cells were co-cultured with HEK 293-hCD19+targets and treated with AP20187 for two days to suppress cytotoxicity, and then subjected to ligand washout followed by continued coculture (FIG. 7C). During CSKdim activation, CAR-T -mediated killing of HEK 293-hCD I9 cells was significantly reduced. Strikingly, removal of AP20187 restored CAR-T cytotoxic activity, resulting in a marked increase in CD19+target cell killing relative to the CSKdim-ON condition. Minimal killing was observed against CD 19’ HEK 293 controls under all conditions, confirming antigen specificity. Collectively, these results establish that CSKdim functions as a reversible molecular brake on CAR-T cytotoxicity across multiple target cell types and experimental timescales. This reversibility distinguishes CSKdim from irreversible suicide switches and supports its utility as a dynamic safety and control module for engineered T cell therapies.

[0240] Referring now to FIG. 8A-8C, the present example further demonstrates that cytoplasmic-to-extracellular reprogramming of the CSKdim switch enables membrane-proximal control of TCR signaling

[0241] While the original CSKdim design functions as a cytoplasmic, membrane-targeted kinase switch, therapeutic control of engineered T cells would benefit from modular architectures that more closely resemble receptor-like signaling systems. We therefore re-engineered CSKdim to transition from a cytoplasmic configuration to extracellularly addressable, transmembrane formats, enabling future coupling to extracellular sensing modules while preserving inducible CSK activation.

[0242] To this end, we designed a series of CSKdim variants in which the myristoylation-based membrane anchor was replaced with a signal peptide (SP), CD8 hinge region, and CD28 transmembrane domain, positioning the CSK kinase domain at the inner leaflet of the plasma membrane while exposing regulatory elements extracellularly. Three architectures were evaluated: the original intracellular CSKdim (inCSKdim), an extracellular CSKdim containing a flexible (G4S)a linker (exCSKdim-G4S), and an extracellular CSKdim incorporating a truncated ETV6 (TEL) linkerAttorney Docket No. 0073605-001129 (exCSKdim-TEL). All constructs retained FKBP12F36Xsensing domains and the CSK kinase domain containing the Y304 autophosphorylation site.

[0243] We first assessed the functional impact of these designs on TCR-driven cytokine output using an IL-2 ELISA in Jurkat T cells. As expected, wild-type cells and control conditions exhibited robust IL-2 secretion following CD3 / CD28 stimulation, which was unaffected by the dimerizer alone. Knockdown of LCK (shLCKl) markedly reduced IL-2 production, validating dependence on proximal Src-family kinase signaling. Expression of intracellular CSKdim (inCSKdim) significantly suppressed IL-2 secretion upon dimerizer addition, consistent with cytoplasmic CSK activation.

[0244] Notably, extracellular CSKdim variants also retained inducible inhibitory function. Both exCSKdim-G4S and exCSKdim-TEL constructs significantly reduced CD3 / CD28-induced IL-2 secretion upon B / B dimerizer treatment, with the TEL-linked configuration exhibiting stronger suppression, consistent with enhanced proximity-driven CSK activation. These results demonstrate that transmembrane reconfiguration does not impair CSKdim function and that linker architecture influences inhibitory potency.

[0245] To directly assess proximal signaling, we analyzed phosphorylation of LCK at the activating residue Y394 by flow cytometry. CD3 / CD28 stimulation induced strong pLCK-Y394 signals in control Jurkat cells, whereas activation of intracellular CSKdim reduced Y394 phosphorylation. Importantly, extracellular CSKdim constructs similarly suppressed pLCK-Y394 upon dimerizer treatment, confirming that membrane-spanning CSKdim variants effectively inhibit proximal TCR signaling. Quantification of mean fluorescence intensity (MFI) further confirmed robust suppression of LCK activation across intracellular and extracellular CSKdim formats.

[0246] Collectively, these data demonstrate that CSKdim can be successfully reprogrammed from a cytoplasmic switch into extracellularly configurable, transmembrane architectures while preserving inducible inhibition of TCR signaling. This design transition establishes a modular foundation for future closed-loop control systems in which extracellular cues can dynamically regulate intracellular kinase activity. See FIG. 8A-8C.

[0247] Referring now to FIG. 9A-9E, the present example further shows that reprogramming CSKdim into an ZL-6-responsive transmembrane kinase enables closed-loop suppression of TCR signaling

[0248] While chemical dimerization enables precise experimental control of CSK activity, therapeutic regulation of engineered T cells requires autonomous, stimulus-responsive control mechanisms. Because interleukin-6 (IL-6) is a key inflammatory cytokine associated with cytokine release syndrome (CRS), we sought to convert CSKdim from an open-loop, drug-dependent switchAttorney Docket No. 0073605-001129 into a closed-loop IL-6-responsive inhibitory kinase capable of dynamically suppressing TCR signaling in response to inflammatory cues.

[0249] To achieve this goal, we re-engineered the CSKdim architecture in three conceptual stages (FIG. 9A). First, we established an intracellular, chemically inducible CSK dimerization system in which FKBP12F36V-mediated homodimerization activates CSK and suppresses Src-family kinases (LCK and FYN). Second, this system was converted into a transmembrane configuration by incorporating a CD8 hinge and CD28 transmembrane domain, positioning the CSK kinase domain at the inner leaflet of the plasma membrane while preserving dimerization-dependent activation.Finally, the extracellular FKBP-based dimerization module was replaced with the extracellular domains of IL-6 receptor a (IL-6Ra) and gpl30, thereby coupling CSK activation directly to IL-6-induced receptor complex formation. In this configuration, IL-6 binding enforces proximity of CSK kinase domains, converting cytokine sensing into intracellular inhibitory signaling.

[0250] We first assessed whether IL-6-dependent CSK activation suppresses TCR-driven cytokine production. Jurkat T cells expressing IL6Ra-TEL-CSK and gpl30-TEL-CSK constructs were stimulated with CD3 / CD28 antibodies in the presence of increasing concentrations of IL-6. Whereas wild-type cells maintained robust fENy and IL-2 production upon stimulation, IL-6 treatment reduced both IFNy and IL-2 secretion in cells expressing the IL-6-coupled CSK constructs (FIG. 9B). These results indicate that IL-6 sensing autonomously suppresses TCR-driven cytokine output through CSK activation.

[0251] To confirm that this functional suppression is mediated by canonical CSK-LCK signaling, we next examined phosphorylation of LCK at the inhibitory residue Y505. Immunoblot analysis revealed that IL-6 stimulation induced a marked increase in pLCK-Y505 levels specifically in cells expressing the IL6Ra-CSK / gpl30-CSK constructs, whereas wild-type cells showed minimal changes across the same IL-6 concentrations (FIG. 9C). This result demonstrates that IL-6 receptor engagement directly activates CSK kinase activity and enforces inhibitory LCK phosphorylation.

[0252] We further analyzed proximal signaling dynamics by flow cytometry. IL-6 stimulation increased pLCK-Y505 levels in IL6Ra-CSK / gpl30-CSK-expressing cells, mirroring the effects observed with chemically induced CSKdim activation (FIG. 9D). Conversely, IL-6 treatment reduced phosphorylation of LCK at the activating residue Y394 during CD3 / CD28 stimulation (FIG. 9E), confirming that IL-6-dependent CSK activation suppresses early TCR signaling events.

[0253] Together, these data establish that CSKdim can be successfully reprogrammed into an IL-6-responsive, transmembrane inhibitory kinase, converting an inflammatory cytokine signal into a negative feedback loop that suppresses TCR signaling. This closed-loop design provides aAttorney Docket No. 0073605-001129 mechanistic foundation for autonomous regulation of engineered T cell activity in response to inflammatory cues associated with CRS.

[0254] Referring now to FIG. 10A-10D, the present example further demonstrates that L-6-responsive split inhibitory receptors can modulate LCK phosphorylation within a CRS-relevant concentration range.

[0255] Elevated interleukin-6 (IL-6) levels are a defining feature of cytokine release syndrome (CRS), with reported serum concentrations spanning from low or undetectable levels in healthy individuals to markedly increased levels in severe disease. To explore whether the IL-6-responsive CSK system operates within a clinically relevant concentration range, we examined the effects of increasing IL-6 concentrations on proximal TCR signaling in cells expressing IL-6-coupled split inhibitory receptors (IL6-SIRs).

[0256] The IL6-SIR design includes separate IL-6 receptor a (IL-6Ra) and gpl30 extracellular domains fused to transmembrane anchors and intracellular CSK kinase domains. In this configuration, IL-6 binding promotes receptor association, bringing CSK kinase domains into proximity and enabling inhibitory signaling. This architecture is intended to convert extracellular inflammatory cues into intracellular suppression of Src -family kinase activity.

[0257] We first assessed phosphorylation of LCK at the inhibitory residue Y505 following exposure to increasing IL-6 concentrations spanning levels reported in CRS. Immunoblot analysis showed changes in pLCK-Y505 levels in IL6-SIR-expressing cells across the tested IL-6 concentrations, while total LCK expression remained comparable across conditions. Quantification of pLCK-Y505 normalized to total LCK indicated increased inhibitory phosphorylation at higher IL-6 concentrations relative to baseline, although variability was observed across conditions. We next examined phosphorylation of LCK at the activating residue Y394 under CD3 / CD28 stimulation in the presence of IL-6. As expected, CD3 / CD28 stimulation increased pLCK-Y394 in the absence of IL-6. Addition of IL-6 was associated with a reduction in pLCK-Y394 levels in cells expressing IL6-SIRs compared to stimulated controls, particularly at higher IL-6 concentrations. These observations are consistent with IL-6-mediated activation of CSK leading to attenuation of proximal TCR signaling, although further studies may be required to fully define the sensitivity and dynamic range of this response. Together, these data suggest that IL-6-responsive CSK activation can modulate LCK phosphorylation within an IL-6 concentration range relevant to CRS. While not necessarily demonstrating precise quantitative control, these results provide initial evidence that inflammatory IL-6 signaling can be coupled to inhibitory regulation of TCR signaling through the IL6-SIR-CSK system.Attorney Docket No. 0073605-001129

[0258] Referring now to FIG. 11 A-l ID, mutational analysis of IL-6 split inhibitory receptors suggests residue-specific contributions to CSK-mediated LCK inhibition.

[0259] To further explore how IL-6 receptor engagement contributes to CSK activation in the split inhibitory receptor (IL6-SIR) system, we performed a targeted mutational analysis of the IL-6Ra extracellular domain. Based on prior structural and biochemical studies of IL-6 receptor signaling, we selected three point mutations (W134A, S186A, and Y188A) that have been reported to affect IL-6 binding or receptor complex stability. These mutations were introduced individually into the IL-6Ra extracellular domain while preserving the overall IL6-SIR architecture, including the gpl30 partner and intracellular CSK kinase domains (FIG. 11A).

[0260] We first examined inhibitory phosphorylation of LCK at Y505 under basal conditions and following IL-6 exposure. In the absence of IL-6, pLCK-Y505 levels were comparable across constructs expressing wild-type or mutant IL-6R01 domains, indicating minimal ligand-independent CSK activation. Upon addition of IL-6, differences in pLCK-Y505 levels were observed between the unmutated receptor and selected IL-6Ra mutants (FIG. 1 IB). In particular, constructs harboring the Y188A mutation exhibited pLCK-Y505 levels similar to the unmutated receptor under these conditions, whereas reduced pLCK-Y5O5 levels were observed with the S186A and W134A mutations.

[0261] We next evaluated pLCK-Y505 levels at a higher IL-6 concentration relevant to severe inflammatory conditions. Under these conditions, the unmutated IL6-SIR construct showed increased inhibitory LCK phosphorylation compared to control cells lacking IL6-SIR expression (FIG. 11C-1 ID). The Y188A mutant again displayed pLCK-Y505 levels comparable to the unmutated construct, whereas the S186A and W134A mutants exhibited lower pLCK-Y505 signals. Together, these observations suggest that specific residues within the IL-6Ra extracellular domain contribute to the ability of the IL6-SIR system to engage CSK and modulate LCK phosphorylation following IL-6 exposure. While these results do not necessarily define a precise structure-function relationship, they provide initial evidence that IL-6 receptor interface integrity influences the efficiency of CSK-mediated inhibitory signaling in this system.

[0262] Referring now to FIG. 12A-12C, the present example further shows that a single-chain IL-6 inhibitory receptor architecture modulates can proximal TCR signaling in the presence of IL-6.

[0263] In addition to the split inhibitory receptor (IL6-SIR) configuration, we explored whether IL-6-responsive CSK activation could be implemented using a single-chain inhibitory receptor (scIR) architecture. This design is consistent with prior work demonstrating that IL-6 receptor components can be repurposed in engineered T cells. Notably, a study published in Cell described aAttorney Docket No. 0073605-001129 chimeric cytokine receptor in CAR-T cells in which the extracellular domains of IL-6Ra and gpl30 were fused to the intracellular domain of the IL-7 receptor, enabling IL-6-dependent modulation of CAR-T cell behavior in inflammatory environments (Kagoya etal., 2018). Inspired by this precedent, we investigated whether a related IL-6Ra / gpl30 single-chain configuration could be adapted to couple IL-6 sensing to CSK-mediated inhibitory signaling rather than cytokine-driven activation.

[0264] In the scIR design, the extracellular domains of gpl30 and IL-6Ra are linked in cis, followed by a transmembrane domain and an intracellular CSK kinase domain. This configuration is intended to promote receptor association upon IL-6 engagement, bringing CSK kinase domains into proximity.

[0265] To assess the impact of this architecture on proximal TCR signaling, we examined phosphorylation of LCK at the activating residue Y394 following CD3 / CD28 stimulation in the presence of IL-6. As expected, CD3 / CD28 stimulation increased pLCK-Y394 relative to unstimulated controls. In cells expressing the scIR construct, the presence of IL-6 was associated with reduced pLCK-Y394 compared to CD3 / CD28 stimulation alone.

[0266] We next examined inhibitory phosphorylation of LCK at Y505 in scIR-expressing cells following IL-6 exposure. Immunoblot analysis showed higher pLCK-Y505 levels in the presence of IL-6 compared to no-IL-6 controls, while total LCK levels remained comparable across samples. Quantification of pLCK-Y505 normalized to total LCK indicated increased inhibitory phosphorylation in IL-6-treated conditions, though the magnitude of this effect varied. Together, these data suggest that a single-chain IL-6 receptor configuration incorporating CSK can influence LCK phosphorylation in the presence of IL-6. While this architecture is less modular than the split receptor design and requires further validation, these results provide initial evidence that IL-6Ra / gp 130-based single-chain receptors — previously used to enhance CAR-T function — can also be repurposed to engage inhibitory signaling pathways. See FIG. 12A-12C.Discussion

[0267] In this study, we investigated whether CSK activity could be synthetically modulated through engineered proximity and whether such modulation could be coupled to extracellular cues relevant to inflammatory T cell states. Prior structural and biochemical studies have shown that CSK catalytic activity can be enhanced through dimerization or scaffold-mediated clustering, which promotes autophosphorylation at Y304 and stabilizes the active kinase conformation. These observations provided a conceptual foundation for our initial chemically inducible CSK design. Consistent with this framework, we observed that enforced CSK proximity was associated withAttorney Docket No. 0073605-001129 increased inhibitory phosphorylation of LCK and attenuation of proximal TCR signaling. At the same time, the magnitude of these effects varied across experimental conditions, and we do not necessarily interpret these data as evidence of precise or quantitatively calibrated control of CSK activity.

[0268] Extending this approach, we explored whether CSK-mediated inhibition could be repositioned from a cytoplasmic configuration to transmembrane and extracellularly addressable architectures. Previous work in synthetic receptor engineering has demonstrated that intracellular signaling modules can remain functional when repositioned within membrane-spanning scaffolds, provided that proximity to their substrates is maintained. Our observations that transmembrane CSK configurations can influence LCK phosphorylation are consistent with this literature; however, variability across constructs and stimulation conditions suggests that spatial organization, linker composition, and expression levels likely contribute to the observed effects and will require further optimization.

[0269] A rationale for coupling CSK activity to extracellular cytokine sensing was the well-established role of interleukin-6 (IL-6) in inflammatory T cell responses and cytokine release syndrome (CRS). Prior work has demonstrated that IL-6 receptor components can be repurposed in engineered T cells to drive non-canonical signaling outcomes. Notably, Kagoya and colleagues reported a chimeric cytokine receptor in CAR-T cells in which the extracellular domains of IL-6Ra and gpl30 were fused to the intracellular domain of the IL-7 receptor, enabling IL-6-rich environments to support CAR-T cell persistence (Cell, 2018). This study provided an important precedent that IL-6Ra / gpl30 architectures can be functionally integrated into engineered receptors in T cells. Building on this work, we examined whether IL-6 receptor configurations could be adapted to engage inhibitory signaling through CSK rather than cytokine-driven activation. Our data suggest that IL-6-responsive CSK constructs can influence LCK phosphorylation in the presence of IL-6, consistent with the biochemical relationship between CSK and Src-family kinases.Importantly, these designs do not necessarily confer precise cytokine sensing, defined activation thresholds, or proportional signal transduction. Rather, the observed changes in proximal signaling should be interpreted as initial evidence that inflammatory cytokine cues can be coupled to negative regulation of TCR signaling through engineered receptor architectures.

[0270] We further explored both split and single-chain IL-6 receptor configurations, drawing on prior receptor engineering strategies that balance modularity and simplicity. While split receptor designs offer conceptual advantages in terms of tunability and geometric control, single-chain architectures have been successfully employed in CAR-T contexts to reduce complexity. OurAttorney Docket No. 0073605-001129 comparative analyses indicate that both configurations can modulate CSK-dependent signaling, though differences in consistency and magnitude highlight the need for additional structure-function studies. We do not establish design rules that would favor one architecture over the other, and further optimization can be used to clarify their respective advantages and limitations.

[0271] Several aspects of this work may also underscore the need for cautious interpretation. Our analyses focus primarily on proximal signaling events, such as LCK phosphorylation, which serve as established indicators of TCR activation state but do not capture the full spectrum of T cell functional outcomes. Moreover, the IL-6-responsive systems were evaluated under controlled in vitro conditions and do not necessarily account for the complexity of in vivo inflammatory environments where multiple cytokines, receptor cross-talk, and feedback loops coexist. As a result, the specificity of the observed effects to IL-6 signaling and their behavior in more physiologically relevant contexts remain open questions. Taken together, this study builds on extensive prior research in CSK regulation, Src -family kinase signaling, and cytokine receptor engineering to explore a new design space in which inflammatory cues are linked to inhibitory control of T cell signaling. Rather than necessarily demonstrating a finalized or optimized system, our findings provide proof-of-concept evidence that CSK activity can be modulated through both chemical and cytokine-responsive mechanisms. More broadly, this work suggests a potential framework for integrating negative feedback into engineered T cells, complementing existing strategies that primarily emphasize enhancement of activation or persistence. Future studies may be needed to refine these designs, define their operating boundaries, and assess their behavior in more complex biological settings.Methods

[0272] Cell Lines and Culture

[0273] Jurkat T cells (Clone E6-1; ATCC, Cat. #TIB-152), Jurkat NF AT -luciferase reporter cells, and Jurkat IL-2-luciferase reporter cells were maintained in RPMI-1640 medium (Gibco, Cat. #11875093) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS; Gibco, Cat. #10438026), 100 U mL’1penicillin and 100 pg mL'1streptomycin (Gibco, Cat. #15140122), and 2 mM L-glutamine (Gibco, Cat. #25030081).

[0274] The human B-acute lymphoblastic leukemia cell line BV173 (DSMZ, Cat. #ACC-21), Burkitt lymphoma RAJI cells (ATCC, Cat. #CCL-86), erythroleukemia K562 cells (ATCC, Cat. #CCL-243), and CD19-transduced K562-hCD19+cells were cultured in complete RPMI-1640 medium as described above and used as target cells for cytotoxicity, signaling, and co-culture experiments.Attorney Docket No. 0073605-001129

[0275] All cell lines were maintained at 37 °C in a humidified incubator with 5% CO2 and were passaged every 2-3 days to maintain logarithmic growth. Cell density was routinely kept below 1 x 106cells mL'1to prevent nutrient depletion and spontaneous activation. All parental and engineered cell lines were regularly screened for mycoplasma contamination using the MycoAlert™ Mycoplasma Detection Kit (Lonza, Cat. #LT07-318) and confirmed negative prior to experimentation. Only low-passage cells (passages 3-7 after thawing) were used throughout the study.

[0276] Primary Human T-Cell Isolation, Thawing, and Expansion

[0277] Cryopreserved human peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained from Stemcell Technologies (Cat. #70025.1). Vials were equilibrated on ice for approximately 30 seconds and rapidly thawed in a 37 °C water bath until a small ice crystal remained. Cells were immediately transferred dropwise into pre-warmed RPMI-1640 medium supplemented with 10% FBS while gently swirling to minimize osmotic shock.

[0278] When visible cell clumping was observed, DNase I (Sigma-Aldrich) was added to a final concentration of 100 pg mL'1and incubated for 15 minutes at room temperature. Cells were subsequently filtered through a 70-pm cell strainer, washed twice with RPMI-1640 medium, and counted using trypan blue exclusion.

[0279] Human T cells were isolated from PBMCs by negative selection using the EasySep™ Human T-Cell Isolation Kit (Stemcell Technologies, Cat. #17951), following the manufacturer’s protocol. This approach depletes non-T cells through magnetic labeling with a cocktail of biotinylated antibodies. Purity was routinely assessed by flow cytometry using anti-CD3-FITC (BioLegend, Cat. #300306) and consistently exceeded 95%.

[0280] Isolated T cells were cultured in OpTmizer™ T-Cell Expansion Serum-Free Medium (Gibco, Cat. #A1048501) supplemented with recombinant human IL-2, IL-7, and IL-15 (each at 10 ng mL'1; PeproTech, Cat. #200-02, #200-07, #200-15). Cells were maintained at densities between 2 x 105and 1 * 106cells mL'1.

[0281] T-Cell Activation Prior to Transduction

[0282] For lentiviral transduction, freshly isolated T cells were activated for 48 hours using anti-CD3 / CD28 antibody complexes (Stemcell Technologies, Cat. #10971), according to the manufacturer’s instructions. Activation was performed in cytokine-supplemented OpTmizer™ medium to promote efficient viral entry and early expansion while minimizing excessive differentiation.

[0283] Lentiviral Vector DesignAttorney Docket No. 0073605-001129

[0284] All lentiviral constructs encoding CSKdim, extracellular CSK variants, IL-6-responsive CSK receptors, CAR constructs, and fluorescent reporters were cloned into third-generation lentiviral backbones under the control of the EFla promoter. A complete list of constructs, domain boundaries, and tags is provided in Table 1 below. All constructs were sequence-verified prior to virus production. All plasmid were synthesized at Gene Universal company.

[0285] Lentiviral Particle Production

[0286] Lentiviral particles were generated by transient transfection of HEK 293T cells (ATCC, Cat. #CRL-3216) using a third-generation packaging system. HEK 293T cells were seeded in 10-cm tissue culture dishes at approximately 70% confluence in DMEM (Gibco, Cat. #11965092) supplemented with 10% FBS and 1% Penicillin-Streptomycin.

[0287] Cells were co-transfected with the lentiviral transfer plasmid encoding the construct of interest, the packaging plasmid psPAX2 (Addgene plasmid #12260), and the envelope plasmid pMD2.G (Addgene plasmid #12259). DNA plasmids were diluted in Opti-MEM™ Reduced-Serum Medium (Gibco, Cat. #31985070) and complexed with Lipofectamine™ 2000 (Invitrogen, Cat. #11668-019) at a ratio of 2.5 pL Lipofectamine per pg total DNA. Complexes were incubated for 5 minutes at room temperature before being added dropwise to the cells.

[0288] Viral supernatants were collected at 48 and 72 hours post-transfection, clarified by centrifugation at 300 x g for 5 minutes, and filtered through 0.45-pm PVDF filters (Millipore). When indicated, virus was concentrated using Lenti-X™ Concentrator (Takara, Cat. #631231) according to the manufacturer’s instructions.

[0289] Viral Titer Estimation

[0290] Functional viral titers were estimated by infecting Jurkat T cells with serial dilutions of viral supernatant in the presence of 8 pg mL-1polybrene (Sigma-Aldrich, Cat. #TR-1003). After 72 hours, the percentage of GFP -positive cells was quantified by flow cytometry using a BD LSRFortessa™ analyzer. Infectious units per milliliter (IFU mL'1) were calculated, and all experimental transductions were performed at an MOI between 1 and 2 to favor predominantly single-copy integration.

[0291] Lentiviral Transduction of Jurkat and K562 Cells

[0292] Jurkat and K562-hCD19+cells were transduced by incubation with lentiviral supernatant. Cells (1x106per well in 6-well plates) were resuspended in complete RPML1640 medium containing 8 pg mL'1polybrene and the appropriate volume of viral supernatant. Cells were incubated for 24 hours at 37 °C without centrifugation. Medium was then replaced with fresh complete medium, and cells were allowed to recover for 48 hours.Attorney Docket No. 0073605-001129

[0293] Puromycin selection was initiated at 0.5 jug ml1and continued for 3-5 days until uninfected control cells were eliminated. Cells expressing fluorescent reporters were enriched by fluorescence-activated cell sorting and expanded for at least one week prior to downstream assays.

[0294] RetroNectin-Mediated Lentiviral Transduction of Primary T Cells

[0295] Primary human T cells were transduced using RetroNectin-coated plates to enhance lentiviral delivery efficiency. Non-tissue-culture-treated 6-well plates (Corning, Cat. #3736) were coated overnight at 4 °C with 20 pg ml / 1RetroNectin (Takara, Cat. #T100A / B) diluted in sterile PBS. Plates were washed twice with PBS and blocked with 2% (w / v) bovine serum albumin (BSA; Sigma-Aldrich, Cat. #A9647) for 30 minutes at room temperature.

[0296] Concentrated lentiviral supernatant was added to the coated wells and centrifuged at 2,000 x g for 2 hours at 32 °C to allow viral adsorption. Activated T cells were gently added to virus-bound wells and centrifuged at 500 * g for 10 minutes to promote cell-virus contact. Cells were incubated at 37 °C and expanded in cytokine-supplemented medium. After 48-72 hours, cells were washed extensively with PBS and expanded further. Transduction efficiency and viability (>90%) were confirmed by flow cytometry. To reduce or minimize activation-induced exhaustion, T cells were rested for 48 hours prior to functional assays.

[0297] Western Blotting

[0298] Cells were treated as indicated with AP20187 (MedChemExpress, Cat. #HY-13992), recombinant human IL-6 (PeproTech, Cat. #200-06), or stimulation antibodies. Cells were lysed in PhosphoSafe™ Extraction Buffer (Millipore, Cat. #71296) supplemented with protease and phosphatase inhibitors. Equal protein amounts were resolved on 4-12% Bis-Tris gels, transferred to PVDF membranes, blocked in 5% BSA, and probed with phospho-specific and total protein antibodies. Signals were detected using enhanced chemiluminescence and quantified using ImageJ.

[0299] Flow Cytometry

[0300] Phospho-Flow

[0301] Cells were fixed, permeabilized, and stained with antibodies against phosphorylated LCK Tyr394 and Tyr5O5. Data were acquired on a BD LSRFortessa™ and analyzed using FlowJo™ software.

[0302] Intracellular Cytokine Staining

[0303] Following stimulation, Brefeldin A (BioLegend, Cat. #420601) was added for the final 4 hours. Cells were surface-stained, fixed, permeabilized, and stained intracellularly for IL-2, IFN-y, and TNF-a. Compensation and FMO controls were included for all experiments.

[0304] Chemical Inducers and CytokinesAttorney Docket No. 0073605-001129

[0305] The B / B homodimerizer AP20187 was purchased from TAKARA- diluted in ethanol 0.5mm. Aliquots were stored at -20 °C. Unless otherwise indicated, AP20187 was used at a final concentration of 10 nM, and DMSO vehicle controls were included in all experiments.

[0306] Recombinant human IL-6 was obtained from PeproTech (Cat. #200-06) and reconstituted in sterile PBS containing 0.1% BSA. Cytokine stocks were aliquoted and stored at -80 °C to avoid repeated freeze-thaw cycles.

[0307] TCR Stimulation

[0308] For Jurkat and primary T-cell stimulation, anti-CD3 / CD28 Dynabeads™ (Thermo Fisher Scientific, Cat. #1113 ID) or antibody complexes (Stemcell Technologies, Cat. #10971) were used at a bead-to-cell ratio of 1:5 unless otherwise indicated. For phospho-signaling assays, stimulation times ranged from 5 to 30 minutes. For cytokine secretion assays, stimulation was performed for 16-24 hours.

[0309] Luciferase Reporter Assays

[0310] Jurkat NFAT-luciferase and Jurkat IL-2-luciferase reporter cells were seeded at 1 x 105cells per well in white, opaque 96-well plates (Corning). Cells were pre-treated with AP20187 or vehicle control for 12 hours prior to stimulation. Cells were stimulated with anti-CD3 / CD28 antibodies for 6 hours unless otherwise specified. Luciferase activity was quantified using the Bright-Glo™ Luciferase Assay System (Promega, Cat. #E2610) according to the manufacturer’s instructions. Luminescence was measured on a SpectraMax i3x plate reader.

[0311] Raw relative light units (RLU) were background-subtracted and normalized to unstimulated controls within each experiment.

[0312] Enzyme-Linked Immunosorbent Assays (ELISA)

[0313] Supernatants from stimulated T-cell or CAR-T cell cultures were collected, centrifuged at 500 x for 5 minutes to remove cellular debris, and stored at -80 °C until analysis.

[0314] Human IL-2 and IFN-y concentrations were quantified using ELISA kits (BioLegend ELISA MAX1MDeluxe Sets; IL-2 Cat. #431801; IFN-y Cat. #430104). Assays were performed according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader, and cytokine concentrations were calculated using standard curves generated with recombinant cytokine standards.

[0315] For experiments involving AP20187 washout, supernatants were collected before treatment, during treatment, and after ligand removal to assess reversibility.

[0316] Intracellular Cytokine Reversibility AssaysAttorney Docket No. 0073605-001129

[0317] For reversibility experiments, primary T cells or CAR-T cells were stimulated with anti-CD3 / CD28 antibodies in the presence of AP20187 for 16 hours. The ligand was then removed by washing cells three times with cytokine-supplemented medium. Cells were rested for 24-48 hours before restimulation in the absence of AP20187.

[0318] Intracellular cytokine production was assessed by flow cytometry as described above.

[0319] CAR-T Cell Generation

[0320] Second- and third-generation FMC63-based anti-CD19 CAR constructs containing CD28 or 4-1BB costimulatory domains were cloned into lentiviral vectors. CAR-T cells were generated by lentiviral transduction of activated primary human T cells using RetroNectin-assisted methods as described above.

[0321] CAR expression was confirmed by flow cytometry using GFP+. Only CAR-T populations with >60% CAR expression were used for downstream assays or cells were sorted and expanded for experiments

[0322] In Vitro Cytotoxicity Assays

[0323] Short-Term Killing Assays (Flow Cytometry-Based)

[0324] CAR-T cells were co-cultured with BV173-CD19+, RAJI-CD19+, or K562-hCD19+target cells at indicated effector-to-target (E:T) ratios (ranging from 1 : 10 to 10: 1) in complete RPMI medium.

[0325] After 48 hours of co-culture, cells were stained with Zombie Aqua™ Fixable Viability Dye (BioLegend) and anti-CD3 antibodies to distinguish T cells from target cells. Target cell killing was quantified as the percentage reduction in viable CD19+target cells relative to target-only controls.

[0326] Long-Term Killing and Reversibility Assays

[0327] For long-term killing assays, CAR-T cells were co-cultured with adherent HEK 293-hCD19+or suspension targets in the presence or absence of AP20187. In washout experiments, AP20187 was removed after 48 hours, and fresh target cells were added to assess recovery of cytotoxic function.

[0328] Target cell numbers were quantified by flow cytometry or manual counting using counting beads (BioLegend).

[0329] IL-6-responsive CSK Constructs

[0330] Extracellular IL-6-responsive CSK receptors were generated by fusing FL-6Ra and gpl30 extracellular domains to transmembrane domains and intracellular CSK kinase domains via the TEL linker. Both split-receptor and single-chain receptor architectures were evaluated. CellsAttorney Docket No. 0073605-001129 expressing these constructs were treated with recombinant human IL-6 prior to stimulation.Downstream signaling was assessed by phospho-LCK immunoblotting and phospho-flow cytometry.

[0331] Exemplary amino acid sequences referenced throughout the present disclosure are provided below in Table 1, SEQ ID NOs: 1-26.

[0332] Exemplary full sequences of the amino acids, nucleic acids, and plasmid maps described herein are provided in SEQ ID NOs: 27-33.Table 1. Exemplary Amino Acid Sequences Referenced in the Present Disclosure.Attorney Docket No. 0073605-001129Attorney Docket No. 0073605-001129Attorney Docket No. 0073605-001129<<<Attorney Docket No. 0073605-001129Attorney Docket No. 0073605-001129Attorney Docket No. 0073605-001129Attorney Docket No. 0073605-001129Attorney Docket No. 0073605-001129Attorney Docket No. 0073605-001129Attorney Docket No. 0073605-001129Attorney Docket No. 0073605-001129

[0333] It should be understood that modifications to the embodiments disclosed herein can be made to meet a particular set of design criteria. For instance, the number of or configuration of components or parameters may be used to meet a particular objective.

[0334] It will be apparent to those skilled in the art that numerous modifications and variations of the described examples and embodiments are possible in light of the above teachings of the disclosure. The disclosed examples and embodiments are presented for purposes of illustration only. Other alternative embodiments may include some or all the features of the various embodiments disclosed herein. For instance, it is contemplated that a particular feature described, either individually or as part of an embodiment, can be combined with other individually described features, or parts of other embodiments. The elements and acts of the various embodiments described herein can therefore be combined to provide further embodiments.

[0335] It i s the intent to cover all such modifications and alternative embodiments as may come within the true scope of this invention, which is to be given the full breadth thereof. Additionally, the disclosure of a range of values is a disclosure of every numerical value within that range, including the end points. Thus, while certain exemplary embodiments of the device and methods of making and using the same have been discussed and illustrated herein, it is to be distinctly understood thatAttorney Docket No. 0073605-001129 the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

Claims

Attorney Docket No. 0073605-001129CLAIMS1. A synthetic protein switch having an active state and an inactive state, the synthetic protein switch comprising:(I) a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11; and(II) a sensing domain,wherein the synthetic protein switch(i) reversibly converts from the inactive state to the active state upon interaction with an inducer; and(ii) reversibly converts from the active state to the inactive state upon removal of the inducer.

2. The synthetic protein switch according to claim 1, wherein the sensing domain comprises a FKBP12F36Vsensing domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 9.

3. The synthetic protein switch according to claim 1, wherein the CSK domain contains a Y304 autophosphorylation site.

4. The synthetic protein switch according to claim 1, wherein:when the synthetic protein switch is under the active state, the sensing domain is in a dimeric state; andwhen the synthetic protein switch is under the inactive state, the sensing domain is in a monomeric state.

5. The synthetic protein switch according to claim 1, wherein, when the synthetic protein switch is under the active state, the CSK domain catalyzes phosphorylation of lymphocyte-specific protein tyrosine kinase (LCK), thereby attenuating or suppressing T-cell signaling.

6. The synthetic protein switch according to claim 5, wherein, when the synthetic protein switch is under the active state, the CSK domain enhances phosphorylation at Y505 position of the LCK and suppresses phosphorylation at Y394 position of the LCK, thereby stabilizing the LCK in an inactive conformation.

7. The synthetic protein switch according to claim 1, wherein, when the synthetic protein switch is under the active state, the CSK domain suppresses production or secretion of an inflammatory cytokine.

8. The synthetic protein switch according to claim 7, wherein the inflammatory cytokine comprises an extracellular inflammatory cytokine.Attorney Docket No. 0073605-001129 9. The synthetic protein switch according to claim 7, wherein the inflammatory cytokine comprises an intracellular cytokine.

10. The synthetic protein switch according to any one of claims 7-9, wherein the inflammatory cytokine comprises one or more of interleukin-2 (IL-2), interferon-y (IFNy), and tumor necrosis factor-a (TNFa).

11. The synthetic protein switch according to any one of claims 1-9, wherein, when the synthetic protein switch is under the active state, the CSK domain suppresses CD3(^ phosphorylation.

12. The synthetic protein switch according to any one of claims 1-9, wherein, when the synthetic protein switch is under the active state, the CSK domain suppresses cytotoxic killing of a target cell.

13. The synthetic protein switch according to any one of claims 1-9, wherein, when the synthetic protein switch is under the active state, the CSK domain suppresses antigen-dependent CAR- T activation without causing nonspecific cellular dysfunction.

14. The synthetic protein switch according to claim 4, wherein the inducer comprises a dimerizer that converts the sensing domain from the monomeric state to the dimeric state.

15. The synthetic protein switch according to claim 14, wherein the dimerizer comprises AP20187.

16. The synthetic protein switch according to any one of claims 1-9, wherein the inducer comprises a cytokine.

17. The synthetic protein switch according to claim 16, wherein the cytokine comprises interleukin-6 (IL-6).

18. The synthetic protein switch according to any one of claims 1-9, wherein the CSK domain comprises an interleukin-6 (IL-6) receptor.

19. The synthetic peptide according to claim 1, having a transmembrane configuration.

20. The synthetic protein switch according to claim 19, wherein the CSK domain is to be positioned at an inner leaflet of a plasma membrane.

21. The synthetic protein switch according to claim 19, further comprising a CD8 hinge region that is at least 70% identical to, or a functional variant of, SEQ ID NO: 26.

22. The synthetic protein switch according to claim 19, further comprising a CD28 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 25.Attorney Docket No. 0073605-001129 23. The synthetic protein switch according to claim 19, wherein the sensing domain comprises an hIL6Ra extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 13.

24. The synthetic protein switch according to claim 19, wherein the sensing domain comprises an hIL6Ra transmembrane domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 14.

25. The synthetic protein switch according to claim 19, wherein the sensing domain comprises a gpl30 extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 16.

26. The synthetic protein switch according to claim 19, wherein the sensing domain comprises a gpl30 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 22.

27. The synthetic protein switch according to claim 19, wherein the sensing domain comprises a GP130 domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 17.

28. The synthetic protein switch according to claim 19, wherein the sensing domain comprises an IL6Ra domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 19.

29. The synthetic protein switch according to claim 19, wherein the sensing domain comprises an IL7TM domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 21.

30. The synthetic protein switch according to claim 19, further comprising a signal peptide having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 15.

31. The synthetic protein switch according to any one of claims 1-9, further comprising an N- terminal myristoylation signal having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 8.

32. The synthetic protein switch according to any one of claims 1-9, further comprising one or more of:(A) a TEL linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 10; and(B) a (G3S)4linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 12.Attorney Docket No. 0073605-001129 33. The synthetic protein switch according to any one of claims 1-9, wherein the T-cell signaling is mediated by a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

34. The synthetic protein switch according to any one of claims 1-9, having a molar mass of approximately 80 kDa.

35. The synthetic protein switch according to any one of claims 1-9, wherein the CSK domain contains one or more point mutations and one or more of S186A, Y188A, and W134A positions.

36. The synthetic protein switch according to any one of claims 1-9, having an amino acid sequence that is at least 70% identical to, or a functional variant of, any one of SEQ ID NOs: 1-7.

37. Amethod for reversible modulation or regulation of T-cell signaling in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic protein switch having an active state and an inactive state, the synthetic protein switch comprising:(I) a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11; and(II) a sensing domain,wherein an interaction between the synthetic protein switch and an inducer reversibly converts the synthetic protein switch from the inactive state to the active state.

38. A method of treating a cytokine release syndrome (CRS) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a synthetic protein switch having an active state and an inactive state, the synthetic protein switch comprising:(I) a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11; and(II) a sensing domain,wherein an interaction between the synthetic protein switch and an inducer reversibly converts the synthetic protein switch from the inactive state to the active state.

39. A method of treating a hematological malignancy, comprising administering to the subject (A) a therapeutically effective amount of T cells; and(B) a therapeutically effective amount of a synthetic protein switch having an active state and an inactive state, the synthetic protein switch comprising:Attorney Docket No. 0073605-001129 (I) a C-terminal Src kinase (CSK) domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 11; and (II) a sensing domain,wherein an interaction between the synthetic protein switch and an inducer reversibly converts the synthetic protein switch from the inactive state to the active state, thereby modulating or regulating an activity of the T cells.

40. The method according to any one of claims 37-39, wherein an attenuation or removal of the inducer reversibly converts the synthetic protein switch from the active state to the inactive state.

41. The method according to any one of claims 37-39, wherein the sensing domain comprises a FKBP12F36Vsensing domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 9.

42. The method according to any one of claims 37-39, wherein the CSK domain contains a Y304 autophosphorylation site.

43. The method according to any one of claims 37-39, wherein:when the synthetic protein switch is under the active state, the sensing domain is in a dimeric state; andwhen the synthetic protein switch is under the inactive state, the sensing domain is in a monomeric state.

44. The method according to any one of claims 37-39, further comprising catalyzing phosphorylation of lymphocyte-specific protein tyrosine kinase (LCK), thereby attenuating or suppressing T-cell signaling.

45. The method according to claim 44, further comprising enhancing phosphorylation at Y5O5 position of the LCK and suppressing phosphorylation at Y394 position of the LCK, thereby stabilizing the LCK in an inactive conformation.

46. The method according to any one of claims 37-39, further comprising suppressing production or secretion of an inflammatory cytokine.

47. The method according to claim 46, wherein the inflammatory cytokine comprises an extracellular inflammatory cytokine.

48. The method according to claim 46, wherein the inflammatory cytokine comprises an intracellular cytokine.

49. The method according to claim 46, wherein the inflammatory cytokine comprises one or more of interleukin-2 (IL-2), interferon-y (IFNy), and tumor necrosis factor-a (TNFa).Attorney Docket No. 0073605-001129 50. The method according to any one of 37-39, further comprising suppressing CD3(^ phosphorylation.

51. The method according to any one of claims 37-39, further comprising suppressing cytotoxic killing of a target cell.

52. The method according to any one of claims 37-39, further comprising suppressing antigendependent CAR-T activation without causing nonspecific cellular dysfunction.

53. The method according to claim 43, further comprising converting the sensing domain from the monomeric state to the dimeric state using a dimerizer.

54. The method according to claim 53, wherein the dimerizer comprises AP20187.

55. The method according to any one of claims 37-39, wherein the inducer comprises a cytokine.

56. The method according to claim 55, wherein the cytokine comprises interleukin-6 (IL-6).

57. The method according to any one of claims 37-39, wherein the CSK domain comprises an interleukin-6 (IL-6) receptor.

58. The method according to any one of claims 37-39, wherein the synthetic protein switch has a transmembrane configuration.

59. The method according to claim 58, wherein the CSK domain is to be positioned at an inner leaflet of a plasma membrane.

60. The method according to claim 58, wherein the synthetic protein switch further comprises a CD8 hinge region that is at least 70% identical to, or a functional variant of, SEQ ID NO: 26.

61. The method according to claim 58, wherein the synthetic protein switch further comprises a CD28 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 25.

62. The method according to claim 58, wherein the sensing domain comprises an hIL6Ra extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 13.

63. The method according to claim 58, wherein the sensing domain comprises an hIL6Ra transmembrane domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 14.

64. The method according to claim 58, wherein the sensing domain comprises a gpl30 extracellular domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 16.Attorney Docket No. 0073605-001129 65. The method according to claim 58, wherein the sensing domain comprises a gpl30 transmembrane domain that is at least 70% identical to, or a functional variant of, SEQ ID NO: 22.

66. The method according to claim 58, wherein the sensing domain comprises a GP130 domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 17.

67. The method according to claim 58, wherein the sensing domain comprises an IL6Ra domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 19.

68. The method according to claim 58, wherein the sensing domain comprises an IL7TM domain having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 21.

69. The method according to claim 58, wherein the synthetic protein switch further comprises a signal peptide having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 15.

70. The method according to any one of claims 37-39, wherein the synthetic protein switch further comprises an N-terminal myristoylation signal having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 8.

71. The method according to any one of claims 37-39, wherein the synthetic protein switch further comprises one or more of:(C) a TEL linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 10; and(D) a (G3S)4linker having an amino acid sequence that is at least 70% identical to, or a functional variant of, SEQ ID NO: 12.

72. The method according to any one of claims 37-39, wherein the T-cell signaling is mediated by a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

73. The method according to any one of claims 37-39, wherein the synthetic protein switch has a molar mass of approximately 80 kDa.

74. The method according to any one of claims 37-39, wherein the CSK domain contains one or more point mutations and one or more of S186A, Y188A, and W134A positions.

75. The method according to any one of claims 37-39, wherein the synthetic protein switch has an amino acid sequence that is at least 70% identical to, or a functional variant of, any one of SEQ IDNOs: 1-7.Attorney Docket No. 0073605-001129 76. Use of the synthetic protein switch according to any one of claims 1-9 in the manufacture of a medicament for reversible modulation or regulation of T-cell signaling in a subject in need thereof.

77. Use of the synthetic protein switch according to any one of claims 1-9 in the manufacture of a medicament for treating cytokine release syndrome (CRS) in a subject in need thereof.

78. Use of the synthetic protein switch according to any one of claims 1-9 in combination with a T cell therapy in the manufacture of a medicament for treating a hematological malignancy in a subject in need thereof.

79. The use according to any one of claims 76-78, wherein the T cell therapy comprises a chimeric antigen receptor (CAR) T cell therapy or a transgenic T-cell receptor (TCR) therapy.

80. A method of producing the synthetic protein switch according to any one of claims 1-9, comprising transferring to a host cell or transfecting the host cell with a viral vector having a nucleic acid encoding the synthetic protein switch.

81. The method according to claim 80, wherein the viral vector comprises a lentiviral vector.

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